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Yeung N, Ouwehand AC. Comparison of qRT-PCR and ddPCR for multi-strain probiotic detection after a randomized human clinical trial. Front Microbiol 2025; 16:1579797. [PMID: 40356649 PMCID: PMC12066471 DOI: 10.3389/fmicb.2025.1579797] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2025] [Accepted: 04/14/2025] [Indexed: 05/15/2025] Open
Abstract
The ability to detect probiotic consumption during a human clinical trial is crucial to verify and validate placebo and verum groups in post hoc analysis. While bacterial plating is still a common method for detecting and counting bacteria, when dealing with complex matrices like fecal samples, and given that most probiotics share genera or even species with commensal bacteria, plate counting is not a precise or accurate enough method. Species-specific quantitative real-time polymerase chain reaction (qRT-PCR) has been the most cited method in the literature and when properly validated and optimized remains the high watermark for detecting probiotics from fecal samples. Recent advancements in PCR technology have given rise to a parallel platform, droplet digital PCR (ddPCR). In this work we aimed to detect the components of a multi-strain probiotic product from a human clinical trial and compare both methods. This work dually demonstrates a process for determining multi-strain detection criteria as well as directly comparing the methods through the lens of sensitivity and specificity or the ability to properly discern true positives and true negatives. We described the optimization and validation of three assays for use in our detection panel and observed that, between qRT-PCR and ddPCR. The two methods were found to be quite congruent with ddPCR demonstrating a 10-100 fold lower limit of detection. Moreover, we discovered that most of the sensitivity and specificity had come from a single assay alone (Bifidobacterium animalis subsp. lactis Bl-04). This is despite all three assays performing well in optimization and validation. This suggests that more work needs to be done in the validation stage when developing novel probiotic detection assays. Taken together we can recommend ddPCR as a method for detecting probiotics from human clinical trials, but that qRT-PCR still performs well and comparably to ddPCR, when properly optimized and validated. However, when novel assays or those with unknown performance in a given biological matrix are needed, employing a strategy that combines multiple assays in a layered discrimination approach can help mitigate the potential underperformance of any single assay.
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Luo Y, Hu Q, Yu Y, Lyu W, Shen F. Experimental investigation of confinement effect in single molecule amplification via real-time digital PCR on a multivolume droplet array SlipChip. Anal Chim Acta 2024; 1304:342541. [PMID: 38637051 DOI: 10.1016/j.aca.2024.342541] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2024] [Revised: 03/19/2024] [Accepted: 03/25/2024] [Indexed: 04/20/2024]
Abstract
BACKGROUND Digital polymerase chain reaction (digital PCR) is an important quantitative nucleic acid analysis method in both life science research and clinical diagnostics. One important hypothesis is that by physically constraining a single nucleic acid molecule in a small volume, the relative concentration can be increased therefore further improving the analysis performance, and this is commonly defined as the confinement effect in digital PCR. However, experimental investigation of this confinement effect can be challenging since it requires a microfluidic device that can generate partitions of different volumes and an instrument that can monitor the kinetics of amplification. (96). RESULTS Here, we developed a real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip) that can generate droplet of 125 nL, 25 nL, 5 nL, and 1 nL by a simple "load-slip" operation. In the digital region, by reducing the volume, the relative concentration is increased, the amplification kinetic can be accelerated, and the time to reach the fluorescence threshold, or Cq value, can be reduced. When the copy number per well is much higher than one, the relative concentration is independent of the partition volume, thus the amplification kinetics are similar in different volume partitions. This system is not limited to studying the kinetics of digital nucleic acid amplification, it can also extend the dynamic range of the digital nucleic acid analysis by additional three orders of magnitude by combining a digital and an analog quantification algorithm. (140). SIGNIFICANCE In this study, we experimentally investigated for the first time the confinement effect in the community of digital PCR via a new real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip). And a wider dynamic range of quantification methods compared to conventional digital PCR was validated by this system. This system provides emerging opportunities for life science research and clinical diagnostics. (63).
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Affiliation(s)
- Yang Luo
- School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Hua Shan Road, Shanghai, 200030, PR China
| | - Qixin Hu
- School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Hua Shan Road, Shanghai, 200030, PR China
| | - Yan Yu
- School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Hua Shan Road, Shanghai, 200030, PR China
| | - Weiyuan Lyu
- School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Hua Shan Road, Shanghai, 200030, PR China
| | - Feng Shen
- School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Hua Shan Road, Shanghai, 200030, PR China.
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Shin JC, Jeong JY, Son SG, Choi SH, Nam HC, Yoon TH, Kim HJ, Choi DG, Lee H, Lee U, Yang SM, Kang I, Jung DY, Lee HW, Lee MK, Lee TJ, Kim G, Park HO, Lee SW. Developing centrifugal force real-time digital PCR for detecting extremely low DNA concentration. Sci Rep 2024; 14:11522. [PMID: 38769102 PMCID: PMC11636855 DOI: 10.1038/s41598-024-62199-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2024] [Accepted: 05/14/2024] [Indexed: 05/22/2024] Open
Abstract
Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/μL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | - Ukyeol Lee
- RevoSketch Inc., Daejeon, Republic of Korea
| | | | - Il Kang
- RevoSketch Inc., Daejeon, Republic of Korea
| | | | | | - Moon-Keun Lee
- Center for Nano Bio Development, National NanoFab Center (NNFC), Daejeon, Republic of Korea
| | - Tae Jae Lee
- Center for Nano Bio Development, National NanoFab Center (NNFC), Daejeon, Republic of Korea
| | - Geehong Kim
- Nano-Convergence Systems Research Division, Korea Institute of Machinery & Materials, Daejeon, Republic of Korea
| | - Han-Oh Park
- Bioneer Corporation, Daejeon, Republic of Korea
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Ganguly R, Lee CS. A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets. ACS APPLIED BIO MATERIALS 2024; 7:3441-3451. [PMID: 38658190 DOI: 10.1021/acsabm.4c00350] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/26/2024]
Abstract
Digital PCR (dPCR) has become indispensable in nucleic acid (NA) detection across various fields, including viral diagnostics and mutant detection. However, misclassification of partitions in dPCR can significantly impact accuracy. Despite existing methods to minimize misclassification bias, accurate classification remains elusive, especially for nonamplified target partitions. To address these challenges, this study introduces an innovative microdroplet-based competitive PCR platform for nucleic acid quantification in microfluidic devices independent of Poisson statistics. In this approach, the target concentration (T) is determined from the concentration of competitor DNA (C) at the equivalence point (E.P.), where C/T is 1. Competitive PCR ensures that the ratio of target to competitor DNA remains constant during amplification, reflected in the resultant fluorescence intensity, allowing the quantification of target DNA concentration at the equivalence point. The unique amplification technique eliminates Poisson distribution, addressing misclassification challenges. Additionally, our approach reduces the need for post-PCR procedures and shortens analytical time. We envision this platform as versatile, reproducible, and easily adaptable for driving significant progress in molecular biology and diagnostics.
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Affiliation(s)
- Reya Ganguly
- Department of Chemical Engineering and Applied Chemistry, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea
| | - Chang-Soo Lee
- Department of Chemical Engineering and Applied Chemistry, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea
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Bustin SA. Improving the quality of quantitative polymerase chain reaction experiments: 15 years of MIQE. Mol Aspects Med 2024; 96:101249. [PMID: 38290180 DOI: 10.1016/j.mam.2024.101249] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2023] [Revised: 01/02/2024] [Accepted: 01/15/2024] [Indexed: 02/01/2024]
Abstract
The quantitative polymerase chain reaction (qPCR) is fundamental to molecular biology. It is not just a laboratory technique, qPCR is a bridge between research and clinical practice. Its theoretical foundations guide the design of experiments, while its practical implications extend to diagnostics, treatment, and research advancements in the life sciences, human and veterinary medicine, agriculture, and forensics. However, the accuracy, reliability and reproducibility of qPCR data face challenges arising from various factors associated with experimental design, execution, data analysis and inadequate reporting details. Addressing these concerns, the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have emerged as a cohesive framework offering a standardised set of recommendations that describe the essential information required for assessing qPCR experiments. By emphasising the importance of methodological rigour, the MIQE guidelines have made a major contribution to improving the trustworthiness, consistency, and transparency of many published qPCR results. However, major challenges related to awareness, resources, and publication pressures continue to affect their consistent application.
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Affiliation(s)
- Stephen A Bustin
- Medical Technology Research Centre, Anglia Ruskin University, Chelmsford, Essex, CM1 1SQ, UK.
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Lee SJ, Lee J, Ahn A, Lee S, Hong Y, Lee GD, Song H, Song M, Shin S, Kim M, Kim Y. Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts. J Clin Lab Anal 2024; 38:e25034. [PMID: 38525919 PMCID: PMC11033343 DOI: 10.1002/jcla.25034] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2023] [Revised: 02/09/2024] [Accepted: 03/09/2024] [Indexed: 03/26/2024] Open
Abstract
BACKGROUND Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. METHODS The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. RESULTS Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. CONCLUSION Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.
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Affiliation(s)
- Soo Jung Lee
- Department of Laboratory MedicineSeoul St. Mary's Hospital, College of Medicine, The Catholic University of KoreaSeoulKorea
| | - Jong‐Mi Lee
- Department of Laboratory MedicineSeoul St. Mary's Hospital, College of Medicine, The Catholic University of KoreaSeoulKorea
| | - Ari Ahn
- Department of Laboratory Medicine, Incheon St. Mary's Hospital, College of MedicineThe Catholic University of KoreaSeoulKorea
| | - Sung‐Eun Lee
- Department of Hematology, Catholic Hematology Hospital, Seoul St. Mary's Hospital, College of MedicineThe Catholic University of KoreaSeoulKorea
| | - Yuna Hong
- Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of MedicineThe Catholic University of KoreaSeoulKorea
- Department of Biomedicine & Health Sciences, Graduate SchoolThe Catholic University of KoreaSeoulKorea
| | - Gun Dong Lee
- Department of Laboratory MedicineSeoul St. Mary's Hospital, College of Medicine, The Catholic University of KoreaSeoulKorea
| | - Hyun‐Woo Song
- Bio Institute, Optolane Technologies Inc.PangyoSeong‐Nam‐SiKorea
| | - Min‐Sik Song
- Bio Institute, Optolane Technologies Inc.PangyoSeong‐Nam‐SiKorea
| | - Seung‐Shick Shin
- Bio Institute, Optolane Technologies Inc.PangyoSeong‐Nam‐SiKorea
| | - Myungshin Kim
- Department of Laboratory MedicineSeoul St. Mary's Hospital, College of Medicine, The Catholic University of KoreaSeoulKorea
- Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of MedicineThe Catholic University of KoreaSeoulKorea
| | - Yonggoo Kim
- Department of Laboratory MedicineSeoul St. Mary's Hospital, College of Medicine, The Catholic University of KoreaSeoulKorea
- Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of MedicineThe Catholic University of KoreaSeoulKorea
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Tanvetthayanont P, Yata T, Boonnil J, Temisak S, Ponglowhapan S. Advancing canine mammary tumor diagnostics: Unraveling the diagnostic potential of Cytokeratin 19 through droplet digital PCR analysis. Theriogenology 2024; 217:127-135. [PMID: 38271766 DOI: 10.1016/j.theriogenology.2024.01.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Revised: 01/13/2024] [Accepted: 01/14/2024] [Indexed: 01/27/2024]
Abstract
Cytokeratin 19 (CK19) is a complex intracytoplasmic cytoskeletal protein primarily localized in the ducts of the mammary gland and skin epithelial cells. In humans, the expression of CK19 gene within circulating tumor cells (CTCs) extracted from blood samples of breast cancer patients reflects tumor cell activity, offering valuable insights for predicting early metastatic relapse or monitoring treatment effectiveness. However, knowledge of serum tumor markers is limited in veterinary oncology. Recently, droplet digital PCR (ddPCR), has been employed to explore rare target genes due to its heightened sensitivity and accuracy as a novel molecular diagnostic tool. The objectives of this study were to investigate the expression of the CK19 mRNA in CTCs, non-neoplastic mammary tissues, and both benign and malignant canine mammary tumors (CMTs) through ddPCR analysis. In Study I, we optimized the discard volume for blood samples to reduce CK19 contamination from skin epithelial cells post-venipuncture. The results revealed that discarding the initial 3 mL of blood was adequate and effective in eliminating CK19 mRNA contamination. In Study II, after the removal of the initial 3 mL of blood, we investigated CK19 mRNA-positive CTCs in the peripheral blood of normal healthy dogs, including those with benign and malignant CMTs. Intriguingly, CK19 mRNA was undetectable in all blood samples. The expression of CK19 mRNA in mammary tissues was investigated in Study III. The copy number (CN) ratios of the CK19 gene in non-neoplastic mammary tissues (14.77 ± 14.65) were significantly higher (P < 0.05) than those in benign (4.23 ± 3.35) and malignant groups (6.56 ± 5.64). Notably, no difference was observed between the benign and malignant groups. In conclusion, CK19 mRNA appeared unlikely to be a suitable candidate as a biomarker in the peripheral blood of CMTs, while the CN ratio in mammary tissues could serve as a potential discriminator between non-neoplastic and CMT groups, complementing the gold standard of histopathological examination.
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Affiliation(s)
- Potsawat Tanvetthayanont
- Department of Obstetric Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand
| | - Teerapong Yata
- Unit of Biochemistry, Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand
| | - Jiranun Boonnil
- National Institute of Metrology (NIMT), Pathumthani, 12120, Thailand
| | - Sasithon Temisak
- National Institute of Metrology (NIMT), Pathumthani, 12120, Thailand.
| | - Suppawiwat Ponglowhapan
- Department of Obstetric Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand.
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Parkins MD, Lee BE, Acosta N, Bautista M, Hubert CRJ, Hrudey SE, Frankowski K, Pang XL. Wastewater-based surveillance as a tool for public health action: SARS-CoV-2 and beyond. Clin Microbiol Rev 2024; 37:e0010322. [PMID: 38095438 PMCID: PMC10938902 DOI: 10.1128/cmr.00103-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/16/2024] Open
Abstract
Wastewater-based surveillance (WBS) has undergone dramatic advancement in the context of the coronavirus disease 2019 (COVID-19) pandemic. The power and potential of this platform technology were rapidly realized when it became evident that not only did WBS-measured SARS-CoV-2 RNA correlate strongly with COVID-19 clinical disease within monitored populations but also, in fact, it functioned as a leading indicator. Teams from across the globe rapidly innovated novel approaches by which wastewater could be collected from diverse sewersheds ranging from wastewater treatment plants (enabling community-level surveillance) to more granular locations including individual neighborhoods and high-risk buildings such as long-term care facilities (LTCF). Efficient processes enabled SARS-CoV-2 RNA extraction and concentration from the highly dilute wastewater matrix. Molecular and genomic tools to identify, quantify, and characterize SARS-CoV-2 and its various variants were adapted from clinical programs and applied to these mixed environmental systems. Novel data-sharing tools allowed this information to be mobilized and made immediately available to public health and government decision-makers and even the public, enabling evidence-informed decision-making based on local disease dynamics. WBS has since been recognized as a tool of transformative potential, providing near-real-time cost-effective, objective, comprehensive, and inclusive data on the changing prevalence of measured analytes across space and time in populations. However, as a consequence of rapid innovation from hundreds of teams simultaneously, tremendous heterogeneity currently exists in the SARS-CoV-2 WBS literature. This manuscript provides a state-of-the-art review of WBS as established with SARS-CoV-2 and details the current work underway expanding its scope to other infectious disease targets.
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Affiliation(s)
- Michael D. Parkins
- Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada
- Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
- O’Brien Institute of Public Health, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
| | - Bonita E. Lee
- Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Nicole Acosta
- Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
| | - Maria Bautista
- Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, Alberta, Canada
| | - Casey R. J. Hubert
- Department of Biological Sciences, Faculty of Science, University of Calgary, Calgary, Alberta, Canada
| | - Steve E. Hrudey
- Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
| | - Kevin Frankowski
- Advancing Canadian Water Assets, University of Calgary, Calgary, Alberta, Canada
| | - Xiao-Li Pang
- Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
- Provincial Health Laboratory, Alberta Health Services, Calgary, Alberta, Canada
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Hayden RT, Su Y, Tang L, Zhu H, Gu Z, Glasgow HL, Sam SS, Caliendo AM. Accuracy of quantitative viral secondary standards: a re-examination. J Clin Microbiol 2024; 62:e0166923. [PMID: 38380932 PMCID: PMC10935634 DOI: 10.1128/jcm.01669-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2023] [Accepted: 01/24/2024] [Indexed: 02/22/2024] Open
Abstract
Interlaboratory agreement of viral load assays depends on the accuracy and uniformity of quantitative calibrators. Previous work demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values in both log10 copies/mL and log10 international unit (IU)/mL, with bias from manufacturer-assigned nominal values of 0.0-0.9 log10 units (either copies or IU)/mL. Standards normalized to IU and those values assigned by dPCR rather than by real-time PCR (qPCR) showed better agreement with nominal values. The latter reinforces prior conclusions regarding the utility of using such methods for quantitative value assignment in reference materials. Quantitative standards have improved over the last several years, and the remaining bias from nominal values might be further reduced by universal implementation of dPCR methods for value assignment, normalized to IU. IMPORTANCE Interlaboratory agreement of viral load assays depends on accuracy and uniformity of quantitative calibrators. Previous work, published in JCM several years ago, demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values, indicating a substantial improvement in the production of accurate secondary viral standards, while supporting the need for further work in this area and for the broad adaption of international unit (IU) as a reporting standard for quantitative viral load results.
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Affiliation(s)
- R. T. Hayden
- Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - Y. Su
- Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - L. Tang
- Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - H. Zhu
- Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - Z. Gu
- Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - H. L. Glasgow
- Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - S. S. Sam
- Division of Infectious Diseases, Alpert Medical School of Brown University, Providence, Rhode Island, USA
| | - A. M. Caliendo
- Department of Medicine, Alpert Medical School of Brown University, Providence, Rhode Island, USA
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Takahara H, Tanaka H, Hashimoto M. Fast Thermocycling in Custom Microfluidic Cartridge for Rapid Single-Molecule Droplet PCR. SENSORS (BASEL, SWITZERLAND) 2023; 23:9884. [PMID: 38139729 PMCID: PMC10747138 DOI: 10.3390/s23249884] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/18/2023] [Revised: 12/13/2023] [Accepted: 12/14/2023] [Indexed: 12/24/2023]
Abstract
The microfluidic droplet polymerase chain reaction (PCR), which enables simultaneous DNA amplification in numerous droplets, has led to the discovery of various applications that were previously deemed unattainable. Decades ago, it was demonstrated that the temperature holding periods at the denaturation and annealing stages in thermal cycles for PCR amplification could be essentially eliminated if a rapid change of temperature for an entire PCR mixture was achieved. Microfluidic devices facilitating the application of such fast thermocycling protocols have significantly reduced the time required for PCR. However, in microfluidic droplet PCR, ensuring successful amplification from single molecules within droplets has limited studies on accelerating assays through fast thermocycling. Our developed microfluidic cartridge, distinguished for its convenience in executing single-molecule droplet PCR with common laboratory equipment, features droplets positioned on a thin glass slide. We hypothesized that applying fast thermocycling to this cartridge would achieve single-molecule droplet PCR amplification. Indeed, the application of this fast protocol demonstrated successful amplification in just 22 min for 30 cycles (40 s/cycle). This breakthrough is noteworthy for its potential to expedite microfluidic droplet PCR assays, ensuring efficient single-molecule amplification within a remarkably short timeframe.
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Affiliation(s)
| | | | - Masahiko Hashimoto
- Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, 1-3 Tataramiyakodani, Kyotanabe 610-0321, Kyoto, Japan
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Boyte ME, Benkowski A, Pane M, Shehata HR. Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques. Front Microbiol 2023; 14:1304621. [PMID: 38192285 PMCID: PMC10773886 DOI: 10.3389/fmicb.2023.1304621] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2023] [Accepted: 11/20/2023] [Indexed: 01/10/2024] Open
Abstract
Probiotics are the largest non-herbal/traditional dietary supplements category worldwide. To be effective, a probiotic strain must be delivered viable at an adequate dose proven to deliver a health benefit. The objective of this article is to provide an overview of the various technologies available for probiotic enumeration, including a general description of each technology, their advantages and limitations, and their potential for the future of the probiotics industry. The current "gold standard" for analytical quantification of probiotics in the probiotic industry is the Plate Count method (PC). PC measures the bacterial cell's ability to proliferate into detectable colonies, thus PC relies on cultivability as a measure of viability. Although viability has widely been measured by cultivability, there has been agreement that the definition of viability is not limited to cultivability. For example, bacterial cells may exist in a state known as viable but not culturable (VBNC) where the cells lose cultivability but can maintain some of the characteristics of viable cells as well as probiotic properties. This led to questioning the association between viability and cultivability and the accuracy of PC in enumerating all the viable cells in probiotic products. PC has always been an estimate of the number of viable cells and not a true cell count. Additionally, newer probiotic categories such as Next Generation Probiotics (NGPs) are difficult to culture in routine laboratories as NGPs are often strict anaerobes with extreme sensitivity to atmospheric oxygen. Thus, accurate quantification using culture-based techniques will be complicated. Another emerging category of biotics is postbiotics, which are inanimate microorganisms, also often referred to as tyndallized or heat-killed bacteria. Obviously, culture dependent methods are not suitable for these products, and alternative methods are needed for their quantification. Different methodologies provide a more complete picture of a heterogeneous bacterial population versus PC focusing exclusively on the eventual multiplication of the cells. Alternative culture-independent techniques including real-time PCR, digital PCR and flow cytometry are discussed. These methods can measure viability beyond cultivability (i.e., by measuring cellular enzymatic activity, membrane integrity or membrane potential), and depending on how they are designed they can achieve strain-specific enumeration.
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Affiliation(s)
- Marie-Eve Boyte
- NutraPharma Consulting Services Inc., Sainte-Anne-des-Plaines, QC, Canada
| | | | - Marco Pane
- Probiotical Research s.r.l., Novara, Italy
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Rao A, Lin J, Parsons R, Greenleaf M, Westbrook A, Lai E, Bowers HB, McClendon K, O’Sick W, Baugh T, Sifford M, Sullivan JA, Lam WA, Bassit L. Standardization and Comparison of Emergency Use Authorized COVID-19 Assays and Testing Laboratories. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2023:2023.11.08.23297633. [PMID: 37986832 PMCID: PMC10659510 DOI: 10.1101/2023.11.08.23297633] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2023]
Abstract
Motivation The motivation for this work was the need to establish a predefined cutoff based on genome copies per ml (GE/ml) rather than Ct, which can vary depending on the laboratory and assay used. A GE/ml-based threshold was necessary to define what constituted 'low positives" for samples that were included in data sets submitted to the FDA for emergency use approval for SARS-CoV-2 antigen tests. Summary SARS-CoV-2, the causal agent of the global COVID-19 pandemic, made its appearance at the end of 2019 and is still circulating in the population. The pandemic led to an urgent need for fast, reliable, and widely available testing. After December 2020, the emergence of new variants of SARS-CoV-2 led to additional challenges since new and existing tests had to detect variants to the same extent as the original Wuhan strain. When an antigen-based test is submitted to the Food and Drug Administration (FDA) for Emergency Use Authorization (EUA) consideration it is benchmarked against PCR comparator assays, which yield cycle threshold (CT) data as an indirect indicator of viral load - the lower the CT, the higher the viral load of the sample and the higher the CT, the lower the viral load. The FDA mandates that 10-20% of clinical samples used to evaluate the antigen test have to be low positive. Low positive, as defined by the FDA, are clinical samples in which the CT of the SARS-CoV-2 target gene is within 3 CT of the mean CT value of the approved comparator test's Limit of Detection (LOD). While all comparator tests are PCR-based, the results from different PCR assays used are not uniform. Results vary depending on assay platform, target gene, LOD and laboratory methodology. The emergence and dominance of the Omicron variant further challenged this approach as the fraction of low positive clinical samples dramatically increased as compared to earlier SARS-CoV-2 variants. This led to 20-40% of clinical samples having high CT values and therefore assays vying for an EUA were failing to achieve the 80% Percent Positive Agreement (PPA) threshold required. Here we describe the methods and statistical analyses used to establish a predefined cutoff, based on genome copies per ml (GE/ml) to classify samples as low positive (less than the cutoff GE/ml) or high positive (greater than the cutoff GE/mL). CT 30 for the E gene target using Cobas® SARS-CoV-2-FluA/B platform performed at TriCore Reference Laboratories, and this low positive cutoff value was used for two EUA authorizations. Using droplet digital PCR and methods described here, a value 49,447.72 was determined as the GE/ml equivalent for the low positive cutoff. The CT cutoff corresponding to 49447.72 GE/ml was determined across other platforms and laboratories. The methodology and statistical analysis described here can now be used for standardization of all comparators used for FDA submissions with a goal towards establishing uniform criteria for EUA authorization.
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Affiliation(s)
- Anuradha Rao
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Jessica Lin
- Wallace H. Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA, USA
| | - Richard Parsons
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Nell Hodgson Woodruff School of Nursing, Emory University, Atlanta, GA USA
| | - Morgan Greenleaf
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Emory University School of Medicine, Atlanta, GA, USA
| | - Adrianna Westbrook
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Eric Lai
- Personalized Science San Diego CA 05403 USA
| | - Heather B. Bowers
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
- Laboratory of Biochemical Pharmacology, Emory University, Atlanta, Georgia
| | - Kaleb McClendon
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Emory/Children’s Laboratory for Innovative Assay Development, Atlanta, Georgia, USA
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine Atlanta, GA USA
| | - William O’Sick
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Emory/Children’s Laboratory for Innovative Assay Development, Atlanta, Georgia, USA
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine Atlanta, GA USA
| | - Tyler Baugh
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Emory/Children’s Laboratory for Innovative Assay Development, Atlanta, Georgia, USA
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine Atlanta, GA USA
| | - Markayla Sifford
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Emory/Children’s Laboratory for Innovative Assay Development, Atlanta, Georgia, USA
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine Atlanta, GA USA
| | - Julie A. Sullivan
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Wilbur A. Lam
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Wallace H. Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA, USA
- Children’s Healthcare of Atlanta, Atlanta, Georgia, USA
- Aflac Cancer and Blood Disorders Center at Children’s Healthcare of Atlanta, Atlanta, Georgia, USA
- Wallace H. Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA, USA
| | - Leda Bassit
- The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, United States of America
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
- Laboratory of Biochemical Pharmacology, Emory University, Atlanta, Georgia
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13
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Calabrese S, Markl AM, Neugebauer M, Krauth SJ, Borst N, von Stetten F, Lehnert M. Reporter emission multiplexing in digital PCRs (REM-dPCRs): direct quantification of multiple target sequences per detection channel by population specific reporters. Analyst 2023; 148:5243-5254. [PMID: 37727114 DOI: 10.1039/d3an00191a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/21/2023]
Abstract
Digital PCRs (dPCRs) are widely used methods for the detection and quantification of rare abundant sequences relevant to fields such as liquid biopsy or oncology. In order to increase the information content and save valuable sample materials, there is a significant need for digital multiplexing methods that are easy to establish, analyse, and interpret, and ideally allow the usage of existing lab equipment. Herein, we present a novel reporter emission multiplexing approach for the digital PCR method (REM-dPCR), which meets these requirements. It further increases the multiplexing capacity of commercial dPCR devices. For example, we present a stepwise increase in multiplexing degrees from a monochrome two-plex assay in one detection channel to a six-plex REM-dPCR assay in a three-color dPCR device for KRAS/BRAF single nucleotide polymorphism (SNP) target sequences. The guidelines for the REM-dPCR design are presented, and the process from duplex to six-plex assay establishment, taking into account the target sequence-dependent effects on assay performance, is discussed. Furthermore, the assay-specific, sensitive and precise quantification of different fractions of KRAS mutant and wild-type DNA sequences in different ratios is demonstrated. To increase the device capacitance and the degree of multiplexing, the REM-dPCR uses the advantage of n target-independent reporter molecules in combination with target sequence-specific mediator probes. Different reporter types are labelled with fluorophores of different signal intensities but not necessarily different emission spectra. This leads to the generation of n independent single-positive populations in the dataspace, created by k detection channels, whereby n > k and n ≥ 2. By usage of target-independent but population-specific reporter types, a fixed set of six optimized signalling molecules could be defined. This reporter set enables the robust generation and precise differentiation of multiple fluorescence signals in dPCRs and can be transferred to new target panels. The set which enables stable signal generation and differentiation in a specified device would allow easy transfer to new target panels.
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Affiliation(s)
| | - Anja M Markl
- Hahn-Schickard, 79110 Freiburg, Germany.
- Faculty of Chemistry and Pharmacy, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany
| | - Maximilian Neugebauer
- Hahn-Schickard, 79110 Freiburg, Germany.
- Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany
| | - Stefanie J Krauth
- School of Biodiversity, One Health, and Veterinary Medicine, University of Glasgow, Glasgow, UK
- School of Health and Wellbeing, General Practice and Primary Care, University of Glasgow, Glasgow, UK
| | - Nadine Borst
- Hahn-Schickard, 79110 Freiburg, Germany.
- Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany
| | - Felix von Stetten
- Hahn-Schickard, 79110 Freiburg, Germany.
- Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany
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14
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Liu DD, Muliaditan D, Viswanathan R, Cui X, Cheow LF. Melt-Encoded-Tags for Expanded Optical Readout in Digital PCR (METEOR-dPCR) Enables Highly Multiplexed Quantitative Gene Panel Profiling. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2301630. [PMID: 37485651 PMCID: PMC10520687 DOI: 10.1002/advs.202301630] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/13/2023] [Revised: 06/27/2023] [Indexed: 07/25/2023]
Abstract
Digital PCR (dPCR) is an important tool for precise nucleic acid quantification in clinical setting, but the limited multiplexing capability restricts its applications for quantitative gene panel profiling. Here, this work describes melt-encoded-tags for expanded optical readout in digital PCR (METEOR-dPCR), a simple two-step assay that enables simultaneous quantification of a large panel of arbitrary genes in a dPCR platform. Target genes are quantitatively converted into DNA tags with unique melting temperatures through a ligation approach. These tags are then counted and distinguished by their melt-curve profiles on a dPCR platform. A multiplexing capacity of M^N, where M is the number of resolvable melting temperature and N is the number of fluorescence channel, can be achieved. This work validates METEOR-dPCR with simultaneous DNA copy number profiling of 60 targets using dPCR in cancer cells, and demonstrates its sensitivity for estimating tumor fraction in mixed tumor and normal DNA samples. The rapid, quantitative, and highly multiplexed METEOR-dPCR assay will have wide appeal for many clinical applications.
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Affiliation(s)
- Dong Dong Liu
- Institute for Health Innovation and TechnologyNational University of SingaporeSingapore117599Singapore
| | - Daniel Muliaditan
- Department of Biomedical EngineeringFaculty of EngineeringNational University of SingaporeSingapore117583Singapore
- Genome institute of SingaporeAgency for ScienceTechnology and ResearchSingapore138672Singapore
| | - Ramya Viswanathan
- Institute for Health Innovation and TechnologyNational University of SingaporeSingapore117599Singapore
- Department of Biomedical EngineeringFaculty of EngineeringNational University of SingaporeSingapore117583Singapore
| | - Xu Cui
- Department of Biomedical EngineeringFaculty of EngineeringNational University of SingaporeSingapore117583Singapore
| | - Lih Feng Cheow
- Institute for Health Innovation and TechnologyNational University of SingaporeSingapore117599Singapore
- Department of Biomedical EngineeringFaculty of EngineeringNational University of SingaporeSingapore117583Singapore
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15
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Gu Z, Chen L, Zhang W, Su P, Zhang D, Du X, Peng Q, Liu Z, Liao X, Liu Y. A Sensitive Method for Detecting Beauveria bassiana, an Insecticidal Biocontrol Agent, Population Dynamics, and Stability in Different Substrates. THE CANADIAN JOURNAL OF INFECTIOUS DISEASES & MEDICAL MICROBIOLOGY = JOURNAL CANADIEN DES MALADIES INFECTIEUSES ET DE LA MICROBIOLOGIE MEDICALE 2023; 2023:9933783. [PMID: 37663453 PMCID: PMC10473894 DOI: 10.1155/2023/9933783] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/15/2023] [Revised: 07/18/2023] [Accepted: 08/01/2023] [Indexed: 09/05/2023]
Abstract
Beauveria bassiana is a well-known insecticidal biocontrol agent. Despite its broad field applications, its survival, colonization, and stability under field conditions remained unclear, mainly due to the lack of a quick and reliable detection method. In this study, we developed a quantitative real-time PCR technology to monitor the stability and population dynamics of B. bassiana in different substrates (water, soil, and on the cotton leaves surface), different spores of B. bassiana applied on Chinese cabbage leaves surface, and the lethality of Pieris rapae spraying with different spores of B. bassiana. Our results showed a decreased concentration of B. bassiana DNA in all three substrates from the 1st day till 9th day of post inoculation (dpi) period, possibly due to the death of B. bassiana. After this decrease, a quick and significant rebound of B. bassiana DNA concentration was observed, starting from the 11th dpi in all three substrates. The B. bassiana DNA concentration reached the plateau at about 13th dpi in water and 17th dpi in the soil. On cotton leaves surface, the B. bassiana DNA concentration reached the highest level at the 17th dpi followed by a small decline and then stabilized. This increase of DNA concentration suggested recovery of B. bassiana growth in all three substrates. We found that the most suitable killing effectiveness of P. rapae was the 1.0 × 107 spores/mL of B. bassiana. In summary, we have established a detection technology that allows a fast and reliable monitoring for the concentration and stability of B. bassiana under different conditions. This technology can benefit and help us in the development of proper management strategies for the application of this biocontrol agent in the field.
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Affiliation(s)
- Zepei Gu
- College of Plant Protection, Hunan Agricultural University, Changsha 410128, China
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
| | - Lijie Chen
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
- Longping Branch, Graduate School of Hunan University, Changsha 410125, China
| | - Weixing Zhang
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
| | - Pin Su
- College of Plant Protection, Hunan Agricultural University, Changsha 410128, China
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
- Longping Branch, Graduate School of Hunan University, Changsha 410125, China
| | - Deyong Zhang
- College of Plant Protection, Hunan Agricultural University, Changsha 410128, China
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
- Longping Branch, Graduate School of Hunan University, Changsha 410125, China
| | - Xiaohua Du
- College of Plant Protection, Hunan Agricultural University, Changsha 410128, China
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
| | - Qianze Peng
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
| | - Zhuoxin Liu
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
- Longping Branch, Graduate School of Hunan University, Changsha 410125, China
| | - Xiaolan Liao
- College of Plant Protection, Hunan Agricultural University, Changsha 410128, China
| | - Yong Liu
- Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
- Longping Branch, Graduate School of Hunan University, Changsha 410125, China
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16
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Flütsch S, Wiestner F, Butticaz L, Moor D, Stölting KN. Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp. BMC Genomics 2023; 24:375. [PMID: 37403035 DOI: 10.1186/s12864-023-09429-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Accepted: 06/05/2023] [Indexed: 07/06/2023] Open
Abstract
BACKGROUND Vibrio spp. are a diverse group of ecologically important marine bacteria responsible for several foodborne outbreaks of gastroenteritis around the world. Their detection and characterization are moving away from conventional culture-based methods towards next generation sequencing (NGS)-based approaches. However, genomic methods are relative in nature and suffer from technical biases arising from library preparation and sequencing. Here, we introduce a quantitative NGS-based method that enables the quantitation of Vibrio spp. at the limit of quantification (LOQ) through artificial DNA standards and their absolute quantification via digital PCR (dPCR). RESULTS We developed six DNA standards, called Vibrio-Sequins, together with optimized TaqMan assays for their quantification in individually sequenced DNA libraries via dPCR. To enable Vibrio-Sequin quantification, we validated three duplex dPCR methods to quantify the six targets. LOQs were ranging from 20 to 120 cp/µl for the six standards, whereas the limit of detection (LOD) was ~ 10 cp/µl for all six assays. Subsequently, a quantitative genomics approach was applied to quantify Vibrio-DNA in a pooled DNA mixture derived from several Vibrio species in a proof-of-concept study, demonstrating the increased power of our quantitative genomic pipeline through the coupling of NGS and dPCR. CONCLUSIONS We significantly advance existing quantitative (meta)genomic methods by ensuring metrological traceability of NGS-based DNA quantification. Our method represents a useful tool for future metagenomic studies aiming at quantifying microbial DNA in an absolute manner. The inclusion of dPCR into sequencing-based methods supports the development of statistical approaches for the estimation of measurement uncertainties (MU) for NGS, which is still in its infancy.
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Affiliation(s)
- Sabrina Flütsch
- Swiss Federal Institute of Metrology METAS, Lindenweg 50, Bern-Wabern, 3003, Switzerland.
- Swiss Federal Institute of Metrology METAS, Campus Liebefeld, Schwarzenburgstrasse 165, Bern-Köniz, 3097, Switzerland.
| | - Fabian Wiestner
- Swiss Federal Institute of Metrology METAS, Lindenweg 50, Bern-Wabern, 3003, Switzerland
- Swiss Federal Institute of Metrology METAS, Campus Liebefeld, Schwarzenburgstrasse 165, Bern-Köniz, 3097, Switzerland
| | - Lisa Butticaz
- Federal Food Safety and Veterinary Office FSVO, Schwarzenburgstrasse 165, Bern-Köniz, 3003, Switzerland
- Swiss Federal Institute of Metrology METAS, Campus Liebefeld, Schwarzenburgstrasse 165, Bern-Köniz, 3097, Switzerland
| | - Dominik Moor
- Federal Food Safety and Veterinary Office FSVO, Schwarzenburgstrasse 165, Bern-Köniz, 3003, Switzerland
- Swiss Federal Institute of Metrology METAS, Campus Liebefeld, Schwarzenburgstrasse 165, Bern-Köniz, 3097, Switzerland
| | - Kai N Stölting
- Swiss Federal Institute of Metrology METAS, Lindenweg 50, Bern-Wabern, 3003, Switzerland.
- Swiss Federal Institute of Metrology METAS, Campus Liebefeld, Schwarzenburgstrasse 165, Bern-Köniz, 3097, Switzerland.
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17
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Ding R, Liu L, Zhang J, Lv P, Zhou L, Zhang T, Li S, Zhao R, Yang Z, Xiong P, Chen H, Wang W, Wang H, Tian Z, Liu B, Chen C. Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule resolution. Nucleic Acids Res 2023; 51:e65. [PMID: 37194709 PMCID: PMC10287937 DOI: 10.1093/nar/gkad388] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2023] [Revised: 04/04/2023] [Accepted: 05/01/2023] [Indexed: 05/18/2023] Open
Abstract
Despite the need in various applications, accurate quantification of nucleic acids still remains a challenge. The widely-used qPCR has reduced accuracy at ultralow template concentration and is susceptible to nonspecific amplifications. The more recently developed dPCR is costly and cannot handle high-concentration samples. We combine the strengths of qPCR and dPCR by performing PCR in silicon-based microfluidic chips and demonstrate high quantification accuracy in a large concentration range. Importantly, at low template concentration, we observe on-site PCR (osPCR), where only certain sites of the channel show amplification. The sites have almost identical ct values, showing osPCR is a quasi-single molecule phenomenon. Using osPCR, we can measure both the ct values and the absolute concentration of templates in the same reaction. Additionally, osPCR enables identification of each template molecule, allowing removal of nonspecific amplification during quantification and greatly improving quantification accuracy. We develop sectioning algorithm that improves the signal amplitude and demonstrate improved detection of COVID in patient samples.
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Affiliation(s)
- Ruihua Ding
- Shanghai Industrial μTechnology Research Institute (SITRI), Shanghai201800, China
| | - Liying Liu
- Shanghai Si-Gene Biotech Co., Ltd, Shanghai201800, China
| | - Jiali Zhang
- School of Microelectronics, Shanghai University, Shanghai201800, China
| | - Pengxiao Lv
- Shanghai Industrial μTechnology Research Institute (SITRI), Shanghai201800, China
| | - Lin Zhou
- Shanghai Industrial μTechnology Research Institute (SITRI), Shanghai201800, China
| | - Tinglu Zhang
- Shanghai Industrial μTechnology Research Institute (SITRI), Shanghai201800, China
| | - Shenwei Li
- Shanghai International Travel Healthcare Center, Shanghai200335, China
| | - Ran Zhao
- Shanghai Center for Clinical Laboratory, Shanghai200126, China
| | - Zhuo Yang
- School of Microelectronics, Shanghai University, Shanghai201800, China
| | - Peng Xiong
- Shanghai Si-Gene Biotech Co., Ltd, Shanghai201800, China
| | - Hu Chen
- Shanghai Si-Gene Biotech Co., Ltd, Shanghai201800, China
| | - Wei Wang
- Shanghai International Travel Healthcare Center, Shanghai200335, China
| | - Hualiang Wang
- Shanghai Center for Clinical Laboratory, Shanghai200126, China
| | - Zhengan Tian
- Shanghai International Travel Healthcare Center, Shanghai200335, China
| | - Bo Liu
- Shanghai Industrial μTechnology Research Institute (SITRI), Shanghai201800, China
- Shanghai Si-Gene Biotech Co., Ltd, Shanghai201800, China
- School of Microelectronics, Shanghai University, Shanghai201800, China
| | - Chang Chen
- Shanghai Industrial μTechnology Research Institute (SITRI), Shanghai201800, China
- Shanghai Si-Gene Biotech Co., Ltd, Shanghai201800, China
- School of Microelectronics, Shanghai University, Shanghai201800, China
- State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai200050, China
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18
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Sun Y, Wu Y, Ma D, Li JJ, Liu X, You Y, Lu J, Liu Z, Cheng X, Du Y. Digital microfluidics-engaged automated enzymatic degradation and synthesis of oligosaccharides. Front Bioeng Biotechnol 2023; 11:1201300. [PMID: 37415787 PMCID: PMC10320006 DOI: 10.3389/fbioe.2023.1201300] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2023] [Accepted: 06/08/2023] [Indexed: 07/08/2023] Open
Abstract
Glycans are an important group of natural biopolymers, which not only play the role of a major biological energy resource but also as signaling molecules. As a result, structural characterization or sequencing of glycans, as well as targeted synthesis of glycans, is of great interest for understanding their structure-function relationship. However, this generally involves tedious manual operations and high reagent consumptions, which are the main technical bottlenecks retarding the advances of both automatic glycan sequencing and synthesis. Until now, automated enzymatic glycan sequencers or synthesizers are still not available on the market. In this study, to promote the development of automation in glycan sequencing or synthesis, first, programmed degradation and synthesis of glycans catalyzed by enzymes were successfully conducted on a digital microfluidic (DMF) device by using microdroplets as microreactors. In order to develop automatic glycan synthesizers and sequencers, a strategy integrating enzymatic oligosaccharide degradation or synthesis and magnetic manipulation to realize the separation and purification process after enzymatic reactions was designed and performed on DMF. An automatic process for enzymatic degradation of tetra-N-acetyl chitotetraose was achieved. Furthermore, the two-step enzymatic synthesis of lacto-N-tetraose was successfully and efficiently completed on the DMF platform. This work demonstrated here would open the door to further develop automatic enzymatic glycan synthesizers or sequencers based on DMF.
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Affiliation(s)
- Yunze Sun
- School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing, China
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China
| | - Yiran Wu
- School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing, China
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China
| | - Dachuan Ma
- Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning, China
| | - Jian-Jun Li
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China
| | - Xianming Liu
- Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning, China
| | - Yuanjiang You
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China
| | - Jun Lu
- Institute of Physics, Chinese Academy of Sciences, Beijing, China
| | - Zhen Liu
- Department of Materials Science and Engineering, Southern University of Science and Technology, Shenzhen, China
| | - Xin Cheng
- Department of Materials Science and Engineering, Southern University of Science and Technology, Shenzhen, China
| | - Yuguang Du
- State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China
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Jiang L, Lan X, Ren L, Yang M, Wei B, Wang Y. Design of a Digital LAMP Detection Platform Based on Droplet Microfluidic Technology. MICROMACHINES 2023; 14:mi14051077. [PMID: 37241700 DOI: 10.3390/mi14051077] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Revised: 05/08/2023] [Accepted: 05/15/2023] [Indexed: 05/28/2023]
Abstract
Loop-mediated isothermal amplification (LAMP) is a rapid and high-yield amplification technology for specific DNA or RNA molecules. In this study, we designed a digital loop-mediated isothermal amplification (digital-LAMP)-functioning microfluidic chip to achieve higher sensitivity for detection of nucleic acids. The chip could generate droplets and collect them, based on which we could perform Digital-LAMP. The reaction only took 40 min at a constant temperature of 63 °C. The chip enabled highly accurate quantitative detection, with the limit of detection (LOD) down to 102 copies μL-1. For better performance while reducing the investment of money and time in chip structure iterations, we used COMSOL Multiphysics to simulate different droplet generation ways by including flow-focusing structure and T-junction structure. Moreover, the linear structure, serpentine structure, and spiral structure in the microfluidic chip were compared to study the fluid velocity and pressure distribution. The simulations provided a basis for chip structure design while facilitating chip structure optimization. The digital-LAMP-functioning chip proposed in the work provides a universal platform for analysis of viruses.
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Affiliation(s)
- Liying Jiang
- School of Electrical and Information Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China
- Academy for Quantum Science and Technology, Zhengzhou University of Light Industry, Zhengzhou 450002, China
| | - Xianghao Lan
- School of Electrical and Information Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China
| | - Linjiao Ren
- School of Electrical and Information Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China
| | - Mingzhu Yang
- Beijing Research Institute of Mechanical Equipment, Beijing 100143, China
| | - Bo Wei
- Department of Thoracic Surgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China
| | - Yang Wang
- Beijing Advanced Innovation Center for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology, School of Engineering Medicine, Beihang University, Beijing 100083, China
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20
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Baettig CG, Zirngibl M, Smith KF, Lear G, Tremblay LA. Comparison between droplet digital PCR and reverse transcription-quantitative PCR methods to measure ecotoxicology biomarkers. MARINE POLLUTION BULLETIN 2023; 190:114829. [PMID: 36958116 DOI: 10.1016/j.marpolbul.2023.114829] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/24/2023] [Revised: 03/06/2023] [Accepted: 03/08/2023] [Indexed: 06/18/2023]
Abstract
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently the gold-standard technique for detecting and quantifying messenger RNA. However, without proper validation, the method may produce artefactual and non-reproducible cycle threshold values generating poor-quality data. The newer droplet digital PCR (ddPCR) method allows for the absolute quantification of targeted nucleic acids providing more sensitive and accurate measurements without requiring external standards. This study compared these two PCR-based methods to measure the expression of well-documented genes used in ecotoxicology studies. We exposed Mediterranean mussels (Mytilus galloprovincialis) to copper and analyzed gene expression in gills and digestive glands using RT-qPCR and ddPCR assays. A step-by-step methodology to optimize and compare the two technologies is described. After ten-fold serial complementary DNA dilution, both RT-qPCR and ddPCR exhibited comparable linearity and efficiency and produced statistically similar results. We conclude that ddPCR is a suitable method to assess gene expression in an ecotoxicological context. However, RT-qPCR has a shorter processing time and remains more cost-effective.
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Affiliation(s)
- Camille G Baettig
- School of Biological Sciences, University of Auckland, Auckland, New Zealand; Cawthron Institute, Nelson, New Zealand.
| | | | - Kirsty F Smith
- School of Biological Sciences, University of Auckland, Auckland, New Zealand; Cawthron Institute, Nelson, New Zealand
| | - Gavin Lear
- School of Biological Sciences, University of Auckland, Auckland, New Zealand
| | - Louis A Tremblay
- School of Biological Sciences, University of Auckland, Auckland, New Zealand; Cawthron Institute, Nelson, New Zealand
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21
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Van Poelvoorde LAE, Dufrasne FE, Van Gucht S, Saelens X, Roosens NHC. Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants. Curr Issues Mol Biol 2023; 45:2521-2532. [PMID: 36975535 PMCID: PMC10047791 DOI: 10.3390/cimb45030165] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Revised: 03/10/2023] [Accepted: 03/14/2023] [Indexed: 03/29/2023] Open
Abstract
The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.
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Affiliation(s)
- Laura A E Van Poelvoorde
- Transversal Activities in Applied Genomics, Sciensano, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
- National Influenza Centre, Department of Infectious Diseases in Humans, Laboratory of Viral Diseases, Sciensano, Engelandstraat 642, 1180 Brussels, Belgium
- Department of Biochemistry and Microbiology, Ghent University, 9052 Ghent, Belgium
- VIB-UGent Center for Medical Biotechnology, VIB, 9052 Ghent, Belgium
| | - François E Dufrasne
- National Influenza Centre, Department of Infectious Diseases in Humans, Laboratory of Viral Diseases, Sciensano, Engelandstraat 642, 1180 Brussels, Belgium
| | - Steven Van Gucht
- National Influenza Centre, Department of Infectious Diseases in Humans, Laboratory of Viral Diseases, Sciensano, Engelandstraat 642, 1180 Brussels, Belgium
| | - Xavier Saelens
- Department of Biochemistry and Microbiology, Ghent University, 9052 Ghent, Belgium
- VIB-UGent Center for Medical Biotechnology, VIB, 9052 Ghent, Belgium
| | - Nancy H C Roosens
- Transversal Activities in Applied Genomics, Sciensano, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
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22
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Zhou H, Liu X, Lu Z, Hu A, Ma W, Shi C, Bie X, Cheng Y, Wu H, Yang J. Quantitative detection of Vibrio parahaemolyticus in aquatic products by duplex droplet digital PCR combined with propidium monoazide. Food Control 2023. [DOI: 10.1016/j.foodcont.2022.109353] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/14/2022]
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23
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Primary Quantitative Reference Standards for Viral Nucleic Acids Should Be Developed Using Digital Polymerase Chain Reaction Instead of Consensus Testing. J Clin Microbiol 2023; 61:e0133822. [PMID: 36475837 PMCID: PMC9879095 DOI: 10.1128/jcm.01338-22] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
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24
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Wang D, Liu E, Liu H, Jin X, Niu C, Gao Y, Su X. A droplet digital PCR assay for detection and quantification of Verticillium nonalfalfae and V. albo-atrum. Front Cell Infect Microbiol 2023; 12:1110684. [PMID: 36710974 PMCID: PMC9874294 DOI: 10.3389/fcimb.2022.1110684] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Accepted: 12/20/2022] [Indexed: 01/13/2023] Open
Abstract
Verticillium nonalfalfae and V. albo-atrum are notorious pathogenic fungi that cause a destructive vascular disease called Verticillium wilt worldwide. Thus, timely and quantitative monitoring of fungal progression is highly desirable for early diagnosis and risk assessment. In this study, we developed a droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify V. nonalfalfae and V. albo-atrum. The performance of this assay was validated in comparison with that of a quantitative real-time polymerase chain reaction (qPCR) assay. The standard curve analysis of the ddPCR assay showed good linearity. The ddPCR assay indicated similar detection sensitivity to that of qPCR on pure genomic DNA, while it enhanced the positive rate for low-abundance fungi, especially in alfalfa stems. Receiver operating characteristic analysis revealed that ddPCR provided superior diagnostic performance on field tissues compared to qPCR, and the area under curve values were 0.94 and 0.90 for alfalfa roots and stems, respectively. Additionally, the quantitative results of the two methods were highly concordant (roots: R2 = 0.91; stems: R2 = 0.76); however, the concentrations determined by ddPCR were generally higher than those determined by qPCR. This discrepancy was potentially caused by differing amplification efficiencies for qPCR between cultured and field samples. Furthermore, the ddPCR assays appreciably improved quantitative precision, as reflected by lower coefficients of variation. Overall, the ddPCR method enables sensitive detection and accurate quantification of V. nonalfalfae and V. albo-atrum, providing a valuable tool for evaluating disease progression and enacting effective disease control.
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Affiliation(s)
- Di Wang
- 1Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China
| | - Enliang Liu
- 2Institute of Grain Crops, Xinjiang Academy of Agricultural Sciences, Urumqi, China
| | - Haiyang Liu
- 3Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences, Urumqi, China
| | - Xi Jin
- 4Hebei Technology Innovation Center for Green Management of Soil-Borne Diseases, Baoding University, Hebei, China
| | - Chunyan Niu
- 1Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China
| | - Yunhua Gao
- 1Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China,*Correspondence: Yunhua Gao, ; Xiaofeng Su,
| | - Xiaofeng Su
- 5Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China,*Correspondence: Yunhua Gao, ; Xiaofeng Su,
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25
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Della Starza I, De Novi LA, Elia L, Bellomarino V, Beldinanzi M, Soscia R, Cardinali D, Chiaretti S, Guarini A, Foà R. Optimizing Molecular Minimal Residual Disease Analysis in Adult Acute Lymphoblastic Leukemia. Cancers (Basel) 2023; 15:374. [PMID: 36672325 PMCID: PMC9856386 DOI: 10.3390/cancers15020374] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 01/02/2023] [Accepted: 01/03/2023] [Indexed: 01/09/2023] Open
Abstract
Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.
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Affiliation(s)
- Irene Della Starza
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
- GIMEMA Foundation, 00182 Rome, Italy
| | - Lucia Anna De Novi
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Loredana Elia
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Vittorio Bellomarino
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Marco Beldinanzi
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Roberta Soscia
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Deborah Cardinali
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Sabina Chiaretti
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Anna Guarini
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Robin Foà
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
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26
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Takousis P, Devonshire AS, Redshaw N, von Baumgarten L, Whale AS, Jones GM, Fernandez-Gonzalez A, Martin J, Foy CA, Alexopoulos P, Huggett JF, Perneczky R. A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer's disease and brain cancers. N Biotechnol 2022; 72:97-106. [PMID: 36202346 DOI: 10.1016/j.nbt.2022.10.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Revised: 09/23/2022] [Accepted: 10/01/2022] [Indexed: 11/27/2022]
Abstract
Cerebrospinal fluid (CSF) is a source of diagnostic biomarkers for a range of neurological conditions. Cell-free DNA (cfDNA) is detected in CSF and differences in the concentration of cell-free mitochondrial DNA have been reported in studies of neurodegenerative disorders including Alzheimer's disease (AD). However, the influence of pre-analytical steps has not been investigated for cfDNA in CSF and there is no standardised approach for quantification of total cfDNA (copies of nuclear genome or mitochondria-derived gene targets). In this study, the suitability of four extraction methods was evaluated: QIAamp Circulating Nucleic Acid (Qiagen), Quick-cfDNA Serum & Plasma (Zymo), NucleoSnap® DNA Plasma (Macherey-Nagel) and Plasma/Serum Circulating DNA Purification Mini (Norgen) kits, for cfDNA extraction from CSF of controls and AD dementia patients, utilising a spike-in control for extraction efficiency and fragment size. One of the optimal extraction methods was applied to a comparison of cfDNA concentrations in CSF from control subjects, AD dementia and primary and secondary brain tumour patients. Extraction efficiency based on spike-in recovery was similar in all three groups whilst both endogenous mitochondrial and nucleus-derived cfDNA was significantly higher in CSF from cancer patients compared to control and AD groups, which typically contained < 100 genome copies/mL. This study shows that it is feasible to measure low concentration nuclear and mitochondrial gene targets in CSF and that normalisation of extraction yield can help control pre-analytical variability influencing biomarker measurements.
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Affiliation(s)
- Petros Takousis
- Ageing Epidemiology (AGE) Research Unit, School of Public Health, Imperial College London, London, UK
| | - Alison S Devonshire
- Molecular and Cell Biology Team, National Measurement Laboratory, LGC, Teddington, Middlesex, UK.
| | - Nicholas Redshaw
- Molecular and Cell Biology Team, National Measurement Laboratory, LGC, Teddington, Middlesex, UK
| | - Louisa von Baumgarten
- Department of Neurology, University Hospital, LMU Munich, Munich, Germany; Department of Neurosurgery, University Hospital, LMU Munich, Munich, Germany
| | - Alexandra S Whale
- Molecular and Cell Biology Team, National Measurement Laboratory, LGC, Teddington, Middlesex, UK
| | - Gerwyn M Jones
- Molecular and Cell Biology Team, National Measurement Laboratory, LGC, Teddington, Middlesex, UK
| | - Ana Fernandez-Gonzalez
- Molecular and Cell Biology Team, National Measurement Laboratory, LGC, Teddington, Middlesex, UK
| | - Jan Martin
- Department of Anaesthesiology and Intensive Care Medicine, Technical University Munich, Munich, Germany
| | - Carole A Foy
- Molecular and Cell Biology Team, National Measurement Laboratory, LGC, Teddington, Middlesex, UK
| | - Panagiotis Alexopoulos
- Department of Psychiatry, University of Patras, Rion Patras, Greece; Department of Psychiatry and Psychotherapy, Technical University Munich, Munich, Germany
| | - Jim F Huggett
- Molecular and Cell Biology Team, National Measurement Laboratory, LGC, Teddington, Middlesex, UK; School of Biosciences and Medicine, University of Surrey, Guildford, UK
| | - Robert Perneczky
- Ageing Epidemiology (AGE) Research Unit, School of Public Health, Imperial College London, London, UK; Department of Psychiatry and Psychotherapy, University Hospital, LMU Munich, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE) Munich, Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK
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27
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Miyata M, Yoshida J, Takagishi I, Horie K. Comparison of CRISPR-Cas9-mediated megabase-scale genome deletion methods in mouse embryonic stem cells. DNA Res 2022; 30:6854440. [PMID: 36448318 PMCID: PMC9847339 DOI: 10.1093/dnares/dsac045] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2022] [Revised: 10/30/2022] [Accepted: 11/28/2022] [Indexed: 12/02/2022] Open
Abstract
The genome contains large functional units ranging in size from hundreds of kilobases to megabases, such as gene clusters and topologically associating domains. To analyse these large functional units, the technique of deleting the entire functional unit is effective. However, deletion of such large regions is less efficient than conventional genome editing, especially in cultured cells, and a method that can ensure success is anticipated. Here, we compared methods to delete the 2.5-Mb Krüppel-associated box zinc finger protein (KRAB-ZFP) gene cluster in mouse embryonic stem cells using CRISPR-Cas9. Three methods were used: first, deletion by non-homologous end joining (NHEJ); second, homology-directed repair (HDR) using a single-stranded oligodeoxynucleotide (ssODN); and third, HDR employing targeting vectors with a selectable marker and 1-kb homology arms. NHEJ-mediated deletion was achieved in 9% of the transfected cells. Inversion was also detected at similar efficiency. The deletion frequency of NHEJ and HDR was found to be comparable when the ssODN was transfected. Deletion frequency was highest when targeting vectors were introduced, with deletions occurring in 31-63% of the drug-resistant clones. Biallelic deletion was observed when targeting vectors were used. This study will serve as a benchmark for the introduction of large deletions into the genome.
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Affiliation(s)
- Masayuki Miyata
- Department of Physiology II, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Junko Yoshida
- Department of Physiology II, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Itsuki Takagishi
- Department of Physiology II, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Kyoji Horie
- To whom correspondence should be addressed. Tel: +81 744 23 4696. Fax: +81 744 23 4696.
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28
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Berden P, Wiederkehr RS, Lagae L, Michiels J, Stakenborg T, Fauvart M, Van Roy W. Amplification Efficiency and Template Accessibility as Distinct Causes of Rain in Digital PCR: Monte Carlo Modeling and Experimental Validation. Anal Chem 2022; 94:15781-15789. [DOI: 10.1021/acs.analchem.2c03534] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Pieter Berden
- Imec, Leuven 3001, Belgium
- Department of Physics and Astronomy, KU Leuven, Leuven 3001, Belgium
- Department of Molecular and Microbial Systems, Centre of Microbial and Plant Genetics, KU Leuven, Leuven 3001, Belgium
- Center for Microbiology, Flanders Institute for Biotechnology, VIB, Leuven 3001, Belgium
| | | | - Liesbet Lagae
- Imec, Leuven 3001, Belgium
- Department of Physics and Astronomy, KU Leuven, Leuven 3001, Belgium
| | - Jan Michiels
- Department of Molecular and Microbial Systems, Centre of Microbial and Plant Genetics, KU Leuven, Leuven 3001, Belgium
- Center for Microbiology, Flanders Institute for Biotechnology, VIB, Leuven 3001, Belgium
| | | | - Maarten Fauvart
- Imec, Leuven 3001, Belgium
- Department of Molecular and Microbial Systems, Centre of Microbial and Plant Genetics, KU Leuven, Leuven 3001, Belgium
- Center for Microbiology, Flanders Institute for Biotechnology, VIB, Leuven 3001, Belgium
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29
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Xia Y, Chu X, Zhao C, Wang N, Yu J, Jin Y, Sun L, Ma S. A Glass-Ultra-Thin PDMS Film-Glass Microfluidic Device for Digital PCR Application Based on Flexible Mold Peel-Off Process. MICROMACHINES 2022; 13:mi13101667. [PMID: 36296020 PMCID: PMC9608979 DOI: 10.3390/mi13101667] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/14/2022] [Revised: 09/29/2022] [Accepted: 10/01/2022] [Indexed: 05/27/2023]
Abstract
The microfluidic device (MFD) with a glass−PDMS−glass (G-P-G) structure is of interest for a wide range of applications. However, G-P-G MFD fabrication with an ultra-thin PDMS film (especially thickness less than 200 μm) is still a big challenge because the ultra-thin PDMS film is easily deformed, curled, and damaged during demolding and transferring. This study aimed to report a thickness-controllable and low-cost fabrication process of the G-P-G MFD with an ultra-thin PDMS film based on a flexible mold peel-off process. A patterned photoresist layer was deposited on a polyethylene terephthalate (PET) film to fabricate a flexible mold that could be demolded softly to achieve a rigid structure of the glass−PDMS film. The thickness of ultra-thin patterned PDMS could reach less than 50 μm without damage to the PDMS film. The MFD showcased the excellent property of water evaporation inhibition (water loss < 10%) during PCR thermal cycling because of the ultra-thin PDMS film. Its low-cost fabrication process and excellent water evaporation inhibition present extremely high prospects for digital PCR application.
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Affiliation(s)
- Yanming Xia
- Department of Mechanical & Electrical Engineering, Xiamen University, Xiamen 361005, China
- Institute for Chemical and Bioengineering, ETH Zürich, 8093 Zürich, Switzerland
| | - Xianglong Chu
- Department of Mechanical & Electrical Engineering, Xiamen University, Xiamen 361005, China
| | - Caiming Zhao
- Department of Mechanical & Electrical Engineering, Xiamen University, Xiamen 361005, China
| | - Nanxin Wang
- School of Electronic and Computer Engineering, Peking University Shenzhen Graduate School, Shenzhen 518055, China
| | - Juan Yu
- Department of Mechanical & Electrical Engineering, Xiamen University, Xiamen 361005, China
| | - Yufeng Jin
- School of Electronic and Computer Engineering, Peking University Shenzhen Graduate School, Shenzhen 518055, China
| | - Lijun Sun
- School of Mechatronical Engineering, Beijing Institute of Technology, Beijing 100081, China
| | - Shenglin Ma
- Department of Mechanical & Electrical Engineering, Xiamen University, Xiamen 361005, China
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30
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Shekhawat DS, Sharma C, Singh K, Singh P, Bhardwaj A, Patwa P. Critical appraisal of droplet digital polymerase chain reaction application for noninvasive prenatal testing. Congenit Anom (Kyoto) 2022; 62:188-197. [PMID: 35662261 DOI: 10.1111/cga.12481] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 04/13/2022] [Accepted: 04/21/2022] [Indexed: 11/29/2022]
Abstract
Maternal-fetal medicine (FM) is currently a highly demanding branch and is gaining importance as increasing number of genetic disorders rise in incidence. Prenatal testing helps to detect such abnormalities that could affect the health status of the developing fetus like birth defects or genetic disorders. Considering the rising trend of genetic disorders, there is a need for a highly sensitive way of noninvasive prenatal testing (NIPT) that may reduce the incidence of unnecessary invasive procedures and iatrogenic fetal loss. The concept of NIPT for screening of genetic disorders is continuously evolving over the last two decades and multiple techniques have come up to utilize this in the field of FM. The crucial factor which decides the accuracy of NIPS is cell free fetal DNA (cffDNA) that is present in extremely low fraction (10%-15%) in the maternal plasma. Among the available methods, the next generation sequencing (NGS) is considered as the gold standard. However, the higher cost diminishes its utility in low-resource settings. Droplet digital Polymerase chain reaction (ddPCR), a type of digital PCR is a novel technique that is frugal, equally sensitive, less labor intensive, less time-consuming and plain algorithm dependent method for detecting cffDNA fraction. Considering these impressive attributes of ddPCR, we decided to critically review the existing literature on ddPCR for NIPT whilst highlighting the clinical utility, challenges and its advantages over NGS.
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Affiliation(s)
| | - Charu Sharma
- Department of Obstetrics & Gynecology, AIIMS, Jodhpur, India
| | | | - Pratibha Singh
- Department of Obstetrics & Gynecology, AIIMS, Jodhpur, India
| | - Abhishek Bhardwaj
- Department of Dermatology, Venereology and Leprology, AIIMS, Jodhpur, India
| | - Payal Patwa
- Department of Obstetrics & Gynecology, AIIMS, Jodhpur, India
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31
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Wang K, Li B, Guo Y, Wu Y, Li Y, Wu W. An integrated digital PCR system with high universality and low cost for nucleic acid detection. Front Bioeng Biotechnol 2022; 10:947895. [PMID: 36061433 PMCID: PMC9437218 DOI: 10.3389/fbioe.2022.947895] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Accepted: 07/25/2022] [Indexed: 11/13/2022] Open
Abstract
Digital PCR is the most advanced PCR technology. However, due to the high price of the digital PCR analysis instrument, this powerful nucleic acid detection technology is still difficult to be popularized in the general biochemistry laboratory. Moreover, one of the biggest disadvantages of commercial digital PCR systems is the poor versatility of reagents: each instrument can only be used for a few customized kits. Herein, we built a low-cost digital PCR system. The system only relies on low-cost traditional flat-panel PCR equipment to provide temperature conditions for commercial dPCR chips, and the self-made fluorescence detection system is designed and optically optimized to meet a wide range of reagent requirements. More importantly, our system not only has a low cost (<8000 US dollars) but also has a much higher universality for nucleic acid detection reagents than the traditional commercial digital PCR system. In this study, several samples were tested. The genes used in the experiment were plasmids containing UPE-1a fragment, TP53 reference DNA, hepatitis B virus DNA, leukemia sample, SARS-COV-2 DNA, and SARS-COV-2 RNA. Under the condition that DNA can be amplified normally, the function of the dPCR system can be realized with simpler and low-price equipment. Some DNA cannot be detected by using the commercial dPCR system because of the special formula when it is configured as the reaction solution, but these DNA fluorescence signals can be clearly detected by our system, and the concentration can be calculated. Our system is more applicable than the commercial dPCR system to form a new dPCR system that is smaller and more widely applicable than commercially available machinery.
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Affiliation(s)
- Kangning Wang
- Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China
| | - Bin Li
- Institute of Microbiology Chinese Academy of Sciences, Beijing, China
| | - Yu Guo
- School of Mechanical and Electrical Engineering, Guangdong University of Technology, Guangzhou, China
| | - Yanqi Wu
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, China
| | - Yan Li
- Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China
| | - Wenming Wu
- Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China
- *Correspondence: Wenming Wu,
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32
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Tanvetthayanont P, Yata T, Boonnil J, Temisak S, Ponglowhapan S. Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland. Sci Rep 2022; 12:13623. [PMID: 35948591 PMCID: PMC9365843 DOI: 10.1038/s41598-022-17493-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2021] [Accepted: 07/26/2022] [Indexed: 11/09/2022] Open
Abstract
In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (Ta) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues.
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Affiliation(s)
- Potsawat Tanvetthayanont
- Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand
| | - Teerapong Yata
- Unit of Biochemistry, Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand
| | - Jiranun Boonnil
- National Institute of Metrology (NIMT), Pathumthani, 12120, Thailand
| | - Sasithon Temisak
- National Institute of Metrology (NIMT), Pathumthani, 12120, Thailand.
| | - Suppawiwat Ponglowhapan
- Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand.
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Zhao Z, Wu T, Wang M, Chen X, Liu T, Si Y, Zhou Y, Ying B. A new droplet digital PCR assay: improving detection of paucibacillary smear-negative pulmonary tuberculosis. Int J Infect Dis 2022; 122:820-828. [PMID: 35870796 DOI: 10.1016/j.ijid.2022.07.041] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Revised: 06/30/2022] [Accepted: 07/16/2022] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Smear-negative pulmonary tuberculosis (PTB) is difficult to diagnose. Current diagnosis and treatment monitoring methods have inherent limitations. Droplet digital PCR (ddPCR) is a new technique with high sensitivity. This study presents a novel ddPCR for rapid and sensitive identification of Mycobacterium tuberculosis (MTB). METHODS MTB DNA was detected in respiratory specimens from suspected PTB cases using ddPCR assay, which was directed at two different locations within IS6110. We, for the first time, evaluated the clinical diagnostic ability of this ddPCR for paucibacillary smear-negative PTB. RESULTS A total of 605 PTB suspects were recruited, including 263 confirmed PTB patients (84.03% from smear-negative PTB) and 342 non-PTB. The sensitivity and specificity of IS6110 ddPCR were 61.22% (95% confidence interval (CI), 55.00%-67.10%) and 95.03% (95% CI, 92.20%-97.10%) for total PTB, and 57.92% (95% CI, 51.10%-64.50%) and 94.57% (95% CI, 91.20%-96.90%) for smear-negative PTB. ddPCR assay outperformed Xpert MTB/RIF (53.08% vs. 28.46%, p = 0.020) in smear-negative PTB detection. Furthermore, effective anti-tuberculosis treatment was linked to significantly lower IS6110 copies detected by ddPCR. CONCLUSIONS Herein, we developed and validated a highly sensitive and robust ddPCR assay for MTB quantification in respiratory specimens, which improve diagnosis and therapeutic effect evaluation of smear-negative PTB.
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Affiliation(s)
- Zhenzhen Zhao
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Tao Wu
- Department of Clinical Laboratory Medicine, People's Hospital of Ningxia Hui Autonomous Region (First Affiliated Hospital of Northwest Minzu University), Yinchuan, Ningxia Hui Autonomous Region, China
| | - Minjin Wang
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Xiaojuan Chen
- Department of Laboratory Medicine, The People's Hospital of Leshan, Leshan, Sichuan, China
| | - Tangyuheng Liu
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Yanjun Si
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Yanhong Zhou
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Binwu Ying
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
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Self-Assembled Inkjet Printer for Droplet Digital Loop-Mediated Isothermal Amplification. CHEMOSENSORS 2022. [DOI: 10.3390/chemosensors10070247] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Developing rapid and inexpensive diagnostic tools for molecular detection has been pushed forward by the advancements of technical aspects. However, attention has rarely been paid to the molecular detection methodology using inkjet printing technique. Herein, we developed an approach that employed a self-assembled inkjet printer as the enabling technology to realize droplet digital loop-mediated isothermal amplification in a low-cost and practical format. An inkjet printer is a self-assembled tool for the generation of discrete droplets in controllable volumes from a picoliter to a nanoliter. A microfluidic chip serves as a droplets reservoir to perform droplet digital LAMP assays. The inkjet printer approach successfully quantified the HPV16 from CaSki cells. This self-assembled and practical inkjet printer device may therefore become a promising tool for rapid molecular detection and can be extended to on-site analysis.
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Digital Droplet PCR Is a Reliable Tool to Improve Minimal Residual Disease Stratification in Adult Philadelphia-Negative Acute Lymphoblastic Leukemia. J Mol Diagn 2022; 24:893-900. [PMID: 35710027 DOI: 10.1016/j.jmoldx.2022.04.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Revised: 04/19/2022] [Accepted: 04/25/2022] [Indexed: 11/20/2022] Open
Abstract
Digital droplet PCR (ddPCR) is an implementation of conventional PCR, with the potential of overcoming some limitations of real-time quantitative PCR (RQ-PCR). To evaluate if ddPCR may improve the quantification of disease levels and refine patients' risk stratification, 116 samples at four time points from 44 (35 B-lineage and 9 T-lineage) adult Philadelphia-negative acute lymphoblastic leukemia patients enrolled in the GIMEMA LAL1913 protocol were analyzed by RQ-PCR and ddPCR. A concordance rate between RQ-PCR and ddPCR of 79% (P < 0.0001) was observed; discordances were identified in 21% of samples, with the majority being RQ-PCR-negative (NEG) or positive not quantifiable (PNQ). ddPCR significantly reduced the proportion of PNQ samples-2.6% versus 14% (P = 0.003)-and allowed disease quantifiability in 6.6% of RQ-PCR-NEG, increasing minimal residual disease quantification in 14% of samples. Forty-seven samples were also investigated by next-generation sequencing, which confirmed the ddPCR results in samples classified as RQ-PCR-PNQ or NEG. By reclassifying samples on the basis of the ddPCR results, a better event-free survival stratification of patients was observed compared with RQ-PCR: indeed, ddPCR captured more true-quantifiable samples, with five relapses occurring in three patients who resulted RQ-PCR-PNQ/NEG but proved ddPCR positive quantifiable. At variance, no relapses were recorded in patients whose follow-up samples were RQ-PCR-PNQ but reclassified as ddPCR-NEG. A broader application of ddPCR in acute lymphoblastic leukemia clinical trials will help to improve patients' stratification.
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D’Alessandra Y, Valerio V, Moschetta D, Massaiu I, Bozzi M, Conte M, Parisi V, Ciccarelli M, Leosco D, Myasoedova VA, Poggio P. Extraction-Free Absolute Quantification of Circulating miRNAs by Chip-Based Digital PCR. Biomedicines 2022; 10:biomedicines10061354. [PMID: 35740375 PMCID: PMC9220272 DOI: 10.3390/biomedicines10061354] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2022] [Revised: 06/01/2022] [Accepted: 06/03/2022] [Indexed: 11/17/2022] Open
Abstract
Circulating microRNAs (miRNA) have been proposed as specific biomarkers for several diseases. Quantitative Real-Time PCR (RT-qPCR) is the gold standard technique currently used to evaluate miRNAs expression from different sources. In the last few years, digital PCR (dPCR) emerged as a complementary and accurate detection method. When dealing with gene expression, the first and most delicate step is nucleic-acid isolation. However, all currently available protocols for RNA extraction suffer from the variable loss of RNA species due to the chemicals and number of steps involved, from sample lysis to nucleic acid elution. Here, we evaluated a new process for the detection of circulating miRNAs, consisting of sample lysis followed by direct evaluation by dPCR in plasma from healthy donors and in the cardiovascular setting. Our results showed that dPCR is able to detect, with high accuracy, low-copy-number as well as highly expressed miRNAs in human plasma samples without the need for RNA extraction. Moreover, we assessed a known myocardial infarction-related miR-133a in acute myocardial infarct patients vs. healthy subjects. In conclusion, our results show the suitability of the extraction-free quantification of circulating miRNAs as disease markers by direct dPCR.
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Affiliation(s)
- Yuri D’Alessandra
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Vincenza Valerio
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Donato Moschetta
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, 20122 Milan, Italy
| | - Ilaria Massaiu
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Michele Bozzi
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Maddalena Conte
- Department of Translational Medical Sciences, University of Naples Federico II, 80138 Naples, Italy; (M.C.); (V.P.); (D.L.)
- Casa di Cura San Michele, 81024 Maddaloni, Italy
| | - Valentina Parisi
- Department of Translational Medical Sciences, University of Naples Federico II, 80138 Naples, Italy; (M.C.); (V.P.); (D.L.)
| | - Michele Ciccarelli
- Department of Medicine, Surgery and Dentistry, University of Salerno, 84084 Fisciano, Italy;
| | - Dario Leosco
- Department of Translational Medical Sciences, University of Naples Federico II, 80138 Naples, Italy; (M.C.); (V.P.); (D.L.)
| | - Veronika A. Myasoedova
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
| | - Paolo Poggio
- Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; (Y.D.); (V.V.); (D.M.); (I.M.); (M.B.); (V.A.M.)
- Correspondence: ; Tel.: +39-02-5800-2853
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Cao J, Wu Q, Wan F, Guo J, Wang R. Reliable and rapid identification of glyphosate-resistance in the invasive weed Amaranthus palmeri in China. PEST MANAGEMENT SCIENCE 2022; 78:2173-2182. [PMID: 35191163 DOI: 10.1002/ps.6843] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Revised: 01/30/2022] [Accepted: 02/22/2022] [Indexed: 06/14/2023]
Abstract
BACKGROUND Glyphosate-resistant invasive plants, including Amaranthus palmeri S. Watson, have greatly challenged management of new invasions. Elucidating their glyphosate resistance levels rapidly and accurately will better inform management strategies. Quantitative real-time PCR (qPCR) has been used to identify glyphosate resistance in A. palmeri by detecting gene copy numbers of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme inhibited by glyphosate. However, qPCR can only indirectly determine copy numbers because it requires a calibrator sample; it also lacks standardization, thus limiting its usefulness. Droplet digital PCR (ddPCR) is a new method to detect copy number directly and precisely. We evaluated ddPCR as a tool to determine glyphosate-resistance level while using qPCR and glyphosate dose response (GDR) assays as reference technologies to compare performance and efficiency between methods. RESULTS We identified seven susceptible and seven resistant populations of A. palmeri using the GDR assay. Resistant levels detected by qPCR and ddPCR were generally consistent with the GDR results. Although detected values obtained by qPCR and ddPCR were highly correlated (R2 = 0.94), ddPCR results had a lower proportion of non-ideal values (36%) with better accuracy (100%) and specificity (100%) than those of qPCR results. CONCLUSIONS Our findings demonstrate that ddPCR offers improved accuracy and specificity in detecting EPSPS gene copy numbers and is a robust and rapid method for glyphosate-resistance identification in A. palmeri. Our research is the first to measure glyphosate resistance in A. palmeri by ddPCR assay and will shed light on future applications of ddPCR in identifying herbicide resistance in other invasive weeds. © 2022 Society of Chemical Industry.
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Affiliation(s)
- Jingjing Cao
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Qianmei Wu
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Fanghao Wan
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Jianying Guo
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Rui Wang
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
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Deng L, Yang X, Xu Z, Li F, Zhao J, Deng H, Jian Z, Sun X, Zhu L. Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection. Braz J Microbiol 2022; 53:625-631. [PMID: 35277851 PMCID: PMC9151951 DOI: 10.1007/s42770-022-00728-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2021] [Accepted: 03/04/2022] [Indexed: 02/01/2023] Open
Abstract
Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/μL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV.
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Affiliation(s)
- Lishuang Deng
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China
| | - Xiaoyu Yang
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China
| | - Zhiwen Xu
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, 611130, China
| | - Fengqin Li
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China
| | - Jun Zhao
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China
| | - Huidan Deng
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China
| | - Zhijie Jian
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China
| | - Xiangang Sun
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China
| | - Ling Zhu
- College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China. .,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, 611130, China.
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Starza ID, Eckert C, Drandi D, Cazzaniga G. Minimal Residual Disease Analysis by Monitoring Immunoglobulin and T-Cell Receptor Gene Rearrangements by Quantitative PCR and Droplet Digital PCR. METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2022; 2453:79-89. [PMID: 35622321 DOI: 10.1007/978-1-0716-2115-8_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Analysis of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative polymerase chain reaction (RQ-PCR) is the gold standard for sensitive and accurate minimal residual disease (MRD) monitoring; it has been extensively standardized and guidelines have been developed within the EuroMRD consortium ( www.euromrd.org ). However, new generations of PCR-based methods are standing out as potential alternatives to RQ-PCR, such as digital PCR technology (dPCR), the third-generation implementation of conventional PCR, which has the potential to overcome some of the limitations of RQ-PCR such as allowing the absolute quantification of nucleic acid targets without the need for a calibration curve. During the last years, droplet digital PCR (ddPCR) technology has been compared to RQ-PCR in several hematologic malignancies showing its proficiency for MRD analysis. So far, no established guidelines for ddPCR MRD analysis and data interpretation have been defined and its potential is still under investigation. However, a major standardization effort is underway within the EuroMRD consortium ( www.euromrd.org ) for future application of ddPCR in standard clinical practice.
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Affiliation(s)
- Irene Della Starza
- Hematology, Department of Translational and Precision Medicine, "Sapienza" University of Rome, Rome, Italy.,GIMEMA Foundation, Rome, Italy
| | - Cornelia Eckert
- Department of Pediatric Oncology Hematology, Charité - Universitätsmedizin Berlin, Berlin, Germany.,German Cancer Consortium, and German Cancer Research Center, Heidelberg, Germany
| | - Daniela Drandi
- Hematology Division, Department of Molecular Biotechnology and Health sciences, University of Torino, Torino, Italy
| | - Giovanni Cazzaniga
- Centro Ricerca Tettamanti, Fondazione Tettamanti, Centro Maria Letizia Verga, Monza, Italy. .,Genetics, Department of Medicine and Surgery, University of Milan Bicocca, Monza, Italy.
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40
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Gowda HN, Kido H, Wu X, Shoval O, Lee A, Lorenzana A, Madou M, Hoffmann M, Jiang SC. Development of a proof-of-concept microfluidic portable pathogen analysis system for water quality monitoring. THE SCIENCE OF THE TOTAL ENVIRONMENT 2022; 813:152556. [PMID: 34952082 PMCID: PMC8837627 DOI: 10.1016/j.scitotenv.2021.152556] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/23/2021] [Revised: 12/15/2021] [Accepted: 12/16/2021] [Indexed: 05/03/2023]
Abstract
Waterborne diseases cause millions of deaths worldwide, especially in developing communities. The monitoring and rapid detection of microbial pathogens in water is critical for public health protection. This study reports the development of a proof-of-concept portable pathogen analysis system (PPAS) that can detect bacteria in water with the potential application in a point-of-sample collection setting. A centrifugal microfluidic platform is adopted to integrate bacterial cell lysis in water samples, nucleic acid extraction, and reagent mixing with a droplet digital loop mediated isothermal amplification assay for bacteria quantification onto a single centrifugal disc (CD). Coupled with a portable "CD Driver" capable of automating the assay steps, the CD functions as a single step bacterial detection "lab" without the need to transfer samples from vial-to-vial as in a traditional laboratory. The prototype system can detect Enterococcus faecalis, a common fecal indicator bacterium, in water samples with a single touch of a start button within 1 h and having total hands-on-time being less than 5 min. An add-on bacterial concentration cup prefilled with absorbent polymer beads was designed to integrate with the pathogen CD to improve the downstream quantification sensitivity. All reagents and amplified products are contained within the single-use disc, reducing the opportunity of cross contamination of other samples by the amplification products. This proof-of-concept PPAS lays the foundation for field testing devices in areas needing more accessible water quality monitoring tools and are at higher risk for being exposed to contaminated waters.
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Affiliation(s)
- Hamsa N Gowda
- Samueli School of Engineering, University of California, Irvine, Irvine, CA 92617, USA
| | - Horacio Kido
- Samueli School of Engineering, University of California, Irvine, Irvine, CA 92617, USA
| | - Xunyi Wu
- Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA 91125, USA
| | - Oren Shoval
- Samueli School of Engineering, University of California, Irvine, Irvine, CA 92617, USA
| | - Adrienne Lee
- Samueli School of Engineering, University of California, Irvine, Irvine, CA 92617, USA
| | - Albert Lorenzana
- Samueli School of Engineering, University of California, Irvine, Irvine, CA 92617, USA
| | - Marc Madou
- Samueli School of Engineering, University of California, Irvine, Irvine, CA 92617, USA
| | - Michael Hoffmann
- Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA 91125, USA
| | - Sunny C Jiang
- Samueli School of Engineering, University of California, Irvine, Irvine, CA 92617, USA.
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Tan LL, Loganathan N, Agarwalla S, Yang C, Yuan W, Zeng J, Wu R, Wang W, Duraiswamy S. Current commercial dPCR platforms: technology and market review. Crit Rev Biotechnol 2022; 43:433-464. [PMID: 35291902 DOI: 10.1080/07388551.2022.2037503] [Citation(s) in RCA: 52] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Digital polymerase chain reaction (dPCR) technology has provided a new technique for molecular diagnostics, with superior advantages, such as higher sensitivity, precision, and specificity over quantitative real-time PCRs (qPCR). Eight companies have offered commercial dPCR instruments: Fluidigm Corporation, Bio-Rad, RainDance Technologies, Life Technologies, Qiagen, JN MedSys Clarity, Optolane, and Stilla Technologies Naica. This paper discusses the working principle of each offered dPCR device and compares the associated: technical aspects, usability, costs, and current applications of each dPCR device. Lastly, up-and-coming dPCR technologies are also presented, as anticipation of how the dPCR device landscape may likely morph in the next few years.
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Affiliation(s)
- Li Ling Tan
- Singapore Institute of Manufacturing Technology, Singapore, Singapore.,Materials Science and Engineering School, Nanyang Technological University, Singapore, Singapore
| | - Nitin Loganathan
- Singapore Institute of Manufacturing Technology, Singapore, Singapore
| | - Sushama Agarwalla
- Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, India
| | - Chun Yang
- Mechanical and Aerospace Engineering School, Nanyang Technological University, Singapore, Singapore
| | - Weiyong Yuan
- Faculty of Materials & Energy, Institute for Clean Energy and Advanced Materials, Southwest University, Chongqing, China.,Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Chongqing, China
| | - Jasmine Zeng
- Singapore Institute of Manufacturing Technology, Singapore, Singapore
| | - Ruige Wu
- Singapore Institute of Manufacturing Technology, Singapore, Singapore
| | - Wei Wang
- Singapore Institute of Manufacturing Technology, Singapore, Singapore
| | - Suhanya Duraiswamy
- Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, India
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Wei C, Yu C, Li S, Meng J, Li T, Cheng J, Pan F, Li J. Easy-to-Operate Co-flow Step Emulsification Device for Droplet Digital Polymerase Chain Reaction. Anal Chem 2022; 94:3939-3947. [PMID: 35200004 DOI: 10.1021/acs.analchem.1c04983] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including high cost, complex operation, and low integration of the instrumental system. Here, in this work, a novel microfluidic chip based on co-flow step emulsification is proposed for droplet digital PCR (ddPCR), which can achieve droplet generation, droplet array self-assembly, PCR amplification, and fluorescence detection on a single device. With the combination of single-layer lithography and punching operation, a step microstructure was constructed and it served as the key element to develop a Laplace pressure gradient at the Rayleigh-Plateau instability interface so as to achieve droplet generation. It is demonstrated that the fabrication of step microstructure is low cost, easy-to-operate, and reliable. In addition, the single droplet volume can be adjusted flexibly due to the co-flow design; thus, the ddPCR chip can get an ultrahigh upper limit of quantification to deal with DNA templates with high concentrations. Furthermore, the volume fraction of the resulting droplets in this ddPCR chip can be up to 72% and it results in closely spaced droplet arrays, makes the best of CCD camera for fluorescence detections, and is beneficial for the minimization of a ddPCR system. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 50 000 copies/μL. Owing to the characteristics of low cost, easy operation, excellent quantitative capability, and minimization, the proposed ddPCR chip meets the requirements of DNA molecule quantification and is expected to be applied in the point-of-care testing field.
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Affiliation(s)
- Chunyang Wei
- Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions, School of Mechanical Engineering, Hebei University of Technology, Tianjin 300132, China.,State Key Laboratory of Reliability and Intelligence of Electrical Equipment, Hebei University of Technology, Tianjin 300130, China
| | - Chengzhuang Yu
- Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions, School of Mechanical Engineering, Hebei University of Technology, Tianjin 300132, China
| | - Shanshan Li
- Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions, School of Mechanical Engineering, Hebei University of Technology, Tianjin 300132, China.,State Key Laboratory of Reliability and Intelligence of Electrical Equipment, Hebei University of Technology, Tianjin 300130, China
| | - Jiyu Meng
- Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions, School of Mechanical Engineering, Hebei University of Technology, Tianjin 300132, China
| | - Tiejun Li
- Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions, School of Mechanical Engineering, Hebei University of Technology, Tianjin 300132, China
| | - Jingmeng Cheng
- Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions, School of Mechanical Engineering, Hebei University of Technology, Tianjin 300132, China
| | - Feng Pan
- Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions, School of Mechanical Engineering, Hebei University of Technology, Tianjin 300132, China
| | - Junwei Li
- Institute of Biophysics, School of Health Science and Biomedical Engineering, Hebei University of Technology, Tianjin 300401, China.,Department of Electronics and Information Engineering, Hebei University of Technology, Langfang 065000, China
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Basanisi MG, La Bella G, Nobili G, Raele DA, Cafiero MA, Coppola R, Damato AM, Fraccalvieri R, Sottili R, La Salandra G. Detection of Coxiella burnetii DNA in sheep and goat milk and dairy products by droplet digital PCR in south Italy. Int J Food Microbiol 2022; 366:109583. [PMID: 35182931 DOI: 10.1016/j.ijfoodmicro.2022.109583] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2021] [Revised: 01/18/2022] [Accepted: 02/09/2022] [Indexed: 12/11/2022]
Abstract
Coxiella burnetii is a Gram-negative obligate intracellular bacterium that is responsible for Q fever, a common zoonosis which is present virtually worldwide. This microorganism infects a wide range of wild and domestic mammals, but the main reservoirs are cattle, goats and sheep, which also represent sources of human infection. A potential route of transmission of this pathogen to humans is the consumption of C. burnetii-contaminated raw milk or dairy products derived from contaminated raw milk, although the role of these foods as possible infection sources is controversial. The aims of this study were (i) to apply two ddPCR based assays targeting the C. burnetii IS1111 and icd genes for the detection and quantification of C. burnetii DNA, and (ii) to evaluate the occurrence of C. burnetii DNA in raw milk and raw milk products from sheep and goats in Apulia and Basilicata regions of Southern Italy. Of 413 milk and cheese samples tested, 78 were positive for the presence of C. burnetii DNA (18.9%), specifically, 68 of 285 milk samples (23.9%) and 10 of 128 cheese samples (7.8%) The presence of both IS1111 and icd genes was detected in only 2 (2.6%) of the 78 positive samples, while the remaining 76 (97.4%) were positive only for IS1111. C. burnetii DNA was specifically detected by the ddPCR method, whereas no cross-amplification was observed with the DNA of other foodborne bacterial pathogens. The sensitivity of the ddPCR method was determined as 0.35 and 0.56 copies/μL for IS1111 and icd genes, respectively. The findings of this study demonstrate the presence of C. burnetii DNA in a significant proportion of raw milk and dairy products. Although there is no conclusive epidemiological evidence that C. burnetii infection occurs via food, the presence of this organism in raw milk and dairy products made of raw milk should be considered a potential hazard. ddPCR is a useful tool to investigate the quality and safety of food products due to its sensitivity and precision, and could be applied to routine testing.
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Affiliation(s)
- Maria Grazia Basanisi
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy.
| | - Gianfranco La Bella
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
| | - Gaia Nobili
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
| | - Donato Antonio Raele
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
| | - Maria Assunta Cafiero
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
| | - Rosa Coppola
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
| | - Annita Maria Damato
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
| | - Rosa Fraccalvieri
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
| | - Roldano Sottili
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
| | - Giovanna La Salandra
- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB), Via Manfredonia 20, 71121 Foggia, Italy
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Gaňová M, Wang X, Yan Z, Zhang H, Lednický T, Korabečná M, Neužil P. Temperature non-uniformity detection on dPCR chips and temperature sensor calibration. RSC Adv 2022; 12:2375-2382. [PMID: 35425215 PMCID: PMC8979175 DOI: 10.1039/d1ra08138a] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2021] [Accepted: 01/08/2022] [Indexed: 12/12/2022] Open
Abstract
A microfluidic-based digital polymerase chain reaction (dPCR) chip requires precise temperature control as well as uniform temperature distribution to ensure PCR efficiency. However, measuring local temperature and its distribution over thousands of μL/nL-volume samples with minimum disturbance is challenging. Here, we present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip. We filled the dPCR chip with a PCR solution containing amplified DNA fragments with a known melting temperature (TM). We then captured fluorescent images of the chip when it was heated from 70 to 99 °C, plotted the fluorescence intensity of each partition as a function of temperature, and calculated measured TM values from each partition. Finally, we created a 3-D map of the dPCR chip with the measured TM as the parameter. Even when the actual TM of the PCR solution was constant, the measured TM value varied between locations due to temperature non-uniformity in the dPCR chip. The method described here thereby characterized the distribution of temperature non-uniformity using a PCR solution with known TM as a temperature sensor. Among the non-contact temperature measurement methods, the proposed TM-based method can determine the temperature distribution within the chip, instead of only at the chip surface. The method also does not suffer from the undesirable photobleaching effect of fluorescein-based temperature measurement method. Temperature determination over the dPCR chip based on TM allowed us to calibrate the temperature sensor and improve the dPCR configuration and precision. This method is also suitable for determining the temperature uniformity of other microarray systems where there is no physical access to the system and thus direct temperature measurement is not possible. We present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip mounted on two different thermal cycling configurations.![]()
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Affiliation(s)
- Martina Gaňová
- Central European Institute of Technology, Brno University of Technology Purkyňova 123 612 00 Brno Czech Republic
| | - Xinlu Wang
- Northwestern Polytechnical University, School of Mechanical Engineering, Department of Microsystem Engineering 127 West Youyi Road Xi'an Shaanxi 710072 PR China
| | - Zhiqiang Yan
- Northwestern Polytechnical University, School of Marine Science and Technology 127 West Youyi Road Xi'an Shaanxi 710072 PR China
| | - Haoqing Zhang
- Northwestern Polytechnical University, School of Mechanical Engineering, Department of Microsystem Engineering 127 West Youyi Road Xi'an Shaanxi 710072 PR China
| | - Tomáš Lednický
- Central European Institute of Technology, Brno University of Technology Purkyňova 123 612 00 Brno Czech Republic
| | - Marie Korabečná
- Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague Albertov 4 128 00 Prague Czech Republic
| | - Pavel Neužil
- Northwestern Polytechnical University, School of Mechanical Engineering, Department of Microsystem Engineering 127 West Youyi Road Xi'an Shaanxi 710072 PR China
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45
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ZHOU W, LI YB, ZHANG YL, LI X, SHI GH, YANG XL. Establishment of ddPCR detection technology system for three contaminants in fermented milk. FOOD SCIENCE AND TECHNOLOGY 2022. [DOI: 10.1590/fst.115721] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Affiliation(s)
- Wei ZHOU
- Hebei Food Inspection and Research Institute, China; Hebei Normal University, China
| | - Yong-Bo LI
- Hebei Food Inspection and Research Institute, China
| | - Ya-Lun ZHANG
- Hebei Food Inspection and Research Institute, China
| | - Xian LI
- Hebei Food Inspection and Research Institute, China
| | - Guo-hua SHI
- Hebei Food Inspection and Research Institute, China
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Yao J, Luo Y, Zhang Z, Li J, Li C, Li C, Guo Z, Wang L, Zhang W, Zhao H, Zhou L. The development of real-time digital PCR technology using an improved data classification method. Biosens Bioelectron 2021; 199:113873. [PMID: 34953301 DOI: 10.1016/j.bios.2021.113873] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2021] [Revised: 11/26/2021] [Accepted: 12/06/2021] [Indexed: 02/09/2023]
Abstract
For digital polymerase chain reaction (PCR), data classification is always a crucial task. The dynamic real-time amplification process information of each partition is always ignored in typical digital PCR analysis, which can easily lead to inaccurate outcomes. In this work, an integrated device that offers real-time chip-based digital PCR analysis was established. In addition, an enhanced process-based classification model (PAM) was built and trained. And then the device and the analytical model were employed in classification tasks for different concentrations of Epstein-Barr Virus (EBV) plasmid quantification assays. The results indicated that the real-time analysis device achieved a linearity of 0.97, the classification method was able to distinguish the false-positive curves, and the recognition error of positive wells was decreased by 64.4% compared with typical static analysis techniques when low concentrations of samples were tested. With these advantages, it is supposed that the real-time digital PCR analysis apparatus and the improved classification method can be employed to enhance the performance of digital PCR technology.
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Affiliation(s)
- Jia Yao
- School of Electronic and Information Engineering, Soochow University, Suzhou, 215006, China; CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China
| | - Yuanyuan Luo
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China
| | - Zhiqi Zhang
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China
| | - Jinze Li
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China
| | - Chuanyu Li
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China
| | - Chao Li
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China
| | - Zhen Guo
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China
| | - Lirong Wang
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China
| | - Wei Zhang
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China.
| | - Heming Zhao
- School of Electronic and Information Engineering, Soochow University, Suzhou, 215006, China.
| | - Lianqun Zhou
- CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China; School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China.
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Tan F, Xiao P, Yang JR, Chen H, Jin L, Yang Y, Lin TF, Willis A, Yang J. Precision early detection of invasive and toxic cyanobacteria: A case study of Raphidiopsis raciborskii. HARMFUL ALGAE 2021; 110:102125. [PMID: 34887005 DOI: 10.1016/j.hal.2021.102125] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/24/2021] [Revised: 10/13/2021] [Accepted: 10/16/2021] [Indexed: 06/13/2023]
Abstract
Blooms of the toxic cyanobacterium, Raphidiopsis raciborskii (basionym Cylindrospermopsis raciborskii), are becoming a major environmental issue in freshwater ecosystems globally. Our precision prevention and early detection of R. raciborskii blooms rely upon the accuracy and speed of the monitoring method. A duplex digital PCR (dPCR) monitoring approach was developed and validated to detect the abundance and toxin-producing potential of R. raciborskii simultaneously in both laboratory spiked and environmental samples. Results of dPCR were strongly correlated with traditional real time quantitative PCR (qPCR) and microscopy for both laboratory and environmental samples. However, discrepancies between methods were observed when measuring R. raciborskii at low abundance (1 - 105 cells L - 1), with dPCR showing a higher precision compared to qPCR at low cell concentration. Furthermore, the dPCR assay had the highest detection rate for over two hundred environmental samples especially under low abundance conditions, followed by microscopy and qPCR. dPCR assay had the advantages of simple operation, time-saving, high sensitivity and excellent reproducibility. Therefore, dPCR would be a fast and precise monitoring method for the early warning of toxic bloom-forming cyanobacterial species and assessment of water quality risks, which can improve prediction and prevention of the impacts of harmful cyanobacterial bloom events in inland waters.
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Affiliation(s)
- Fengjiao Tan
- Aquatic EcoHealth Group, Fujian Key Laboratory of Watershed Ecology, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Peng Xiao
- Aquatic EcoHealth Group, Fujian Key Laboratory of Watershed Ecology, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China
| | - Jun R Yang
- Aquatic EcoHealth Group, Fujian Key Laboratory of Watershed Ecology, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China; Engineering Research Center of Ecology and Agricultural Use of Wetland (Ministry of Education), College of Agriculture, Yangtze University, Jingzhou 434025, China
| | - Huihuang Chen
- Aquatic EcoHealth Group, Fujian Key Laboratory of Watershed Ecology, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Lei Jin
- Aquatic EcoHealth Group, Fujian Key Laboratory of Watershed Ecology, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yigang Yang
- Aquatic EcoHealth Group, Fujian Key Laboratory of Watershed Ecology, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Tsair-Fuh Lin
- Department of Environmental Engineering, National Cheng Kung University, Tainan 70101, Taiwan
| | - Anusuya Willis
- Australian National Algae Culture Collection, CSIRO, Hobart 7000, Tasmania, Australia
| | - Jun Yang
- Aquatic EcoHealth Group, Fujian Key Laboratory of Watershed Ecology, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China.
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Van Poelvoorde LAE, Gand M, Fraiture MA, De Keersmaecker SCJ, Verhaegen B, Van Hoorde K, Cay AB, Balmelle N, Herman P, Roosens N. Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution. Curr Issues Mol Biol 2021; 43:1937-1949. [PMID: 34889894 PMCID: PMC8928932 DOI: 10.3390/cimb43030134] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2021] [Revised: 10/08/2021] [Accepted: 11/02/2021] [Indexed: 12/12/2022] Open
Abstract
The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.
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Affiliation(s)
- Laura A. E. Van Poelvoorde
- Transversal Activities in Applied Genomics, Sciensano, 1050 Brussels, Belgium; (L.A.E.V.P.); (M.G.); (M.-A.F.); (S.C.J.D.K.)
| | - Mathieu Gand
- Transversal Activities in Applied Genomics, Sciensano, 1050 Brussels, Belgium; (L.A.E.V.P.); (M.G.); (M.-A.F.); (S.C.J.D.K.)
| | - Marie-Alice Fraiture
- Transversal Activities in Applied Genomics, Sciensano, 1050 Brussels, Belgium; (L.A.E.V.P.); (M.G.); (M.-A.F.); (S.C.J.D.K.)
| | - Sigrid C. J. De Keersmaecker
- Transversal Activities in Applied Genomics, Sciensano, 1050 Brussels, Belgium; (L.A.E.V.P.); (M.G.); (M.-A.F.); (S.C.J.D.K.)
| | - Bavo Verhaegen
- Food Pathogens, Sciensano, 1050 Brussels, Belgium; (B.V.); (K.V.H.)
| | | | - Ann Brigitte Cay
- Enzootic, Vector-Borne and Bee Diseases, Sciensano, 1180 Brussels, Belgium; (A.B.C.); (N.B.)
| | - Nadège Balmelle
- Enzootic, Vector-Borne and Bee Diseases, Sciensano, 1180 Brussels, Belgium; (A.B.C.); (N.B.)
| | - Philippe Herman
- Expertise and Service Provision, Sciensano, 1050 Brussels, Belgium;
| | - Nancy Roosens
- Transversal Activities in Applied Genomics, Sciensano, 1050 Brussels, Belgium; (L.A.E.V.P.); (M.G.); (M.-A.F.); (S.C.J.D.K.)
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49
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Trick AY, Chen FE, Schares JA, Freml BE, Lor P, Yun Y, Wang TH. High resolution estimates of relative gene abundance with quantitative ratiometric regression PCR (qRR-PCR). Analyst 2021; 146:6463-6469. [PMID: 34605831 PMCID: PMC8627783 DOI: 10.1039/d1an01397a] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Quantification of the relative abundance of genetic traits has broad applications for biomarker discovery, diagnostics, and assessing gene expression in biological research. Relative quantification of genes is traditionally done with the 2-ΔΔCT method using quantitative real-time polymerase chain reaction (qPCR) data, which is often limited in resolution beyond orders of magnitude difference. The latest techniques for quantification of nucleic acids employ digital PCR or microarrays which involve lengthy sample preparation and complex instrumentation. In this work, we describe a quantitative ratiometric regression PCR (qRR-PCR) method for computing relative abundance of genetic traits in a sample with high resolution from a single duplexed real-time quantitative PCR assay. Instead of comparing the individual cycle threshold (Ct) values as is done for the 2-ΔΔCT method, our qRR-PCR algorithm leverages the innate relationship of co-amplified PCR targets to measure their relative quantities using characteristic curves derived from the normalized ratios of qPCR fluorescence curves. We demonstrate the utility of this technique for discriminating the fractional abundance of mixed alleles with resolution below 5%.
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Affiliation(s)
- Alexander Y Trick
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
| | - Fan-En Chen
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
| | | | | | - Pa Lor
- Corteva Agriscience, Johnston, IA 50131, USA
| | - Yue Yun
- Corteva Agriscience, Johnston, IA 50131, USA
| | - Tza-Huei Wang
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
- Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
- Institute for NanoBiotechnology, Johns Hopkins University, Baltimore, MD 21218, USA
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50
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Diagnostic Techniques for COVID-19: A Mini-review of Early Diagnostic Methods. JOURNAL OF ANALYSIS AND TESTING 2021; 5:314-326. [PMID: 34631199 PMCID: PMC8488931 DOI: 10.1007/s41664-021-00198-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Accepted: 08/25/2021] [Indexed: 12/26/2022]
Abstract
The outbreak of severe pneumonia at the end of 2019 was proved to be caused by the SARS-CoV-2 virus spreading out the world. And COVID-19 spread rapidly through a terrible transmission way by human-to-human, which led to many suspected cases waiting to be diagnosed and huge daily samples needed to be tested by an effective and rapid detection method. With an increasing number of COVID-19 infections, medical pressure is severe. Therefore, more efficient and accurate diagnosis methods were keen urgently established. In this review, we summarized several methods that can rapidly and sensitively identify COVID-19; some of them are widely used as the diagnostic techniques for SARS-CoV-2 in various countries, some diagnostic technologies refer to SARS (Severe Acute Respiratory Syndrome) or/and MERS (Middle East Respiratory Syndrome) detection, which may provide potential diagnosis ideas.
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