Digital polymerase chain reaction detection of telomerase reverse transcriptase promoter mutations in hepatitis B virus related hepatocellular carcinoma

Figure 2 Optimized digital polymerase chain reaction system for ultra-sensitive detection of the guanine-cytosine-rich telomerase reverse transcriptase promoter C228T mutation. A: Workflow schematic: Illustration of the digital polymerase chain reaction (dPCR) process, including sample loading, droplet partitioning, thermal cycling, fluorescence signal detection, and Poisson-based absolute quantification. This system enables precise molecular counting through endpoint fluorescence analysis; B: Optimization strategies: The guanine-cytosine-rich telomerase reverse transcriptase promoter region [> 80% guanine-cytosine (GC) content] forms stable secondary structures that hinder amplification. Optimization with 7-deaza-dPCR (reducing GC stacking), CviQ1 restriction enzyme digestion (simplifying templates), and Mg²⁺/ethylenediaminetetraacetic acid balancing (enhancing specificity and signal strength) effectively overcomes GC-barriers and improves amplification efficiency; C: Performance validation: Comparative analysis between dPCR and Sanger sequencing demonstrates superior performance of dPCR, achieving 100% sensitivity, 90% specificity, and a detection limit of 0.55 cp/μL. Representative fluorescence droplet images illustrate clear separation of positive (green) and negative (gray) droplets at the endpoint. dPCR: Digital polymerase chain reaction; GER: G-quadruplex-forming element-rich; GC: Guanine-cytosine; LOD: Limit of detection; PCR: Polymerase chain reaction; TERT: Telomerase reverse transcriptase; dGTP: Deoxyguanosine triphosphate; EDTA: Ethylenediaminetetraacetic acid.