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1
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Tayyab M, Gandotra N, Sui J, Scharfe C, Javanmard M. Allele-specific electrical genotyping for diagnosis of transthyretin amyloidosis. COMMUNICATIONS ENGINEERING 2025; 4:47. [PMID: 40082707 PMCID: PMC11906782 DOI: 10.1038/s44172-025-00385-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/19/2024] [Accepted: 02/27/2025] [Indexed: 03/16/2025]
Abstract
Clinical genetic testing often takes days to weeks, but rapid, affordable tests during outpatient visits could significantly benefit patients. This is crucial for detecting common, actionable point mutations, such as those linked to hereditary transthyretin (TTR) amyloidosis, which is often underdiagnosed in individuals of West African ancestry with congestive heart failure. Here we developed a method for detecting known DNA variants using allele-specific polymerase chain reaction (ASPCR) and electrical impedance. Oligonucleotide primers were designed to selectively amplify the ancestral and variant allele of TTR c.424G>A (p.Val142Ile). PCR products were detected in solution using passive-flow microfluidic impedance cytometry across multiple excitation frequencies. Impedance responses correlated with DNA concentration, allowing for the calculation of a robust DNA quantification score. TTR V142I genotypes from six patients (four heterozygous, two wildtype) matched results from gel electrophoresis and sequencing. Future improvements will focus on reducing DNA input requirements and enabling multiplex variant detection.
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Affiliation(s)
- Muhammad Tayyab
- Department of Electrical and Computer Engineering, Rutgers University, New Brunswick, NJ, USA
| | - Neeru Gandotra
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- SUNY Upstate Medical University, Syracuse, NY, USA
| | - Jianye Sui
- Department of Electrical and Computer Engineering, Rutgers University, New Brunswick, NJ, USA
| | - Curt Scharfe
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
| | - Mehdi Javanmard
- Department of Electrical and Computer Engineering, Rutgers University, New Brunswick, NJ, USA.
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2
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Mai Z, Zhou T, Lin Z. Detecting CYP2C19 genes through an integrated CRISPR/Cas13a-assisted system. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2025; 17:1382-1388. [PMID: 39836103 DOI: 10.1039/d4ay01930j] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/22/2025]
Abstract
CYP2C19 gene single nucleotide polymorphisms (SNPs) should be considered in the clinical use of clopidogrel as they have important guiding value for predicting the risk of bleeding and thrombosis after clopidogrel treatment. The CRISPR/Cas system is increasingly used for SNP detection owing to its single-nucleotide mismatch specificity. Simultaneous detection of multiple SNPs for rapid identification of the CYP2C19 genotype is important, but there is no method to detect a wide variety of CYP2C19 SNPs. This study proposes a new integrated system that integrates the PCR reaction and CRISPR/Cas detection of three CYP2C19 genes on a device, achieving rapid, sensitive, and specific detection. In our design, magnetic beads with three different sizes capture target nucleic acid from the sample, which are dragged through different areas by magnetic force, for PCR amplification reaction and CRISPR/Cas13a detection of CYP2C19*2, CYP2C19*3 and CYP2C19*17 genes. Note that magnetic beads were sorted via microporous PC membranes of different apertures. This study exhibits a broad clinical application prospect and provides a favorable tool for clinical clopidogrel administration.
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Affiliation(s)
- Zhaokang Mai
- Department of Cardiology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, China.
| | - Tao Zhou
- Department of Cardiology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, China.
| | - Zhun Lin
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China.
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3
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Guo C, Ding R, Zhao Z, Guo J, Li F. Enrichment Strategies for Low-Abundant Single Nucleotide Mutations. Chemistry 2025; 31:e202402872. [PMID: 39448543 DOI: 10.1002/chem.202402872] [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: 07/30/2024] [Revised: 10/20/2024] [Accepted: 10/24/2024] [Indexed: 10/26/2024]
Abstract
Over the past three decades, significant advancements have been made in mutation enrichment methods, driven by the increasing need for precise and efficient identification of rare genetic variants associated with diseases. Mutation-enrichment methods have emerged to boost sensitivity and enable easy detection of low-frequency mutations. These methods are crucial in genomics research and clinical diagnostics, allowing for the detection of low-frequency mutations within large genomic datasets. This review presents a summary of technological developments in rare mutation enrichment and emphasizes their mechanisms and applications in liquid biopsies.
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Affiliation(s)
- Chen Guo
- Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, Chengdu, Sichuan, 610064, China
| | - Ruolin Ding
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, 610064, China
| | - Zhihe Zhao
- State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, 610064, China
| | - Jian Guo
- Department of Neurology, West China Hospital, Sichuan University, Chengdu, Sichuan, 610064, China
| | - Feng Li
- Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, Chengdu, Sichuan, 610064, China
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Choi H, Choi EJ, Kim HJ, Baek IC, Won A, Park SJ, Kim TG, Chung YJ. A walk through the development of human leukocyte antigen typing: from serologic techniques to next-generation sequencing. CLINICAL TRANSPLANTATION AND RESEARCH 2024; 38:294-308. [PMID: 39658458 PMCID: PMC11732764 DOI: 10.4285/ctr.24.0055] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/29/2024] [Revised: 11/11/2024] [Accepted: 11/12/2024] [Indexed: 12/12/2024]
Abstract
Human leukocyte antigen (HLA) is a group of glycoproteins encoded by the major histocompatibility complex (MHC) that plays a pivotal role in the host's immune defense. Given that the MHC represents the most polymorphic region in the human genome, HLA typing is crucial in organ transplantation. It significantly influences graft rejection, graft-versus-host disease, and the overall patient outcome by mediating the discrimination between self and nonself. HLA typing technology began with serological methods and has evolved rapidly alongside advances in molecular technologies, progressing from DNA-based typing to next- or third-generation sequencing. These advancements have increased the accuracy of HLA typing and reduced ambiguities, leading to marked improvements in transplantation outcomes. Additionally, numerous novel HLA alleles have been identified. In this review, we explore the developmental history and future prospects of HLA typing technology, which promises to further benefit the field of transplantation.
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Affiliation(s)
- Haeyoun Choi
- Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul, Korea
- Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea
- Department of Medical Sciences, Graduate School of The Catholic University of Korea, Seoul, Korea
| | - Eun-Jeong Choi
- Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea
| | - Hyoung-Jae Kim
- Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea
| | - In-Cheol Baek
- Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea
| | - Aegyeong Won
- Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea
| | - Su Jin Park
- Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea
| | | | - Yeun-Jun Chung
- Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul, Korea
- Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea
- Department of Medical Sciences, Graduate School of The Catholic University of Korea, Seoul, Korea
- Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, Seoul, Korea
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da Silva SM, Amaral C, Malta-Luís C, Grilo D, Duarte AG, Morais I, Afonso G, Faria N, Antunes W, Gomes I, Sá-Leão R, Miragaia M, Serrano M, Pimentel C. A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva. Biol Methods Protoc 2024; 9:bpae035. [PMID: 38835855 PMCID: PMC11147803 DOI: 10.1093/biomethods/bpae035] [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: 03/22/2024] [Revised: 05/03/2024] [Accepted: 05/17/2024] [Indexed: 06/06/2024] Open
Abstract
The gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.
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Affiliation(s)
- Sofia M da Silva
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Catarina Amaral
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Cláudia Malta-Luís
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Diana Grilo
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Américo G Duarte
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Inês Morais
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Gonçalo Afonso
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Nuno Faria
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Wilson Antunes
- Centro de Investigação da Academia Militar (CINAMIL), Unidade Militar Laboratorial de Defesa Biológica e Química (UMLDBQ), Av. Dr Alfredo Bensaúde, Lisboa, 1849-012, Portugal
| | - Inês Gomes
- Centro de Investigação da Academia Militar (CINAMIL), Unidade Militar Laboratorial de Defesa Biológica e Química (UMLDBQ), Av. Dr Alfredo Bensaúde, Lisboa, 1849-012, Portugal
| | - Raquel Sá-Leão
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Maria Miragaia
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Mónica Serrano
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
| | - Catarina Pimentel
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, 2780-157, Portugal
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6
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Xiong E, Liu P, Deng R, Zhang K, Yang R, Li J. Recent advances in enzyme-free and enzyme-mediated single-nucleotide variation assay in vitro. Natl Sci Rev 2024; 11:nwae118. [PMID: 38742234 PMCID: PMC11089818 DOI: 10.1093/nsr/nwae118] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Revised: 03/14/2024] [Accepted: 03/15/2024] [Indexed: 05/16/2024] Open
Abstract
Single-nucleotide variants (SNVs) are the most common type variation of sequence alterations at a specific location in the genome, thus involving significant clinical and biological information. The assay of SNVs has engaged great awareness, because many genome-wide association studies demonstrated that SNVs are highly associated with serious human diseases. Moreover, the investigation of SNV expression levels in single cells are capable of visualizing genetic information and revealing the complexity and heterogeneity of single-nucleotide mutation-related diseases. Thus, developing SNV assay approaches in vitro, particularly in single cells, is becoming increasingly in demand. In this review, we summarized recent progress in the enzyme-free and enzyme-mediated strategies enabling SNV assay transition from sensing interface to the test tube and single cells, which will potentially delve deeper into the knowledge of SNV functions and disease associations, as well as discovering new pathways to diagnose and treat diseases based on individual genetic profiles. The leap of SNV assay achievements will motivate observation and measurement genetic variations in single cells, even within living organisms, delve into the knowledge of SNV functions and disease associations, as well as open up entirely new avenues in the diagnosis and treatment of diseases based on individual genetic profiles.
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Affiliation(s)
- Erhu Xiong
- Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China
| | - Pengfei Liu
- Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China
| | - Ruijie Deng
- College of Biomass Science and Engineering, Healthy Food Evaluation Research Center, Sichuan University, Chengdu 610065, China
| | - Kaixiang Zhang
- School of Pharmaceutical Sciences, Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001, China
| | - Ronghua Yang
- Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China
| | - Jinghong Li
- Department of Chemistry, Center for Bioanalytical Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China
- Beijing Institute of Life Science and Technology, Beijing 102206, China
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7
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Wang X, Chen T, Ping Y, Dai Y, Yu P, Xie Y, Liu Z, Sun B, Duan X, Tao Z. Sequence-Guided Localization of DNA Hybridization Enables Highly Selective and Robust Genotyping. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2024; 20:e2307985. [PMID: 38084466 DOI: 10.1002/smll.202307985] [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: 11/09/2023] [Revised: 11/28/2023] [Indexed: 05/18/2024]
Abstract
Genetic variations are always related to human diseases or susceptibility to therapies. Nucleic acid probes that precisely distinguish closely related sequences become an indispensable requisite both in research and clinical applications. Here, a Sequence-guided DNA LOCalization for leaKless DNA detection (SeqLOCK) is introduced as a technique for DNA hybridization, where the intended targets carrying distinct "guiding sequences" act selectively on the probes. In silicon modeling, experimental results reveal considerable agreement (R2 = 0.9228) that SeqLOCK is capable of preserving high discrimination capacity at an extraordinarily wide range of target concentrations. Furthermore, SeqLOCK reveals high robustness to various solution conditions and can be directly adapted to nucleic acid amplification techniques (e.g., polymerase chain reaction) without the need for laborious pre-treatments. Benefiting from the low hybridization leakage of SeqLOCK, three distinct variations with a clinically relevant mutation frequency under the background of genomic DNA can be discriminated simultaneously. This work establishes a reliable nucleic acid hybridization strategy that offers great potential for constructing robust and programmable systems for molecular sensing and computing.
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Affiliation(s)
- Xuchu Wang
- Department of Laboratory Medicine, the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310000, China
| | - Tao Chen
- Department of Blood Transfusion, Zhejiang Hospital, Hangzhou, 310052, China
| | - Ying Ping
- Department of Laboratory Medicine, the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310000, China
| | - Yibei Dai
- Department of Laboratory Medicine, the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310000, China
| | - Pan Yu
- Department of Laboratory Medicine, the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310000, China
| | - Yiyi Xie
- Department of Laboratory Medicine, the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310000, China
| | - Zhenping Liu
- Department of Laboratory Medicine, Yuhang Branch of the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310058, China
| | - Bohao Sun
- Department of Pathology, the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310009, China
| | - Xiuzhi Duan
- Department of Laboratory Medicine, the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310000, China
| | - Zhihua Tao
- Department of Laboratory Medicine, the Second Affiliated Hospital of Zhejiang University, Hangzhou, 310000, China
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8
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Daprà V, Giraudo I, Zaniol E, Galliano I, Calvi C, Montanari P, Alliaudi C, Saracco P, Bergallo M. Evaluation of the FCGR2B polymorphism in children with immune thrombocytopenia. Minerva Pediatr (Torino) 2024; 76:93-99. [PMID: 34859644 DOI: 10.23736/s2724-5276.21.05888-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
BACKGROUND Childhood immune thrombocytopenia (ITP) is an immune-mediated disease characterized by an isolated low platelet count. Pathogenesis of ITP is complex but many patients have platelet specific autoantibodies leading to accelerated clearance of opsonized platelets by Fc-gamma receptor (FcγR) bearing phagocytes, particularly in the spleen. In humans, there are three main types of Fcγ receptors: high-affinity FcγRI and low-affinity FcγRII and FcγRIII. About FcγRII, genetic variation of FCGR2B is associated with response to IVIg treatment in patients with Kawasaki disease and ITP. METHODS We used a TaqMAMA genotyping assay for detection of rs1050501 FCGR2B polymorphism in children with chronic ITP. A SNP rs1050501 (GenBank access number NG_023318.1, Homo sapiens Fc fragment of IgG receptor IIb [FCGR2B]) on chromosome 1 showing a T/C transition in position 15894 on FCGRB2 gene was chosen in this study. A series of experiments was conducted to evaluate the performance of the FCGR2B-MAMAPCR real time on a QuantStudio™ 5 Real-Time PCR System as compared to the 7500 Real-Time PCR System. RESULTS Background noise, genotypes discrimination, variability, allele and genotype frequencies and concordance were obtained. About clinical validation, all 60 samples collected from chronic ITP patients were analyzed. We found 53 on the 60 patients (88.4%) were homozygotes (52 TT and 1 CC) and 7/60 (11.6%) heterozygotes (TC). CONCLUSIONS The ability of the FCGR2B-MAMAPCR real time to detect rs1050501 FCGR2B polymorphism in children with chronic ITP on the QuantStudio™ 5 System is comparable to that on the 7500 System.
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Affiliation(s)
- Valentina Daprà
- Department of Public Health and Pediatrics, University of Turin, School of Medicine, Turin, Italy
- BioMole srl, Turin, Italy
| | - Isaac Giraudo
- Department of Public Health and Pediatrics, University of Turin, School of Medicine, Turin, Italy
- Unit of Hematology, Department of Pediatrics, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Turin, Italy
| | - Elena Zaniol
- Department of Public Health and Pediatrics, University of Turin, School of Medicine, Turin, Italy
| | - Ilaria Galliano
- Department of Public Health and Pediatrics, University of Turin, School of Medicine, Turin, Italy
| | - Cristina Calvi
- Department of Public Health and Pediatrics, University of Turin, School of Medicine, Turin, Italy
| | - Paola Montanari
- Department of Public Health and Pediatrics, University of Turin, School of Medicine, Turin, Italy
| | - Carla Alliaudi
- Department of Public Health and Pediatrics, University of Turin, School of Medicine, Turin, Italy
| | - Paola Saracco
- Unit of Hematology, Department of Pediatrics, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Turin, Italy
| | - Massimiliano Bergallo
- Department of Public Health and Pediatrics, University of Turin, School of Medicine, Turin, Italy -
- BioMole srl, Turin, Italy
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9
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Sani A, Idrees Khan M, Shah S, Tian Y, Zha G, Fan L, Zhang Q, Cao C. Diagnosis and screening of abnormal hemoglobins. Clin Chim Acta 2024; 552:117685. [PMID: 38030031 DOI: 10.1016/j.cca.2023.117685] [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: 10/26/2023] [Revised: 11/21/2023] [Accepted: 11/24/2023] [Indexed: 12/01/2023]
Abstract
Hemoglobin (Hb) abnormalities, such as thalassemia and structural Hb variants, are among the most prevalent inherited diseases and are associated with significant mortality and morbidity worldwide. However, there were not comprehensive reviews focusing on different clinical analytical techniques, research methods and artificial intelligence (AI) used in clinical screening and research on hemoglobinopathies. Hence the review offers a comprehensive summary of recent advancements and breakthroughs in the detection of aberrant Hbs, research methods and AI uses as well as the present restrictions anddifficulties in hemoglobinopathies. Recent advances in cation exchange high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), flow cytometry, mass spectrometry (MS) and polymerase chain reaction (PCR) etc have allowed for the definitive detection by using advanced AIand portable point of care tests (POCT) integrating with smartphone microscopic classification, machine learning (ML) model, complete blood counts (CBC), imaging-based method, speedy immunoassay, and electrochemical-, microfluidic- and sensing-related platforms. In addition, to confirm and validate unidentified and novel Hbs, highly specialized genetic based techniques like PCR, reverse transcribed (RT)-PCR, DNA microarray, sequencing of genomic DNA, and sequencing of RT-PCR amplified globin cDNA of the gene of interest have been used. Hence, adequate utilization and improvement of available diagnostic and screening technologies are important for the control and management of hemoglobinopathies.
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Affiliation(s)
- Ali Sani
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Muhammad Idrees Khan
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Saud Shah
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Youli Tian
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China; School of Life Science and Biotechnology, State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Genhan Zha
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
| | - Liuyin Fan
- Student Innovation Center, Shanghai Jiao Tong University, Shanghai 200240, China.
| | - Qiang Zhang
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China.
| | - Chengxi Cao
- School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China; School of Life Science and Biotechnology, State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, 200240, China.
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10
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Adawaye C, Fokam J, Kamangu EN, Ngwese DTA, Susin F, Moussa AM, Hig-Zounet B, Mad-Toingué J, Tidjani A, Vaira D, Moutschen M. Performance characteristics of Allele-Specific PCR (ASPCR) in detecting drug resistance mutations among non-B HIV-1 Variants. J Virol Methods 2024; 323:114856. [PMID: 38000668 DOI: 10.1016/j.jviromet.2023.114856] [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: 11/05/2023] [Accepted: 11/20/2023] [Indexed: 11/26/2023]
Abstract
Allele-Specific Polymerase Chain Reaction (ASPCR) is an affordable point-mutation assay whose validation could improve the detection of HIV-1 drug resistance mutations (DRMs) in resource-limited settings (RLS). We assessed the performance of ASPCR onforty-four non-B HIV-1 plasma samples from patients who were ARV treated in failure in N'Djamena-Chad. Viral RNA was reverse-transcribed and amplified using LightCycler® FastStart DNA MasterPLUS SYBR Green I. Detection of six major DRMs (K70R, K103N, Y181C, M184V, T215F, T215Y) was evaluated on Roche LightCycler®480 automated system (with dilutions 0.01-100%). ASPCR-results were compared to Sanger-sequencing (gold-standard). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02-0.28 vs. 0.12-0.37), K103N (0.08-0.42 vs. 0.12-0.37), Y181C (0.12-0.39 vs. 0.31-0.37), M184V (0.13-0.39 vs. 0.23-0.42), T215F (0.05-0.43 vs. 0.04-0.45) and T215Y (0.13-0.41 vs. 0.19-0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02-0.28 vs. 0.12-0.37), K103N (0.08-0.42 vs. 0.12-0.37), Y181C (0.12-0.39 vs. 0.31-0.37), M184V (0.13-0.39 vs. 0.23-0.42), T215F (0.05-0.43 vs. 0.04-0.45) and T215Y (0.13-0.41 vs. 0.19-0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). ASPCR appears more efficient for detecting DRMs on diverse HIV-1 non-B circulating in RLS like Chad.
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Affiliation(s)
- Chatté Adawaye
- National Institute of Sciences and Techniques of Abeche (INSTA), Abeche, Chad; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium.
| | - Joseph Fokam
- Virology Laboratory, Chantal BIYA International Reference Centre for research on HIV/AIDS prevention and management, Yaoundé, Cameroon; Department of Medical Laboratory Sciences, Faculty of Health Sciences, University of Buea, Cameroon; Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Yaounde, Cameroon; National HIV Drug Resistance Surveillance and Prevention Working Group (HIVDRWG), Ministry of Public Health, Yaounde, Cameroon.
| | - Erick Ntambwe Kamangu
- Department of Basic Sciences, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
| | - Derrick Tambe Ayuk Ngwese
- Virology Laboratory, Chantal BIYA International Reference Centre for research on HIV/AIDS prevention and management, Yaoundé, Cameroon; Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Yaounde, Cameroon; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
| | - Fabrice Susin
- Department of Basic Sciences, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
| | - Ali Mahamat Moussa
- AIDS Reference Laboratory of Liege, CHU de Liege, Liege, Belgium; Faculty of Human Health Sciences, University of N'Djamena, N'Djamena, Chad; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
| | - BertinTchombou Hig-Zounet
- AIDS Reference Laboratory of Liege, CHU de Liege, Liege, Belgium; Faculty of Human Health Sciences, University of N'Djamena, N'Djamena, Chad; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
| | - Joseph Mad-Toingué
- AIDS Reference Laboratory of Liege, CHU de Liege, Liege, Belgium; Faculty of Human Health Sciences, University of N'Djamena, N'Djamena, Chad; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
| | - Abdelsalam Tidjani
- AIDS Reference Laboratory of Liege, CHU de Liege, Liege, Belgium; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
| | - Dolores Vaira
- Department of Basic Sciences, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
| | - Michel Moutschen
- Department of Basic Sciences, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of Congo; National Reference General Hospital, N'Djamena, Chad; Infectious Diseases and Internal Medicine Service, University Hospital Center of Liège, Liège, Belgium
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Alshabeeb MA, Alwadaani D, Al Qahtani FH, Abohelaika S, Alzahrani M, Al Zayed A, Al Saeed HH, Al Ajmi H, Alsomaie B, Rashid M, Daly AK. Impact of Genetic Variations on Thromboembolic Risk in Saudis with Sickle Cell Disease. Genes (Basel) 2023; 14:1919. [PMID: 37895268 PMCID: PMC10606407 DOI: 10.3390/genes14101919] [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: 07/16/2023] [Revised: 10/04/2023] [Accepted: 10/07/2023] [Indexed: 10/29/2023] Open
Abstract
BACKGROUND Sickle cell disease (SCD) is a Mendelian disease characterized by multigenic phenotypes. Previous reports indicated a higher rate of thromboembolic events (TEEs) in SCD patients. A number of candidate polymorphisms in certain genes (e.g., FVL, PRT, and MTHFR) were previously reported as risk factors for TEEs in different clinical conditions. This study aimed to genotype these genes and other loci predicted to underlie TEEs in SCD patients. METHODOLOGY A multi-center genome-wide association study (GWAS) involving Saudi SCD adult patients with a history of TEEs (n = 65) and control patients without TEE history (n = 285) was performed. Genotyping used the 10× Affymetrix Axiom array, which includes 683,030 markers. Fisher's exact test was used to generate p-values of TEE associations with each single-nucleotide polymorphism (SNP). The haplotype analysis software tool version 1.05, designed by the University of Göttingen, Germany, was used to identify the common inherited haplotypes. RESULTS No association was identified between the targeted single-nucleotide polymorphism rs1801133 in MTHFR and TEEs in SCD (p = 0.79). The allele frequency of rs6025 in FVL and rs1799963 in PRT in our cohort was extremely low (<0.01); thus, both variants were excluded from the analysis as no meaningful comparison was possible. In contrast, the GWAS analysis showed novel genome-wide associations (p < 5 × 10-8) with seven signals; five of them were located on Chr 11 (rs35390334, rs331532, rs317777, rs147062602, and rs372091), one SNP on Chr 20 (rs139341092), and another on Chr 9 (rs76076035). The other 34 SNPs located on known genes were also detected at a signal threshold of p < 5 × 10-6. Seven of the identified variants are located in olfactory receptor family 51 genes (OR51B5, OR51V1, OR51A1P, and OR51E2), and five variants were related to family 52 genes (OR52A5, OR52K1, OR52K2, and OR52T1P). The previously reported association between rs5006884-A in OR51B5 and fetal hemoglobin (HbF) levels was confirmed in our study, which showed significantly lower levels of HbF (p = 0.002) and less allele frequency (p = 0.003) in the TEE cases than in the controls. The assessment of the haplotype inheritance pattern involved the top ten significant markers with no LD (rs353988334, rs317777, rs14788626882, rs49188823, rs139349992, rs76076035, rs73395847, rs1368823, rs8888834548, and rs1455957). A haplotype analysis revealed significant associations between two haplotypes (a risk, TT-AA-del-AA-ins-CT-TT-CC-CC-AA, and a reverse protective, CC-GG-ins-GG-del-TT-CC-TT-GG-GG) and TEEs in SCD (p = 0.024, OR = 6.16, CI = 1.34-28.24, and p = 0.019, OR = 0.33, CI = 0.13-0.85, respectively). CONCLUSIONS Seven markers showed novel genome-wide associations; two of them were exonic variants (rs317777 in OLFM5P and rs147062602 in OR51B5), and less significant associations (p < 5 × 10-6) were identified for 34 other variants in known genes with TEEs in SCD. Moreover, two 10-SNP common haplotypes were determined with contradictory effects. Further replication of these findings is needed.
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Affiliation(s)
- Mohammad A. Alshabeeb
- King Abdullah International Medical Research Center (KAIMRC), Riyadh 11426, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), Ministry of National Guard Health Affairs (MNGHA), Riyadh 11426, Saudi Arabia (M.A.)
| | - Deemah Alwadaani
- King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), Ministry of National Guard Health Affairs (MNGHA), Riyadh 11426, Saudi Arabia (M.A.)
- Medical Genomics Research Department, King Abdullah International Medical Research Center (KAIMRC), Riyadh 11481, Saudi Arabia
| | - Farjah H. Al Qahtani
- Hematology/Oncology Center, King Saud University Medical City (KSUMC), Riyadh 11411, Saudi Arabia;
| | - Salah Abohelaika
- Research Department, Qatif Central Hospital (QCH), Qatif 32654, Saudi Arabia;
- Pharmacy Department, Qatif Central Hospital (QCH), Qatif 32654, Saudi Arabia
| | - Mohsen Alzahrani
- King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), Ministry of National Guard Health Affairs (MNGHA), Riyadh 11426, Saudi Arabia (M.A.)
- King Fahad Hospital, Ministry of National Guard Health Affairs (MNGHA), Riyadh 11426, Saudi Arabia
| | - Abdullah Al Zayed
- Hematology Department, Qatif Central Hospital (QCH), Qatif 32654, Saudi Arabia; (A.A.Z.); (H.H.A.S.)
| | - Hussain H. Al Saeed
- Hematology Department, Qatif Central Hospital (QCH), Qatif 32654, Saudi Arabia; (A.A.Z.); (H.H.A.S.)
| | - Hala Al Ajmi
- King Abdullah International Medical Research Center (KAIMRC), Riyadh 11426, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), Ministry of National Guard Health Affairs (MNGHA), Riyadh 11426, Saudi Arabia (M.A.)
| | - Barrak Alsomaie
- King Abdullah International Medical Research Center (KAIMRC), Riyadh 11426, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), Ministry of National Guard Health Affairs (MNGHA), Riyadh 11426, Saudi Arabia (M.A.)
| | - Mamoon Rashid
- King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), Ministry of National Guard Health Affairs (MNGHA), Riyadh 11426, Saudi Arabia (M.A.)
- Department of AI and Bioinformatics, King Abdullah International Medical Research Center (KAIMRC), Riyadh 11481, Saudi Arabia
| | - Ann K. Daly
- Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK
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Li B, Liu J, Huang Q. A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations. Int J Mol Sci 2023; 24:13405. [PMID: 37686219 PMCID: PMC10488114 DOI: 10.3390/ijms241713405] [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: 08/08/2023] [Revised: 08/25/2023] [Accepted: 08/27/2023] [Indexed: 09/10/2023] Open
Abstract
Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe's tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection.
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Affiliation(s)
| | | | - Qilai Huang
- Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao 266237, China
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13
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Wu Y, Liu Y, Chang Y, Liu M. Integration of CRISPR/Cas13a and V-Shape PCR for Rapid, Sensitive, and Specific Genotyping of CYP2C19 Gene Polymorphisms. Anal Chem 2023. [PMID: 37326604 DOI: 10.1021/acs.analchem.3c01968] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/17/2023]
Abstract
Rapid detection of single nucleotide polymorphisms (SNPs) in the CYP2C19 gene is of great significance for clopidogrel-accurate medicine. CRISPR/Cas systems have been increasingly used in SNP detection due to their single-nucleotide mismatch specificity. PCR, as a powerful amplification tool, has been incorporated into the CRISPR/Cas system to improve the sensitivity. However, the complicated three-step temperature control of the conventional PCR impeded rapid detection. The "V" shape PCR can shorten about 2/3 of the amplification time compared with conventional PCR. Herein, we present a new system termed the "V" shape PCR-coupled CRISPR/Cas13a (denoted as VPC) system, achieving the rapid, sensitive, and specific genotyping of CYP2C19 gene polymorphisms. The wild- and mutant-type alleles in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes can be discriminated by using the rationally programmed crRNA. A limit of detection (LOD) of 102 copies/μL was obtained within 45 min. In addition, the clinical applicability was demonstrated by genotyping SNPs in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes from clinical blood samples and buccal swabs within 1 h. Finally, we conducted the HPV16 and HPV18 detections to validate the generality of the VPC strategy.
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Affiliation(s)
- Yunping Wu
- School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), Dalian University of Technology, Dalian 116024, China
- Dalian POCT Laboratory, Dalian 116024, China
| | - Yi Liu
- Department of Neurology, Dalian Municipal Central Hospital Affiliated Hospital of Dalian Medical University, Dalian, 116033, China
| | - Yangyang Chang
- School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), Dalian University of Technology, Dalian 116024, China
- Dalian POCT Laboratory, Dalian 116024, China
| | - Meng Liu
- School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), Dalian University of Technology, Dalian 116024, China
- Dalian POCT Laboratory, Dalian 116024, China
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14
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Liao X, Xia X, Yang H, Zhu Y, Deng R, Ding T. Bacterial drug-resistance and viability phenotyping upon disinfectant exposure revealed by single-nucleotide resolved-allele specific isothermal RNA amplification. JOURNAL OF HAZARDOUS MATERIALS 2023; 448:130800. [PMID: 36716555 PMCID: PMC9883656 DOI: 10.1016/j.jhazmat.2023.130800] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Revised: 12/22/2022] [Accepted: 01/14/2023] [Indexed: 06/18/2023]
Abstract
Disinfectant abuse poses a risk of bacterial evolution against stresses, especially during the coronavirus disease 2019 (COVID-19) pandemic. However, bacterial phenotypes, such as drug resistance and viability, are hard to access quickly. Here, we reported an allele specific isothermal RNA amplification (termed AlleRNA) assay, using an isothermal RNA amplification technique, i.e., nucleic acid sequence-based amplification (NASBA), integrated the amplification refractory mutation system (ARMS), involving the use of sequence-specific primers to allow the amplification of the targets with complete complementary sequences. AlleRNA assay enables rapid and simultaneous detection of the single nucleotide polymorphism (SNP) (a detection limit, a LOD of 0.5 % SNP) and the viability (a LOD of 80 CFU) of the quinolone resistant Salmonella enterica. With the use of AlleRNA assay, we found that the quinolone resistant S. enterica exhibited higher survival ability during exposure toquaternary ammonium salt, 75 % ethanol and peracetic acid, which might be attributed to the upregulation of stress response-associated genescompared with the susceptible counterparts. Additionally, the AlleRNA assay indicated the potential risk in a high-frequency occurrence of viable but nonculturable (VBNC) quinolone resistant S. enterica induced by disinfectants due to the depression of ATP biosynthesis. The excessive usage of disinfectants during the COVID-19 pandemic should be carefully evaluated due to the latent threat to ecological and human health.
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Affiliation(s)
- Xinyu Liao
- Department of Food Science and Nutrition, Zhejiang University, Hangzhou, Zhejiang 310058, China; School of Mechanical and Energy Engineering, NingboTech University, Ningbo, China; Future Food Laboratory, Innovation Center of Yangtze River Delta, Zhejiang University, 314100, Jiashan, China
| | - Xuhan Xia
- College of Biomass Science and Engineering, Healthy Food Evaluation Research Center, Sichuan University, Chengdu, Sichuan 610065, China
| | - Hao Yang
- College of Biomass Science and Engineering, Healthy Food Evaluation Research Center, Sichuan University, Chengdu, Sichuan 610065, China
| | - Yulin Zhu
- College of Biomass Science and Engineering, Healthy Food Evaluation Research Center, Sichuan University, Chengdu, Sichuan 610065, China
| | - Ruijie Deng
- College of Biomass Science and Engineering, Healthy Food Evaluation Research Center, Sichuan University, Chengdu, Sichuan 610065, China.
| | - Tian Ding
- Department of Food Science and Nutrition, Zhejiang University, Hangzhou, Zhejiang 310058, China; Future Food Laboratory, Innovation Center of Yangtze River Delta, Zhejiang University, 314100, Jiashan, China.
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15
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Limanskaya OY, Limanskii OP. Intramolecular Interactions in the Fluorophore–Quencher System in Linear and Hairpin Probes for Real-Time PCR. CYTOL GENET+ 2023. [DOI: 10.3103/s009545272302007x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/08/2023]
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16
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Saito Y, Tada F, Takashina T, Ikegami H. Allele-Specific Mutation Genotyping with Mismatches in Primer Design. Methods Mol Biol 2023; 2638:249-262. [PMID: 36781647 DOI: 10.1007/978-1-0716-3024-2_17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/15/2023]
Abstract
Genotyping technologies for single nucleotide polymorphisms (SNPs) and other mutation types have evolved to become essential tools in various fields. Although high-throughput genotyping technologies occupy a key position in handling large amounts of SNP data, simple, low-cost, and conventional genotyping technologies remain in demand. Allele-specific (AS) polymerase chain reaction (PCR) and its related improved methods can effectively identify target SNPs and allele types using AS primers that introduce instability through mismatched bases at and around the SNP site. In this chapter, we present what is known from the literature on primer design with mismatches for AS-PCR and describe three cases of mutation detection (SNPs and insertions/deletions) associated with functional genes of crop species, which could be useful to guide future AS-PCR experiments.
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Affiliation(s)
- Yutaro Saito
- Yamagata Integrated Agricultural Research Center, Horticultural Research Institute, Yamagata, Japan
| | - Fumito Tada
- Yamagata Integrated Agricultural Research Center, Horticultural Research Institute, Yamagata, Japan
| | - Tadashi Takashina
- Yamagata Integrated Agricultural Research Center, Horticultural Research Institute, Yamagata, Japan
| | - Hidetoshi Ikegami
- Fukuoka Agriculture and Forestry Research Center, Buzen Branch, Yukuhashi, Japan.
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17
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Hassan S, Bahar R, Johan MF, Mohamed Hashim EK, Abdullah WZ, Esa E, Abdul Hamid FS, Zulkafli Z. Next-Generation Sequencing (NGS) and Third-Generation Sequencing (TGS) for the Diagnosis of Thalassemia. Diagnostics (Basel) 2023; 13:diagnostics13030373. [PMID: 36766477 PMCID: PMC9914462 DOI: 10.3390/diagnostics13030373] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2022] [Revised: 01/11/2023] [Accepted: 01/16/2023] [Indexed: 01/20/2023] Open
Abstract
Thalassemia is one of the most heterogeneous diseases, with more than a thousand mutation types recorded worldwide. Molecular diagnosis of thalassemia by conventional PCR-based DNA analysis is time- and resource-consuming owing to the phenotype variability, disease complexity, and molecular diagnostic test limitations. Moreover, genetic counseling must be backed-up by an extensive diagnosis of the thalassemia-causing phenotype and the possible genetic modifiers. Data coming from advanced molecular techniques such as targeted sequencing by next-generation sequencing (NGS) and third-generation sequencing (TGS) are more appropriate and valuable for DNA analysis of thalassemia. While NGS is superior at variant calling to TGS thanks to its lower error rates, the longer reads nature of the TGS permits haplotype-phasing that is superior for variant discovery on the homologous genes and CNV calling. The emergence of many cutting-edge machine learning-based bioinformatics tools has improved the accuracy of variant and CNV calling. Constant improvement of these sequencing and bioinformatics will enable precise thalassemia detections, especially for the CNV and the homologous HBA and HBG genes. In conclusion, laboratory transiting from conventional DNA analysis to NGS or TGS and following the guidelines towards a single assay will contribute to a better diagnostics approach of thalassemia.
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Affiliation(s)
- Syahzuwan Hassan
- Department of Hematology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia
- Institute for Medical Research, Shah Alam 40170, Malaysia
| | - Rosnah Bahar
- Department of Hematology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia
| | - Muhammad Farid Johan
- Department of Hematology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia
| | | | - Wan Zaidah Abdullah
- Department of Hematology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia
| | - Ezalia Esa
- Institute for Medical Research, Shah Alam 40170, Malaysia
| | | | - Zefarina Zulkafli
- Department of Hematology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia
- Correspondence:
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McCloskey D, Boza J, Mason CE, Erickson D. MINI: A high-throughput point-of-care device for performing hundreds of nucleic acid tests per day. Biosens Bioelectron 2022; 216:114654. [PMID: 36084523 PMCID: PMC10960951 DOI: 10.1016/j.bios.2022.114654] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2022] [Revised: 08/11/2022] [Accepted: 08/20/2022] [Indexed: 11/24/2022]
Abstract
There are a variety of infectious diseases with a high incidence and mortality in limited resource settings that could benefit from rapid point of care molecular diagnosis. Global health efforts have sought to implement mass-screening programs to provide earlier detection and subsequent treatment in an effort to control transmission and improve health outcomes. However, many of the current diagnostic technologies under development are limited to fewer than 10 samples per run, which inherently restricts the screening throughput of these devices. We have developed a high throughput device called "MINI" that is capable of testing hundreds of samples per day at the point-of-care. MINI can utilize multiple energy sources - electricity, flame, or solar - to perform loop-mediated isothermal amplification (LAMP) in a portable and robust device which is ideal for use in limited resource settings. The unique opto-electronic design of MINI minimizes the energy and space requirements of the device and maximizes the optical isolation and signal clarity, enabling point-of-care analysis of 96 unique samples at once. We show comparable performance to a commercial instrument using two different LAMP assays for Kaposi's sarcoma-associated herpesvirus and a common housekeeping gene, GAPDH. With a single device capable of running hundreds of samples per day, increased access to modern molecular diagnostics could improve health outcomes for a variety of diseases common in limited resource settings.
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Affiliation(s)
- Duncan McCloskey
- Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Juan Boza
- Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Christopher E Mason
- Institute for Computational Biomedicine and Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA; The WorldQuant Initiative for Quantitative Prediction, New York, NY, USA
| | - David Erickson
- Division of Nutritional Science, Cornell University, Ithaca, NY, USA; Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY, USA.
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19
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Lee WL, Armas F, Guarneri F, Gu X, Formenti N, Wu F, Chandra F, Parisio G, Chen H, Xiao A, Romeo C, Scali F, Tonni M, Leifels M, Chua FJD, Kwok GW, Tay JY, Pasquali P, Thompson J, Alborali GL, Alm EJ. Rapid displacement of SARS-CoV-2 variant Delta by Omicron revealed by allele-specific PCR in wastewater. WATER RESEARCH 2022; 221:118809. [PMID: 35841797 PMCID: PMC9250349 DOI: 10.1016/j.watres.2022.118809] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Revised: 06/18/2022] [Accepted: 07/01/2022] [Indexed: 05/06/2023]
Abstract
On November 26, 2021, the B.1.1.529 COVID-19 variant was classified as the Omicron variant of concern (VOC). Reports of higher transmissibility and potential immune evasion triggered flight bans and heightened health control measures across the world to stem its distribution. Wastewater-based surveillance has demonstrated to be a useful complement for clinical community-based tracking of SARS-CoV-2 variants. Using design principles of our previous assays that detect SARS-CoV-2 variants (Alpha and Delta), we developed an allele-specific RT-qPCR assay which simultaneously targets the stretch of mutations from Q493R to Q498R for quantitative detection of the Omicron variant in wastewater. We report their validation against 10-month longitudinal samples from the influent of a wastewater treatment plant in Italy. SARS-CoV-2 RNA concentrations and variant frequencies in wastewater determined using these variant assays agree with clinical cases, revealing rapid displacement of the Delta variant by the Omicron variant within three weeks. These variant trends, when mapped against vaccination rates, support clinical studies that found the rapid emergence of SARS-CoV-2 Omicron variant being associated with an infection advantage over Delta in vaccinated persons. These data reinforce the versatility, utility and accuracy of these open-sourced methods using allele-specific RT-qPCR for tracking the dynamics of variant displacement in communities through wastewater for informed public health responses.
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Affiliation(s)
- Wei Lin Lee
- Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore; Campus for Research Excellence and Technological Enterprise (CREATE), Singapore
| | - Federica Armas
- Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore; Campus for Research Excellence and Technological Enterprise (CREATE), Singapore
| | - Flavia Guarneri
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" (IZSLER), Italy
| | - Xiaoqiong Gu
- Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore; Campus for Research Excellence and Technological Enterprise (CREATE), Singapore
| | - Nicoletta Formenti
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" (IZSLER), Italy
| | - Fuqing Wu
- Center for Infectious Disease, Department of Epidemiology, Human Genetics, and Environmental Sciences, University of Texas School of Public Health, Houston, TX, USA
| | - Franciscus Chandra
- Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore; Campus for Research Excellence and Technological Enterprise (CREATE), Singapore
| | - Giovanni Parisio
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" (IZSLER), Italy
| | - Hongjie Chen
- Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore; Campus for Research Excellence and Technological Enterprise (CREATE), Singapore
| | - Amy Xiao
- Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, USA; Department of Biological Engineering, Massachusetts Institute of Technology, USA
| | - Claudia Romeo
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" (IZSLER), Italy
| | - Federico Scali
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" (IZSLER), Italy
| | - Matteo Tonni
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" (IZSLER), Italy
| | - Mats Leifels
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore
| | - Feng Jun Desmond Chua
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore
| | - Germaine Wc Kwok
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore
| | - Joey Yr Tay
- Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore; Campus for Research Excellence and Technological Enterprise (CREATE), Singapore
| | - Paolo Pasquali
- Dipartimento di Sicurezza Alimentare, Nutrizione e Sanità Pubblica Veterinaria, Istituto Superiore di Sanità, Italy
| | - Janelle Thompson
- Campus for Research Excellence and Technological Enterprise (CREATE), Singapore; Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore; Asian School of the Environment, Nanyang Technological University, Singapore.
| | - Giovanni Loris Alborali
- Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" (IZSLER), Italy
| | - Eric J Alm
- Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore; Campus for Research Excellence and Technological Enterprise (CREATE), Singapore; Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, USA; Department of Biological Engineering, Massachusetts Institute of Technology, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
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20
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Xu X, Deng Y, Ding J, Zheng X, Li S, Liu L, Chui HK, Poon LLM, Zhang T. Real-time allelic assays of SARS-CoV-2 variants to enhance sewage surveillance. WATER RESEARCH 2022; 220:118686. [PMID: 35679788 PMCID: PMC9148393 DOI: 10.1016/j.watres.2022.118686] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Revised: 05/25/2022] [Accepted: 05/27/2022] [Indexed: 05/21/2023]
Abstract
To effectively control the ongoing outbreaks of fast-spreading SARS-CoV-2 variants, there is an urgent need to add rapid variant detection and discrimination methods to the existing sewage surveillance systems established worldwide. We designed eight assays based on allele-specific RT-qPCR for real-time allelic discrimination of eight SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, Omicron, Lambda, Mu, and Kappa) in sewage. In silico analysis of the designed assays for identifying SARS-CoV-2 variants using more than four million SARS-CoV-2 variant sequences yielded ∼100% specificity and >90% sensitivity. All assays could sensitively discriminate and quantify target variants at levels as low as 10 viral RNA copy/µL with minimal cross-reactivity to the corresponding nontarget genotypes, even for sewage samples containing mixtures of SARS-CoV-2 variants with differential abundances. Integration of this method into the routine sewage surveillance in Hong Kong successfully identified the Beta variant in a community sewage. Complete concordance was observed between the results of viral whole-genome sequencing and those of our novel assays in sewage samples that contained exclusively the Delta variant discharged by a clinically diagnosed COVID-19 patient living in a quarantine hotel. Our assays in this method also provided real-time discrimination of the newly emerging Omicron variant in sewage two days prior to clinical test results in another quarantine hotel in Hong Kong. These novel allelic discrimination assays offer a rapid, sensitive, and specific way for detecting multiple SARS-CoV-2 variants in sewage and can be directly integrated into the existing sewage surveillance systems.
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Affiliation(s)
- Xiaoqing Xu
- Environmental Microbiome Engineering and Biotechnology Lab, Center for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
| | - Yu Deng
- Environmental Microbiome Engineering and Biotechnology Lab, Center for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
| | - Jiahui Ding
- Environmental Microbiome Engineering and Biotechnology Lab, Center for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
| | - Xiawan Zheng
- Environmental Microbiome Engineering and Biotechnology Lab, Center for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
| | - Shuxian Li
- Environmental Microbiome Engineering and Biotechnology Lab, Center for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
| | - Lei Liu
- Environmental Microbiome Engineering and Biotechnology Lab, Center for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
| | - Ho-Kwong Chui
- Environmental Protection Department, The Government of Hong Kong SAR, Tamar, Hong Kong SAR, China
| | - Leo L M Poon
- School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Sassoon Road, Hong Kong SAR, China
| | - Tong Zhang
- Environmental Microbiome Engineering and Biotechnology Lab, Center for Environmental Engineering Research, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
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21
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Bergallo M, Montanari P, Loiacono E, Galliano I. A novel TaqMAMA assay for allelic discrimination of immunoproteasome subunit PSMB8 in pediatric patients. Minerva Pediatr (Torino) 2022; 74:301-307. [DOI: 10.23736/s2724-5276.16.04675-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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22
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Cortez DR, Lima FM, Reis-Cunha JL, Bartholomeu DC, Villacis RAR, Rogatto SR, Costa-Martins AG, Marchiano FS, do Carmo RA, da Silveira JF, Marini MM. Trypanosoma cruzi Genomic Variability: Array Comparative Genomic Hybridization Analysis of Clone and Parental Strain. Front Cell Infect Microbiol 2022; 12:760830. [PMID: 35402315 PMCID: PMC8992781 DOI: 10.3389/fcimb.2022.760830] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Accepted: 02/25/2022] [Indexed: 11/13/2022] Open
Abstract
Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits extensive inter- and intrastrain genetic diversity. As we have previously described, there are some genetic differences between the parental G strain and its clone D11, which was isolated by the limiting dilution method and infection of cultured mammalian cells. Electrophoretic karyotyping and Southern blot hybridization of chromosomal bands with specific markers revealed chromosome length polymorphisms of small size with additional chromosomal bands in clone D11 and the maintenance of large syntenic groups. Both G strain and clone D11 belong to the T. cruzi lineage TcI. Here, we designed intraspecific array-based comparative genomic hybridization (aCGH) to identify chromosomal regions harboring copy-number variations between clone D11 and the G strain. DNA losses were more extensive than DNA gains in clone D11. Most alterations were flanked by repeated sequences from multigene families that could be involved in the duplication and deletion events. Several rearrangements were detected by chromoblot hybridization and confirmed by aCGH. We have integrated the information of genomic sequence data obtained by aCGH to the electrophoretic karyotype, allowing the reconstruction of possible recombination events that could have generated the karyotype of clone D11. These rearrangements may be explained by unequal crossing over between sister or homologous chromatids mediated by flanking repeated sequences and unequal homologous recombination via break-induced replication. The genomic changes detected by aCGH suggest the presence of a dynamic genome that responds to environmental stress by varying the number of gene copies and generating segmental aneuploidy.
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Affiliation(s)
- Danielle Rodrigues Cortez
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
| | - Fabio Mitsuo Lima
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
- Centro Universitário São Camilo, Biomedicina, São Paulo, Brazil
| | - João Luís Reis-Cunha
- Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
| | | | | | - Silvia Regina Rogatto
- Department of Clinical Genetics, Institute of Regional Health Research, University of Southern Denmark, Vejle, Denmark
| | - André Guilherme Costa-Martins
- Department of Clinical and Toxicological Analyses, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil
| | - Fernanda Sycko Marchiano
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
| | - Rafaela Andrade do Carmo
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
| | - Jose Franco da Silveira
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
- *Correspondence: Marjorie Mendes Marini, ; Jose Franco da Silveira,
| | - Marjorie Mendes Marini
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil
- Centro Universitário São Camilo, Biomedicina, São Paulo, Brazil
- *Correspondence: Marjorie Mendes Marini, ; Jose Franco da Silveira,
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23
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Kalendar R, Shustov AV, Akhmetollayev I, Kairov U. Designing Allele-Specific Competitive-Extension PCR-Based Assays for High-Throughput Genotyping and Gene Characterization. Front Mol Biosci 2022; 9:773956. [PMID: 35300118 PMCID: PMC8921500 DOI: 10.3389/fmolb.2022.773956] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2021] [Accepted: 02/02/2022] [Indexed: 12/03/2022] Open
Abstract
Polymerase chain reaction (PCR) is a simple and rapid method that can detect nucleotide polymorphisms and sequence variation in basic research applications, agriculture, and medicine. Variants of PCR, collectively known as allele-specific PCR (AS-PCR), use a competitive reaction in the presence of allele-specific primers to preferentially amplify only certain alleles. This method, originally named by its developers as Kompetitive Allele Specific PCR (KASP), is an AS-PCR variant adapted for fluorescence-based detection of amplification results. We developed a bioinformatic tool for designing probe sequences for PCR-based genotyping assays. Probe sequences are designed in both directions, and both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be targeted. In addition, the tool allows discrimination of up to four-allelic variants at a single SNP site. To increase both the reaction specificity and the discriminative power of SNP genotyping, each allele-specific primer is designed such that the penultimate base before the primer's 3' end base is positioned at the SNP site. The tool allows design of custom FRET cassette reporter systems for fluorescence-based assays. FastPCR is a user-friendly and powerful Java-based software that is freely available (http://primerdigital.com/tools/). Using the FastPCR environment and the tool for designing AS-PCR provides unparalleled flexibility for developing genotyping assays and specific and sensitive diagnostic PCR-based tests, which translates into a greater likelihood of research success.
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Affiliation(s)
- Ruslan Kalendar
- Institute of Biotechnology HiLIFE, University of Helsinki, Helsinki, Finland
- PrimerDigital Ltd., Helsinki, Finland
- National Laboratory Astana, Nazarbayev University, Nur-Sultan, Kazakhstan
| | | | | | - Ulykbek Kairov
- National Laboratory Astana, Nazarbayev University, Nur-Sultan, Kazakhstan
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24
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Moyer AM, Gandhi MJ. Human Leukocyte Antigen (HLA) Testing in Pharmacogenomics. Methods Mol Biol 2022; 2547:21-45. [PMID: 36068459 DOI: 10.1007/978-1-0716-2573-6_2] [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: 06/15/2023]
Abstract
The genetic region on the short arm of chromosome 6 where the human leukocyte antigen (HLA) genes are located is the major histocompatibility complex. The genes in this region are highly polymorphic, and some loci have a high degree of homology with other genes and pseudogenes. Histocompatibility testing has traditionally been performed in the setting of transplantation and involves determining which specific alleles are present. Several HLA alleles have been associated with disease risk or increased risk of adverse drug reaction (ADR) when treated with certain medications. Testing for these applications differs from traditional histocompatibility in that the desired result is simply presence or absence of the allele of interest, rather than determining which allele is present. At present, the majority of HLA typing is done by molecular methods using commercially available kits. A subset of pharmacogenomics laboratories has developed their own methods, and in some cases, query single nucleotide variants associated with certain HLA alleles rather than directly testing for the allele. In this chapter, a brief introduction to the HLA system is provided, followed by an overview of a variety of testing technologies including those specifically used in pharmacogenomics, and the chapter concludes with details regarding specific HLA alleles associated with ADR.
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Affiliation(s)
- Ann M Moyer
- Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.
| | - Manish J Gandhi
- Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
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25
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Carolus H, Jacobs S, Lobo Romero C, Deparis Q, Cuomo CA, Meis JF, Van Dijck P. Diagnostic Allele-Specific PCR for the Identification of Candida auris Clades. J Fungi (Basel) 2021; 7:754. [PMID: 34575792 PMCID: PMC8471779 DOI: 10.3390/jof7090754] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2021] [Revised: 09/08/2021] [Accepted: 09/10/2021] [Indexed: 12/19/2022] Open
Abstract
Candida auris is an opportunistic pathogenic yeast that emerged worldwide during the past decade. This fungal pathogen poses a significant public health threat due to common multidrug resistance (MDR), alarming hospital outbreaks, and frequent misidentification. Genomic analyses have identified five distinct clades that are linked to five geographic areas of origin and characterized by differences in several phenotypic traits such as virulence and drug resistance. Typing of C. auris strains and the identification of clades can be a powerful tool in molecular epidemiology and might be of clinical importance by estimating outbreak and MDR potential. As C. auris has caused global outbreaks, including in low-income countries, typing C. auris strains quickly and inexpensively is highly valuable. We report five allele-specific polymerase chain reaction (AS-PCR) assays for the identification of C. auris and each of the five described clades of C. auris based on conserved mutations in the internal transcribed spacer (ITS) rDNA region and a clade-specific gene cluster. This PCR method provides a fast, cheap, sequencing-free diagnostic tool for the identification of C. auris, C. auris clades, and potentially, the discovery of new clades.
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Affiliation(s)
- Hans Carolus
- Laboratory of Molecular Cell Biology, Department of Biology, Institute of Botany and Microbiology, KU Leuven, 3001 Leuven, Belgium; (H.C.); (S.J.); (C.L.R.)
- VIB-KU Leuven Center for Microbiology, 3001 Leuven, Belgium;
| | - Stef Jacobs
- Laboratory of Molecular Cell Biology, Department of Biology, Institute of Botany and Microbiology, KU Leuven, 3001 Leuven, Belgium; (H.C.); (S.J.); (C.L.R.)
| | - Celia Lobo Romero
- Laboratory of Molecular Cell Biology, Department of Biology, Institute of Botany and Microbiology, KU Leuven, 3001 Leuven, Belgium; (H.C.); (S.J.); (C.L.R.)
- VIB-KU Leuven Center for Microbiology, 3001 Leuven, Belgium;
| | - Quinten Deparis
- VIB-KU Leuven Center for Microbiology, 3001 Leuven, Belgium;
- Laboratory for Genetics and Genomics, Centre for Microbial and Plant Genetics, KU Leuven, 3001 Leuven, Belgium
| | | | - Jacques F. Meis
- Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, 6532 Nijmegen, The Netherlands;
- Centre of Expertise in Mycology Radboudumc/CWZ, 6532 Nijmegen, The Netherlands
| | - Patrick Van Dijck
- Laboratory of Molecular Cell Biology, Department of Biology, Institute of Botany and Microbiology, KU Leuven, 3001 Leuven, Belgium; (H.C.); (S.J.); (C.L.R.)
- VIB-KU Leuven Center for Microbiology, 3001 Leuven, Belgium;
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Bustin S, Kirvell S, Huggett JF, Nolan T. RT-qPCR Diagnostics: The "Drosten" SARS-CoV-2 Assay Paradigm. Int J Mol Sci 2021; 22:ijms22168702. [PMID: 34445406 PMCID: PMC8395416 DOI: 10.3390/ijms22168702] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Revised: 07/31/2021] [Accepted: 08/11/2021] [Indexed: 12/23/2022] Open
Abstract
The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level, and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail, and our findings reveal some limitations but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Nevertheless, whilst our data show that some errors can be tolerated, it is always prudent to confirm that the primer and probe sequences complement their intended target, since, when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case, it is unlikely that a mismatch will result in poor specificity or a significant number of false-positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.
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Affiliation(s)
- Stephen Bustin
- Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University Chelmsford, Chelmsford CM1 1SQ, UK; (S.K.); (T.N.)
- Correspondence:
| | - Sara Kirvell
- Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University Chelmsford, Chelmsford CM1 1SQ, UK; (S.K.); (T.N.)
| | - Jim F. Huggett
- National Measurement Laboratory, LGC, Queens Rd, Teddington, London TW11 0LY, UK;
| | - Tania Nolan
- Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University Chelmsford, Chelmsford CM1 1SQ, UK; (S.K.); (T.N.)
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Meng Q, Wang X, Wang Y, Dang L, Liu X, Ma X, Chi T, Wang X, Zhao Q, Yang G, Liu M, Huang X, Ma P. Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA. Biotechnol J 2021; 16:e2100040. [PMID: 33595922 DOI: 10.1002/biot.202100040] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2021] [Revised: 02/10/2021] [Accepted: 02/11/2021] [Indexed: 12/19/2022]
Abstract
Detection of pathogens with single-nucleotide variations is indispensable for the disease tracing, but remains technically challenging. The D614G mutation in the SARS-CoV-2 spike protein is known to markedly enhance viral infectivity but is difficult to detect. Here, we report an effective approach called "synthetic mismatch integrated crRNA guided Cas12a detection" (symRNA-Cas12a) to detect the D614 and G614 variants effectively. Using this method, we systemically screened a pool of crRNAs that contain all the possible nucleotide substitutions covering the -2 to +2 positions around the mutation and identify one crRNA that can efficiently increase the detection specificity by 13-fold over the ancestral crRNA. With this selected crRNA, the symRNA-Cas12a assay can detect as low as 10 copies of synthetic mutant RNA and the results are confirmed to be accurate by Sanger sequencing. Overall, we have developed the symRNA-Cas12a method to specifically, sensitively and rapidly detect the SARS-CoV-2 D614G mutation.
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Affiliation(s)
- Qingzhou Meng
- Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China
| | - Xinjie Wang
- Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
- Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China
- Guangdong Key Laboratory of Mental Health and Cognitive Science, Institute for Brain Research and Rehabilitation, Centre for Studies of Psychological Application, South China Normal University, Guangzhou, China
| | - Yanqun Wang
- Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Lu Dang
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China
| | - Xinyi Liu
- Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China
| | - Xiaodong Ma
- Guangdong Key Laboratory of Mental Health and Cognitive Science, Institute for Brain Research and Rehabilitation, Centre for Studies of Psychological Application, South China Normal University, Guangzhou, China
| | - Tian Chi
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Xian Wang
- Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Qin Zhao
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, China
| | - Guang Yang
- Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China
| | - Ming Liu
- Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Xingxu Huang
- Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Peixiang Ma
- Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
- Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China
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28
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Techniques for the Detection of Sickle Cell Disease: A Review. MICROMACHINES 2021; 12:mi12050519. [PMID: 34063111 PMCID: PMC8148117 DOI: 10.3390/mi12050519] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Revised: 04/17/2021] [Accepted: 04/25/2021] [Indexed: 12/14/2022]
Abstract
Sickle cell disease (SCD) is a widespread disease caused by a mutation in the beta-globin gene that leads to the production of abnormal hemoglobin called hemoglobin S. The inheritance of the mutation could be homozygous or heterozygous combined with another hemoglobin mutation. SCD can be characterized by the presence of dense, sickled cells that causes hemolysis of blood cells, anemia, painful episodes, organ damage, and in some cases death. Early detection of SCD can help to reduce the mortality and manage the disease effectively. Therefore, different techniques have been developed to detect the sickle cell disease and the carrier states with high sensitivity and specificity. These techniques can be screening tests such as complete blood count, peripheral blood smears, and sickling test; confirmatory tests such as hemoglobin separation techniques; and genetic tests, which are more expensive and need to be done in centralized labs by highly skilled personnel. However, advanced portable point of care techniques have been developed to provide a low-cost, simple, and user-friendly device for detecting SCD, for instance coupling solubility tests with portable devices, using smartphone microscopic classifications, image processing techniques, rapid immunoassays, and sensor-based platforms. This review provides an overview of the current and emerging techniques for sickle cell disease detection and highlights the different potential methods that could be applied to help the early diagnosis of SCD.
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Graham MS, Sudre CH, May A, Antonelli M, Murray B, Varsavsky T, Kläser K, Canas LS, Molteni E, Modat M, Drew DA, Nguyen LH, Polidori L, Selvachandran S, Hu C, Capdevila J, Hammers A, Chan AT, Wolf J, Spector TD, Steves CJ, Ourselin S. Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study. Lancet Public Health 2021; 6:e335-e345. [PMID: 33857453 DOI: 10.1101/2021.03.28.21254404] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2021] [Revised: 03/05/2021] [Accepted: 03/10/2021] [Indexed: 05/27/2023]
Abstract
BACKGROUND The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. METHODS We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. FINDINGS From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6-0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56-0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38-0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02-1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. INTERPRETATION The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. FUNDING Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society.
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Affiliation(s)
- Mark S Graham
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK.
| | - Carole H Sudre
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK; MRC Unit for Lifelong Health and Ageing, Department of Population Science and Experimental Medicine, University College London, London, UK; Centre for Medical Image Computing, Department of Computer Science, University College London, London, UK
| | | | - Michela Antonelli
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
| | - Benjamin Murray
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
| | - Thomas Varsavsky
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
| | - Kerstin Kläser
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
| | - Liane S Canas
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
| | - Erika Molteni
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
| | - Marc Modat
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
| | - David A Drew
- Clinical and Translational Epidemiology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - Long H Nguyen
- Clinical and Translational Epidemiology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | | | | | | | | | - Alexander Hammers
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
| | - Andrew T Chan
- Clinical and Translational Epidemiology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | | | - Tim D Spector
- Department of Twin Research and Genetic Epidemiology, King's College London, London, UK
| | - Claire J Steves
- Department of Twin Research and Genetic Epidemiology, King's College London, London, UK
| | - Sebastien Ourselin
- School of Biomedical Engineering and Imaging Sciences, King's College London, London, UK
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Michiyuki S, Tomita N, Mori Y, Kanda H, Tashiro K, Notomi T. Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification. Biosci Biotechnol Biochem 2021; 85:359-368. [PMID: 33604636 DOI: 10.1093/bbb/zbaa012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Accepted: 09/15/2020] [Indexed: 12/22/2022]
Abstract
Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.
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Affiliation(s)
- Satoru Michiyuki
- Biochemical Research Laboratory, Eiken Chemical Co., Ltd., Otawara, Tochigi, Japan
| | - Norihiro Tomita
- Biochemical Research Laboratory, Eiken Chemical Co., Ltd., Otawara, Tochigi, Japan
| | - Yasuyoshi Mori
- Biochemical Research Laboratory, Eiken Chemical Co., Ltd., Otawara, Tochigi, Japan
| | - Hidetoshi Kanda
- Biochemical Research Laboratory, Eiken Chemical Co., Ltd., Otawara, Tochigi, Japan
| | - Kosuke Tashiro
- Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Nishi-Ku, Fukuoka, Japan
| | - Tsugunori Notomi
- Biochemical Research Laboratory, Eiken Chemical Co., Ltd., Otawara, Tochigi, Japan
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31
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Wannier TM, Ciaccia PN, Ellington AD, Filsinger GT, Isaacs FJ, Javanmardi K, Jones MA, Kunjapur AM, Nyerges A, Pal C, Schubert MG, Church GM. Recombineering and MAGE. NATURE REVIEWS. METHODS PRIMERS 2021; 1:7. [PMID: 35540496 PMCID: PMC9083505 DOI: 10.1038/s43586-020-00006-x] [Citation(s) in RCA: 51] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 11/19/2020] [Indexed: 12/17/2022]
Abstract
Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies. First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells. Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms. In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools. We then look forward to the future of genetic engineering.
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Affiliation(s)
- Timothy M. Wannier
- Department of Genetics, Harvard Medical School, Boston, MA, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
| | - Peter N. Ciaccia
- Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT, USA
- Systems Biology Institute, Yale University, West Haven, CT, USA
| | - Andrew D. Ellington
- Department of Molecular Biosciences, College of Natural Sciences, University of Texas at Austin, Austin, TX, USA
| | - Gabriel T. Filsinger
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
- Department of Systems Biology, Harvard University, Cambridge, MA, USA
| | - Farren J. Isaacs
- Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT, USA
- Systems Biology Institute, Yale University, West Haven, CT, USA
- Department of Biomedical Engineering, Yale University, New Haven, CT, USA
| | - Kamyab Javanmardi
- Department of Molecular Biosciences, College of Natural Sciences, University of Texas at Austin, Austin, TX, USA
| | - Michaela A. Jones
- Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA
| | - Aditya M. Kunjapur
- Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA
| | - Akos Nyerges
- Department of Genetics, Harvard Medical School, Boston, MA, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
| | - Csaba Pal
- Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary
| | - Max G. Schubert
- Department of Genetics, Harvard Medical School, Boston, MA, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
| | - George M. Church
- Department of Genetics, Harvard Medical School, Boston, MA, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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32
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Donovan CV, McElroy P, Adair L, Pence BW, Oloo AJ, Lal A, Bloland P, Nahlen B, Juliano JJ, Meshnick S. Association of Malnutrition with Subsequent Malaria Parasitemia among Children Younger than Three years in Kenya: A Secondary Data Analysis of the Asembo Bay Cohort Study. Am J Trop Med Hyg 2021; 104:243-254. [PMID: 33200723 PMCID: PMC7790106 DOI: 10.4269/ajtmh.20-0002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2020] [Accepted: 08/22/2020] [Indexed: 11/07/2022] Open
Abstract
Malaria and malnutrition remain primary causes of morbidity and mortality among children younger than 5 years in Africa. Studies investigating the association between malnutrition and subsequent malaria outcomes are inconsistent. We studied the effects of malnutrition on incidence and prevalence of malaria parasitemia in data from a cohort studied in the 1990s. Data came from the Asembo Bay cohort study, which collected malaria and health information on children from 1992 to 1996 in western Kenya. Infants were enrolled at birth and followed up until loss to follow-up, death, end of study, or 5 years old. Anthropometric measures and blood specimens were obtained monthly. Nutritional exposures included categorized Z-scores for height-for-age, weight-for-age, and weight-for-height. Febrile parasitemia and afebrile parasitemia were assessed with thick and thin blood films. Multiply imputed and weighted multinomial generalized estimating equation models estimated odds ratios (OR) for the association between exposures and outcomes. The sample included 1,182 children aged 0-30 months who contributed 18,028 follow-up visits. There was no significant association between malnutrition and either incident febrile parasitemia or prevalent febrile parasitemia. Prevalence ORs for afebrile parasitemia increased from 1.07 (95% CI: 0.89, 1.29) to 1.35 (1.03, 1.76) as stunting severity increased from mild to severe, and from 1.16 (1.02, 1.33) to 1.35 (1.09, 1.66) as underweight increased from mild to moderate. Stunting and underweight did not show a significant association with subsequent febrile parasitemia infections, but they did show a modest association with subsequent afebrile parasitemia. Consideration should be given to testing malnourished children for malaria, even if they present without fever.
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Affiliation(s)
- Catherine V. Donovan
- Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina
| | - Peter McElroy
- U.S. President’s Malaria Initiative, Malaria Branch, U.S. Centers for Disease Control and Prevention, Atlanta, Georgia
| | - Linda Adair
- Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina
| | - Brian W. Pence
- Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina
| | - Aggrey James Oloo
- Masinde Muliro University of Science and Technology, Kakamega, Kenya
| | - Altaf Lal
- Malaria Elimination Demonstration Project, Foundation for Disease Elimination and Control of India, Mandla, India
| | - Peter Bloland
- Global Immunization Division, U.S Centers for Disease Control and Prevention, Atlanta, Georgia
| | - Bernard Nahlen
- Eck Institute for Global Health, University of Notre Dame, Notre Dame, Indiana
| | - Jonathan J. Juliano
- Division of Infectious Diseases, School of Medicine, University of North Carolina, Chapel Hill, North Carolina
| | - Steven Meshnick
- Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina
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33
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Zhang K, Deng R, Gao H, Teng X, Li J. Lighting up single-nucleotide variation in situ in single cells and tissues. Chem Soc Rev 2020; 49:1932-1954. [PMID: 32108196 DOI: 10.1039/c9cs00438f] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The ability to 'see' genetic information directly in single cells can provide invaluable insights into complex biological systems. In this review, we discuss recent advances of in situ imaging technologies for visualizing the subtlest sequence alteration, single-nucleotide variation (SNV), at single-cell level. The mechanism of recently developed methods for SNV discrimination are summarized in detail. With recent developments, single-cell SNV imaging methods have opened a new door for studying the heterogenous and stochastic genetic information in individual cells. Furthermore, SNV imaging can be used on morphologically preserved tissue, which can provide information on histological context for gene expression profiling in basic research and genetic diagnosis. Moreover, the ability to visualize SNVs in situ can be further developed into in situ sequencing technology. We expect this review to inspire more research work into in situ SNV imaging technologies for investigating cellular phenotypes and gene regulation at single-nucleotide resolution, and developing new clinical and biomedical applications.
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Affiliation(s)
- Kaixiang Zhang
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China. and School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
| | - Ruijie Deng
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.
| | - Hua Gao
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China. and Department of Pathogeny Biology, Medical College, Zhengzhou University, Zhengzhou 450001, China
| | - Xucong Teng
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.
| | - Jinghong Li
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.
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34
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Touroutine D, Tanis JE. A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans. Genetics 2020; 216:343-352. [PMID: 32817008 PMCID: PMC7536863 DOI: 10.1534/genetics.120.303553] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2020] [Accepted: 08/09/2020] [Indexed: 12/30/2022] Open
Abstract
With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end-point PCR in Caenorhabditis elegans Each SS primer consists of a 5' "anchor" that hybridizes to the template, followed by a noncomplementary "bridge," and a "foot" corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3' nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude C. elegans lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.
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Affiliation(s)
- Denis Touroutine
- Department of Biological Sciences, University of Delaware, Newark, Delaware 19716
| | - Jessica E Tanis
- Department of Biological Sciences, University of Delaware, Newark, Delaware 19716
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35
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Gassner C, Denomme GA, Portmann C, Bensing KM, Mattle-Greminger MP, Meyer S, Trost N, Song YL, Engström C, Jungbauer C, Just B, Storry JR, Forster M, Franke A, Frey BM. Two Prevalent ∼100-kb GYPB Deletions Causative of the GPB-Deficient Blood Group MNS Phenotype S-s-U- in Black Africans. Transfus Med Hemother 2020; 47:326-336. [PMID: 32884505 PMCID: PMC7443675 DOI: 10.1159/000504946] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2019] [Accepted: 11/20/2019] [Indexed: 01/11/2023] Open
Abstract
The U antigen (MNS5) is one of 49 antigens belonging to the MNS blood group system (ISBT002) carried on glycophorins A (GPA) and B (GPB). U is present on the red blood cells in almost all Europeans and Asians but absent in approximately 1.0% of Black Africans. U negativity coincides with negativity for S (MNS3) and s (MNS4) on GPB, thus be called S-s-U-, and is thought to arise from homozygous deletion of GYPB. Little is known about the molecular background of these deletions. Bioinformatic analysis of the 1000 Genomes Project data revealed several candidate regions with apparent deletions in GYPB. Highly specific Gap-PCRs, only resulting in positive amplification from DNAs with deletions present, allowed for the exact genetic localization of 3 different breakpoints; 110.24- and 103.26-kb deletions were proven to be the most frequent in Black Americans and Africans. Among 157 CEPH DNAs, deletions in 6 out of 8 African ethnicities were present. Allele frequencies of the deletions within African ethnicities varied greatly and reached a cumulative 23.3% among the Mbuti Pygmy people from the Congo. Similar observations were made for U+var alleles, known to cause strongly reduced GPB expression. The 110- and 103-kb deletional GYPB haplotypes were found to represent the most prevalent hereditary factors causative of the MNS blood group phenotype S-s-U-. Respective GYPB deletions are now accessible by molecular detection of homo- and hemizygous transmission.
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Affiliation(s)
- Christoph Gassner
- Independent at www.c-gassner.bio, Zurich, Switzerland
- Molecular Diagnostics and Research and Development, Blood Transfusion Service Zurich, Swiss Red Cross (SRC), Schlieren, Switzerland
| | | | - Claudia Portmann
- Molecular Diagnostics and Research and Development, Blood Transfusion Service Zurich, Swiss Red Cross (SRC), Schlieren, Switzerland
| | | | - Maja P. Mattle-Greminger
- Molecular Diagnostics and Research and Development, Blood Transfusion Service Zurich, Swiss Red Cross (SRC), Schlieren, Switzerland
| | - Stefan Meyer
- Molecular Diagnostics and Research and Development, Blood Transfusion Service Zurich, Swiss Red Cross (SRC), Schlieren, Switzerland
| | - Nadine Trost
- Molecular Diagnostics and Research and Development, Blood Transfusion Service Zurich, Swiss Red Cross (SRC), Schlieren, Switzerland
| | - Young-Lan Song
- Molecular Diagnostics and Research and Development, Blood Transfusion Service Zurich, Swiss Red Cross (SRC), Schlieren, Switzerland
| | - Charlotte Engström
- Molecular Diagnostics and Research and Development, Blood Transfusion Service Zurich, Swiss Red Cross (SRC), Schlieren, Switzerland
| | - Christof Jungbauer
- Blood Service for Vienna, Lower Austria, and Burgenland, Austrian Red Cross, Vienna, Austria
| | - Burkhard Just
- German Red Cross Blood Donation Service West, Hagen, Germany
| | - Jill R. Storry
- Division of Laboratory Medicine, Department of Hematology and Transfusion Medicine, Lund University, Lund, Sweden
| | - Michael Forster
- Institute of Clinical Molecular Biology, Christian Albrechts University of Kiel, Kiel, Germany
| | - Andre Franke
- Institute of Clinical Molecular Biology, Christian Albrechts University of Kiel, Kiel, Germany
| | - Beat M. Frey
- Head Office, Blood Transfusion Service Zürich, Swiss Red Cross (SRC), Schlieren, Switzerland
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Daprà V, Galliano I, Alliaudi C, Zaniol E, Graziano E, Calvi C, Montanari P, Bergallo M. Evaluation of the polymorphism NUDT15c.415C>T real-time PCR on the CFX96 real-time PCR system and 7500 real-time PCR system. MINERVA BIOTECNOL 2020. [DOI: 10.23736/s1120-4826.20.02620-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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37
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Tran TV, Dang KX, Pham QH, Nguyen UD, Trinh NTT, Hoang LV, Ho SA, Nguyen BV, Nguyen DT, Trinh DT, Tran DN, Orpana A, Stenman UH, Stenman J, Ho TH. Evaluation of the expression levels of BRAF V600E mRNA in primary tumors of thyroid cancer using an ultrasensitive mutation assay. BMC Cancer 2020; 20:368. [PMID: 32357861 PMCID: PMC7195771 DOI: 10.1186/s12885-020-06862-w] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2020] [Accepted: 04/14/2020] [Indexed: 12/05/2022] Open
Abstract
Background The BRAFV600E gene encodes for the mutant BRAFV600E protein, which triggers downstream oncogenic signaling in thyroid cancer. Since most currently available methods have focused on detecting BRAFV600E mutations in tumor DNA, there is limited information about the level of BRAFV600E mRNA in primary tumors of thyroid cancer, and the diagnostic relevance of these RNA mutations is not known. Methods Sixty-two patients with thyroid cancer and non-malignant thyroid disease were included in the study. Armed with an ultrasensitive technique for mRNA-based mutation analysis based on a two step RT-qPCR method, we analysed the expression levels of the mutated BRAFV600E mRNA in formalin-fixed paraffin-embedded samples of thyroid tissues. Sanger sequencing for detection of BRAFV600E DNA was performed in parallel for comparison and normalization of BRAFV600E mRNA expression levels. Results The mRNA-based mutation detection assay enables detection of the BRAFV600E mRNA transcripts in a 10,000-fold excess of wildtype BRAF counterparts. While BRAFV600E mutations could be detected by Sanger sequencing in 13 out of 32 malignant thyroid cancer FFPE tissue samples, the mRNA-based assay detected mutations in additionally 5 cases, improving the detection rate from 40.6 to 56.3%. Furthermore, we observed a surprisingly large, 3-log variability, in the expression level of the BRAFV600E mRNA in FFPE samples of thyroid cancer tissue. Conclusions The expression levels of BRAFV600E mRNA was characterized in the primary tumors of thyroid cancer using an ultrasensitive mRNA-based mutation assay. Our data inspires further studies on the prognostic and diagnostic relevance of the BRAFV600E mRNA levels as a molecular biomarker for the diagnosis and monitoring of various genetic and malignant diseases.
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Affiliation(s)
- Tien Viet Tran
- 103 Military Hospital, Vietnam Military Medical University, Hanoi, Vietnam
| | - Kien Xuan Dang
- Minerva Foundation Institute for Medical Research, Helsinki, Finland
| | - Quynh Huong Pham
- Department of Genomics and Cytogenetics, Institute of Biomedicine and Pharmacy (IBP), Vietnam Military Medical University, 222 Phung Hung street, Ha Dong district, Hanoi, Vietnam
| | - Ung Dinh Nguyen
- Department of Genomics and Cytogenetics, Institute of Biomedicine and Pharmacy (IBP), Vietnam Military Medical University, 222 Phung Hung street, Ha Dong district, Hanoi, Vietnam
| | - Nhung Thi Trang Trinh
- Department of Genomics and Cytogenetics, Institute of Biomedicine and Pharmacy (IBP), Vietnam Military Medical University, 222 Phung Hung street, Ha Dong district, Hanoi, Vietnam
| | - Luong Van Hoang
- Institute of Biomedicine and Pharmacy (IBP), Vietnam Military Medical University, Hanoi, Vietnam
| | - Son Anh Ho
- Institute of Biomedicine and Pharmacy (IBP), Vietnam Military Medical University, Hanoi, Vietnam
| | - Ba Van Nguyen
- Oncology Centre, 103 Military Hospital, Vietnam Military Medical University, Hanoi, Vietnam
| | - Duc Trong Nguyen
- School of Medicine and Pharmacy, Vietnam National University, Hanoi, Vietnam
| | - Dung Tuan Trinh
- Pathology Department, 108 Military Central Hospital, Hanoi, Vietnam
| | - Dung Ngoc Tran
- Department of Pathology, 103 Military Hospital, Vietnam Military Medical University, Hanoi, Vietnam
| | - Arto Orpana
- Laboratory of Genetics, HUSLAB, Helsinki University Central Hospital, Helsinki, Finland
| | - Ulf-Håkan Stenman
- Department of Clinical Chemistry, Medicum, Helsinki University Hospital, University of Helsinki, Helsinki, Finland
| | - Jakob Stenman
- Minerva Foundation Institute for Medical Research, Helsinki, Finland.,Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden
| | - Tho Huu Ho
- Minerva Foundation Institute for Medical Research, Helsinki, Finland. .,Department of Genomics and Cytogenetics, Institute of Biomedicine and Pharmacy (IBP), Vietnam Military Medical University, 222 Phung Hung street, Ha Dong district, Hanoi, Vietnam. .,Department of Medical Microbiology, 103 Military Hospital, Vietnam Medical University, Hanoi, Vietnam.
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38
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Lin B, Sun J, Fraser IDC. Single-tube genotyping for small insertion/deletion mutations: simultaneous identification of wild type, mutant and heterozygous alleles. Biol Methods Protoc 2020; 5:bpaa007. [PMID: 33782652 DOI: 10.1093/biomethods/bpaa007] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 03/17/2020] [Accepted: 03/27/2020] [Indexed: 01/01/2023] Open
Abstract
Current methods of genotyping small insertion/deletion (indel) mutations are costly, laborious, and can be unreliable. To address this, we have developed a method for small indel genotyping in a single polymerase chain reaction, with wild-type, heterozygous and mutant alleles distinguishable by band pattern in routine agarose gel electrophoresis. We demonstrate this method with multiple genes to distinguish 10 bp, 4 bp and even 1 bp deletions from the wild type. Through systematic testing of numerous primer designs, we also propose guidelines for genotyping small indel mutations. Our method provides a convenient approach to genotyping small indels derived from clustered regularly interspaced short palindromic repeats-mediated gene editing, N-ethyl-N-nitrosourea induced mutagenesis or diagnosis of naturally occurring polymorphisms/mutations.
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Affiliation(s)
- Bin Lin
- Signaling Systems Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Jing Sun
- Signaling Systems Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Iain D C Fraser
- Signaling Systems Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Graphene Oxide Nanoribbons in Chitosan for Simultaneous Electrochemical Detection of Guanine, Adenine, Thymine and Cytosine. BIOSENSORS-BASEL 2020; 10:bios10040030. [PMID: 32230779 PMCID: PMC7236021 DOI: 10.3390/bios10040030] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/20/2020] [Revised: 03/23/2020] [Accepted: 03/24/2020] [Indexed: 12/19/2022]
Abstract
Herein, graphene oxide nanoribbons (GONRs) were obtained from the oxidative unzipping of multi-walled carbon nanotubes. Covalent coupling reaction of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) with amine functional groups (-NH2) of the chitosan natural polymer (CH) was used for entrapping GONRs on the activated glassy carbon electrode (GCE/GONRs-CH). The nanocomposite was characterized by high-resolution transmission electron microscopy (HRTEM), and field-emission scanning electron microscopy (FESEM). In addition, the modification steps were monitored using FTIR. The nanocomposite-modified electrode was used for the simultaneous electrochemical determination of four DNA bases; guanine (G), adenine (A), thymine (T) and cytosine (C). The nanocomposite-modified GCE displayed a strong, stable and continuous four oxidation peaks during electrochemistry detection at potentials 0.63, 0.89, 1.13 and 1.27 V for G, A, T and C, respectively. The calibration curves were linear up to 256, 172, 855 and 342 μM with detection limits of 0.002, 0.023, 1.330 and 0.641 μM for G, A, T and C, respectively. The analytical performance of the GCE/GONRs-CH has been used for the determination of G, A, T and C in real samples and obtained a recovery percentage from 91.1%-104.7%. Our preliminary results demonstrated that GCE/GONRs-CH provided a promising platform to detect all four DNA bases for future studies on DNA damage and mutations.
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Suo W, Shi X, Xu S, Li X, Lin Y. Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics. PLoS One 2020; 15:e0230445. [PMID: 32176732 PMCID: PMC7075562 DOI: 10.1371/journal.pone.0230445] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2019] [Accepted: 02/29/2020] [Indexed: 01/08/2023] Open
Abstract
Single-nucleotide polymorphisms (SNPs) is associated with efficacy of specific drugs. Although there are several methods for SNP genotyping in clinical settings, alternative methods with lower cost, higher throughout and less complexity are still needed. In this study, we modified Kompetitive Allele Specific PCR to enable multiplex SNP genotyping by introducing additional fluorescent cassettes that specifically help to differentiate more amplification signals in a single reaction. This new format of assay achieved a limit of detection down to 310 copies/ reactions for simultaneous detection of 2 SNPs with only standard end-point PCR workflow for synthetic controls, and genotyped 117 clinical samples with results that were in 100% agreement with hospital reports. This study presented a simplified, cost-effective high-throughput SNP genotyping alternative for pharmacogenetic variants, and enabled easier access to pharmaceutical guidance when needed.
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Affiliation(s)
- Wei Suo
- Pharmacy Department, Beijing Anzhen Hospital, Capital Medical University, Beijing, China
| | - Xiujin Shi
- Pharmacy Department, Beijing Anzhen Hospital, Capital Medical University, Beijing, China
| | - Sha Xu
- Pharmacy Department, Beijing Anzhen Hospital, Capital Medical University, Beijing, China
| | - Xiao Li
- Pharmacy Department, Beijing Anzhen Hospital, Capital Medical University, Beijing, China
| | - Yang Lin
- Pharmacy Department, Beijing Anzhen Hospital, Capital Medical University, Beijing, China
- * E-mail:
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41
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Development and identification of three functional markers associated with starch content in lotus (Nelumbo nucifera). Sci Rep 2020; 10:4242. [PMID: 32144321 PMCID: PMC7060276 DOI: 10.1038/s41598-020-60736-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2019] [Accepted: 02/17/2020] [Indexed: 11/15/2022] Open
Abstract
It have been significantly demonstrated that Hexokinase (HXK), Granule-bound starch synthase (GBSS) and ADP-glucose pyrophosphorylase (AGPase) are three critical enzymes in the starch biosynthetic pathway and are related to starch (amylose, amylopectin and total starch) content in lotus. It is important to develop functional markers in marker-assisted selection of lotus breeding. So far there have been few reports about lotus functional markers. In this study, based on insertion-deletions (INDELs) and single-nucleotide polymorphisms (SNPs), we developed three functional markers, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1. FMHXK-E1 was developed based on polymorphisms of two haplotypes of NnHXK. 26 lotus cultivars that the 320-bp fragment presented in NnHXK had a lower content of amylose and a higher content of amylopectin. FMGBSS-I8 was developed based on polymorphisms of two haplotypes of NnGBSS. The group containing 32 lotus cultivars with the 210-bp fragment had less amylose content and more amylopectin content. FMAGPL-I1 was developed based on polymorphisms of two haplotypes of NnAGPL (ADP-glucose pyrophosphorylase large subunit gene). The group containing 40 lotus cultivars with the 362-bp fragment had less amylopectin, total starch content and more amylose content. According to the study, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1 are closely related to lotus starch content. It could be provided research basis for molecular assisted selection of lotus starch content improve breeding efficiency.
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Breyer JP, Smith JR. Practical genotyping by single-nucleotide primer extension. Biol Methods Protoc 2020; 5:bpaa002. [PMID: 32382659 PMCID: PMC7200932 DOI: 10.1093/biomethods/bpaa002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Revised: 01/17/2020] [Accepted: 01/27/2020] [Indexed: 11/20/2022] Open
Abstract
Genome-wide association studies bring into focus specific genetic variants of particular interest for which validation is often sought in large numbers of study subjects. Practical alternative methods are limiting for the application of genotyping few variants in many samples. A common scenario is the need to genotype a study population at a specific high-value single nucleotide polymorphism (SNP) or insertion-deletion (indel). Not all such variants, however, will be amenable to assay by a given approach. We have adapted a single-nucleotide primer extension (SNuPE) method that may be tailored to genotype a required variant, and implemented it as a useful general laboratory protocol. We demonstrate reliable application for production-scale genotyping, successfully converting 87% of SNPs and indels for assay with an estimated error rate of 0.003. Our implementation of the SNuPE genotyping assay is a viable addition to existing alternative methods; it is readily customizable, scalable, and uses standard reagents and a laboratory plate reader.
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Affiliation(s)
- Joan P Breyer
- Division of Genetic Medicine, Department of Medicine, Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Jeffrey R Smith
- Division of Genetic Medicine, Department of Medicine, Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN, USA.,Medical Research Service, VA Tennessee Valley Healthcare System, Nashville, TN, USA
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43
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Galliano I, Daprà V, Ciferri F, Montanari P, Calvi C, Alliaudi C, Savino F, Bergallo M. TaqMAMA assay polymerase chain reaction real time for allelic discrimination of Macrophage receptor with collagenous structure rs1318645 polymorphism. MINERVA BIOTECNOL 2020. [DOI: 10.23736/s1120-4826.19.02567-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Abstract
Quantitative real-time PCR (qPCR) is a widely adopted technique used for scientific, clinical, diagnostic, or quality control purposes. One of the main applications of qPCR is gene expression analysis, although mutation detection, genotyping, DNA detection, and quantification (from pathogens or genetically modified organisms) are also investigated using this technique.Although nonspecific detection based on DNA-binding dyes (including SYBR Green I) offers versatility in qPCR assays, detection of the PCR product using fluorescent probes confers higher specificity and sensitivity to assays, justifying the use of fluorescent probes as a detection method.This chapter seeks to propose a procedure for the design of qPCR assays using fluorescent hydrolysis probe technology. Particular attention will be paid to explaining the steps necessary to ensure the specificity of the oligonucleotides used as primers or fluorescent probes.
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Affiliation(s)
- Florent Busi
- Unité de Biologie Fonctionnelle et Adaptative, Université de Paris, CNRS UMR 8251, Paris, France.
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45
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Li B, Ren N, Yang L, Liu J, Huang Q. A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells. Sci Rep 2019; 9:18877. [PMID: 31827197 PMCID: PMC6906436 DOI: 10.1038/s41598-019-55463-6] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Accepted: 11/27/2019] [Indexed: 01/12/2023] Open
Abstract
CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3' end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.
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Affiliation(s)
- Bo Li
- Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China
| | - Naixia Ren
- Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China
| | - Lele Yang
- Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China
| | - Junhao Liu
- Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China
| | - Qilai Huang
- Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China.
- State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
- The Second Hospital of Shandong University, Jinan, China.
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46
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Deekshit VK, Jazeela K, Chakraborty G, Rohit A, Chakraborty A, Karunasagar I. Mismatch amplification mutation assay-polymerase chain reaction: A method of detecting fluoroquinolone resistance mechanism in bacterial pathogens. Indian J Med Res 2019; 149:146-150. [PMID: 31219078 PMCID: PMC6563742 DOI: 10.4103/ijmr.ijmr_2091_17] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3’ proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.
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Affiliation(s)
- Vijaya Kumar Deekshit
- Division of Infectious Diseases, Nitte University Centre for Science Education & Research, Mangaluru, India
| | - Kadeeja Jazeela
- Division of Infectious Diseases, Nitte University Centre for Science Education & Research, Mangaluru, India
| | - Gunimala Chakraborty
- Division of Infectious Diseases, Nitte University Centre for Science Education & Research, Mangaluru, India
| | - Anusha Rohit
- Department of Microbiology, Madras Medical Mission, Chennai, India
| | - Anirban Chakraborty
- Division of Infectious Diseases, Nitte University Centre for Science Education & Research, Mangaluru, India
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Chen SL, Chen CY, Hsieh JCH, Yu ZY, Cheng SJ, Hsieh KY, Yang JW, Kumar PV, Lin SF, Chen GY. Graphene Oxide-Based Biosensors for Liquid Biopsies in Cancer Diagnosis. NANOMATERIALS (BASEL, SWITZERLAND) 2019; 9:E1725. [PMID: 31816919 PMCID: PMC6956293 DOI: 10.3390/nano9121725] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Revised: 11/25/2019] [Accepted: 11/25/2019] [Indexed: 12/12/2022]
Abstract
Liquid biopsies use blood or urine as test samples, which are able to be continuously collected in a non-invasive manner. The analysis of cancer-related biomarkers such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA, and exosomes provides important information in early cancer diagnosis, tumor metastasis detection, and postoperative recurrence monitoring assist with clinical diagnosis. However, low concentrations of some tumor markers, such as CTCs, ctDNA, and microRNA, in the blood limit its applications in clinical detection and analysis. Nanomaterials based on graphene oxide have good physicochemical properties and are now widely used in biomedical detection technologies. These materials have properties including good hydrophilicity, mechanical flexibility, electrical conductivity, biocompatibility, and optical performance. Moreover, utilizing graphene oxide as a biosensor interface has effectively improved the sensitivity and specificity of biosensors for cancer detection. In this review, we discuss various cancer detection technologies regarding graphene oxide and discuss the prospects and challenges of this technology.
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Affiliation(s)
- Shiue-Luen Chen
- Department of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan; (S.-L.C.); (C.-Y.C.); (Z.-Y.Y.); (S.-J.C.); (K.Y.H.); (J.-W.Y.); (S.-F.L.)
- Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan
| | - Chong-You Chen
- Department of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan; (S.-L.C.); (C.-Y.C.); (Z.-Y.Y.); (S.-J.C.); (K.Y.H.); (J.-W.Y.); (S.-F.L.)
- Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan
| | - Jason Chia-Hsun Hsieh
- Division of Haematology/Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital (Linkou), Taoyuan 333, Taiwan;
| | - Zih-Yu Yu
- Department of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan; (S.-L.C.); (C.-Y.C.); (Z.-Y.Y.); (S.-J.C.); (K.Y.H.); (J.-W.Y.); (S.-F.L.)
| | - Sheng-Jen Cheng
- Department of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan; (S.-L.C.); (C.-Y.C.); (Z.-Y.Y.); (S.-J.C.); (K.Y.H.); (J.-W.Y.); (S.-F.L.)
- Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan
| | - Kuan Yu Hsieh
- Department of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan; (S.-L.C.); (C.-Y.C.); (Z.-Y.Y.); (S.-J.C.); (K.Y.H.); (J.-W.Y.); (S.-F.L.)
- Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan
| | - Jia-Wei Yang
- Department of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan; (S.-L.C.); (C.-Y.C.); (Z.-Y.Y.); (S.-J.C.); (K.Y.H.); (J.-W.Y.); (S.-F.L.)
- Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan
| | - Priyank V Kumar
- School of Chemical Engineering, University of New South Wales, Sydney, NSW 2052, Australia;
| | - Shien-Fong Lin
- Department of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan; (S.-L.C.); (C.-Y.C.); (Z.-Y.Y.); (S.-J.C.); (K.Y.H.); (J.-W.Y.); (S.-F.L.)
- Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan
| | - Guan-Yu Chen
- Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung University, Hsinchu 300, Taiwan
- Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan
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Chin RI, Chen K, Usmani A, Chua C, Harris PK, Binkley MS, Azad TD, Dudley JC, Chaudhuri AA. Detection of Solid Tumor Molecular Residual Disease (MRD) Using Circulating Tumor DNA (ctDNA). Mol Diagn Ther 2019; 23:311-331. [PMID: 30941670 PMCID: PMC6561896 DOI: 10.1007/s40291-019-00390-5] [Citation(s) in RCA: 114] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Circulating tumor DNA (ctDNA) is a component of cell-free DNA that is shed by malignant tumors into the bloodstream and other bodily fluids. Levels of ctDNA are typically low, particularly in patients with localized disease, requiring highly sophisticated methods for detection and quantification. Multiple liquid biopsy methods have been developed for ctDNA analysis in solid tumor malignancies and are now enabling detection and assessment of earlier stages of disease, post-treatment molecular residual disease (MRD), resistance to targeted systemic therapy, and tumor mutational burden. Understanding ctDNA biology, mechanisms of release, and clearance and size characteristics, in conjunction with the application of molecular barcoding and targeted error correction, have increased the sensitivity and specificity of ctDNA detection techniques. Combinatorial approaches including integration of ctDNA data with circulating protein biomarkers may further improve assay sensitivity and broaden the scope of ctDNA applications. Circulating viral DNA may be utilized to monitor disease in some virally induced malignancies. In spite of increasingly accurate methods of ctDNA detection, results need to be interpreted with caution given that somatic mosaicisms such as clonal hematopoiesis of indeterminate potential (CHIP) may give rise to genetic variants in the bloodstream unrelated to solid tumors, and the limited concordance observed between different commercial platforms. Overall, highly precise ctDNA detection and quantification methods have the potential to transform clinical practice via non-invasive monitoring of solid tumor malignancies, residual disease detection at earlier timepoints than standard clinical and/or imaging surveillance, and treatment personalization based on real-time assessment of the tumor genomic landscape.
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Affiliation(s)
- Re-I Chin
- Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA
| | - Kevin Chen
- Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA
| | - Abul Usmani
- Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA
| | - Chanelle Chua
- Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA
| | - Peter K Harris
- Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA
| | - Michael S Binkley
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA
| | - Tej D Azad
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA
| | - Jonathan C Dudley
- Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Aadel A Chaudhuri
- Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA.
- Department of Computer Science and Engineering, Washington University, St. Louis, MO, USA.
- Alvin J. Siteman Cancer Center, Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO, USA.
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Abstract
Advances in nucleic acid sequencing and genotyping technologies have facilitated the discovery of an increasing number of single-nucleotide variations (SNVs) associated with disease onset, progression, and response to therapy. The reliable detection of such disease-specific SNVs can ensure timely and effective therapeutic action, enabling precision medicine. This has driven extensive efforts in recent years to develop novel methods for the fast and cost-effective analysis of targeted SNVs. In this Review, we highlight the most recent and significant advances made toward the development of such methodologies.
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Affiliation(s)
- Alireza Abi
- Department of Chemistry, Faculty of Sciences, Shiraz University, Shiraz 7194684795, Iran
| | - Afsaneh Safavi
- Department of Chemistry, Faculty of Sciences, Shiraz University, Shiraz 7194684795, Iran
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50
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Brito LG, Barbieri FS, Rocha RB, Santos APL, Silva RR, Ribeiro ES, Guerrero F, Foil L, Oliveira MCS. Pyrethroid and organophosphate pesticide resistance in field populations of horn fly in Brazil. MEDICAL AND VETERINARY ENTOMOLOGY 2019; 33:121-130. [PMID: 30125976 DOI: 10.1111/mve.12330] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/16/2017] [Revised: 05/29/2018] [Accepted: 06/11/2018] [Indexed: 06/08/2023]
Abstract
Pesticides are used worldwide to control arthropod parasites in cattle herds. The indiscriminate and/or inappropriate use of pesticides without veterinary guidance is a reality in several countries of South America. Improper pesticide use increases the chances of contamination of food and the environment with chemical pesticides and their metabolites. Reduction of these contamination events is an increasing challenge for those involved in livestock production. The horn fly, Haematobia irritans (Linnaeus) (Diptera: Muscidae), is one of the most economically important parasites affecting cattle herds around the world. As such, horn fly control efforts are often required to promote the best productive performance of herds. Pesticide susceptibility bioassays revealed that pyrethroid resistance was widespread and reached high levels in horn fly populations in the Brazilian state of Rondônia. The knockdown resistance (kdr) sodium channel gene mutation was detected in all horn fly populations studied (n = 48), and the super kdr sodium channel gene mutation was found in all homozygous resistant kdr individuals (n = 204). Organophosphate resistance was not identified in any of the fly populations evaluated.
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Affiliation(s)
- L G Brito
- Embrapa Amazônia Oriental, Belém, Brazil
| | | | - R B Rocha
- Embrapa Rondônia, Porto Velho, Brazil
| | - A P L Santos
- Universidade Federal de Rondônia, Porto Velho, Brazil
| | - R R Silva
- Embrapa Rondônia, Porto Velho, Brazil
| | | | - F Guerrero
- USDA-ARS Knipling-Bushland Livestock Insects Research Laboratory, Kerrville, TX, U.S.A
| | - L Foil
- Louisiana State University Agricultural Center, Baton Rouge, LA, U.S.A
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