Development And Evaluation Of The Real Time Genotyping And Quantitative PCR Method For Genotype B And C Of HBV

Objective: To improve and evaluate the the real time genotyping and quantitative PCR (GQ-PCR) method systematically, and explore the actual distribution of HBV genotypes in Chongqing province by it.Method: Designed a series of experiments to optimize annealing temperaturem and the probe and primer concentraion, and determined the standard of sensitivity, specificity, repeatability and linear range to evaluate the method. Then compared GQ-PCR to direct sequence analysis and the multiplex-PCR method, and used GQ-PCR to detect HBV genotypes of 207 patient samples with HBV-DNA positive.Result: The probe and primer concentration were determined 0.3μM and 0.5μM respectively. The inear range of GQ-PCR was 102-109copies/ml, the specificity was 100%, the intra-assay repeatability CV were 0.58% for genotype B and 0.86% for genotype C, and the inter-assay repeatability CV were 4.09% and 1.28% for genotype B genotype C. There was no cross-reaction between genotype B and C. The detection rate of GQ-PCR and direct sequence analysis are 100%, and the multiplex-PCR is 94.69%. The concordance between RT-GQ-PCR and the multiplex-PCR is perfect (Kappa value = 0.915), and the consistency of RT-GQ-PCR and direct sequence analysis is pretty good (Kappa value = 0.742), specially at detecting single genotype. 28 samples with genotypes B and C dual infections were detected by RT-GQ-PCR, but only 19 samples by the multiplex-PCR and 13 samples by direct sequence analysis.In the HBV distribution research, Genotype B (61.35%) is most prevalent in Chongqing province, and HBV genotype B and C co-infection (23.19%) is more common than that has been previously observed. In The HBV viral load of patients with genotype C infected (8.06±1.20) log10copies/ml was significantly both higher than patients with genotype B infected (7.35±1.31) log10copies/ml, and with mixed genotype infected (7.26±1.10) log10copies/ml (P = 0.01).Conclusion: The GQ-PCR is convenient, rapid and accurate in HBV genotyping, especially more sensitive than direct sequence analysis and the multiplex-PCR for detecting dual genotype infections. The method is applicable for large-scale epidemiological study in China, and clinical detectio.