Establishment of a novel two-probe real-time PCR for simultaneously quantification of hepatitis B virus DNA and distinguishing genotype B from non-B genotypes

• A two-probe real-time PCR for quantification and genotyping of HBV was established.
• The novel method is sensitive, specific, accurate and reproducible.
• 539 serum samples from HBV-infected patients were assayed by the new method.
• 54.0% of the 509 samples were genotype B, 39.5% non-B and 6.5% mixed genotype.
• Genotype C may be associated with more severe liver disease than genotype B.

ObjectiveEstablishment of a simple, rapid and economical method for quantification and genotyping of hepatitis B virus (HBV) is of great importance for clinical diagnosis and treatment of chronic hepatitis B patients. We hereby aim to develop a novel two-probe real-time PCR for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes.MethodsConserved primers and TaqMan probes for genotype B and non-B genotypes were designed. The linear range, detection sensitivity, specificity and repeatability of the method were assessed. 539 serum samples from HBV-infected patients were assayed, and the results were compared with commercial HBV quantification and HBV genotyping kits.ResultsThe detection sensitivity of the two-probe real-time PCR was 500 IU/ml; the linear range was 103–109 IU/ml, and the intra-assay CVs and inter-assay CVs were between 0.84% and 2.80%. No cross-reaction was observed between genotypes B and non-B. Of the 539 detected samples, 509 samples were HBV DNA positive. The results showed that 54.0% (275/509) of the samples were genotype B, 39.5% (201/509) were genotype non-B and 6.5% (33/509) were mixed genotype. The coincidence rate between the method and a commercial HBV DNA genotyping kit was 95.9% (488/509, kappa = 0.923, P < 0.01). In addition, high correlation (R2 = 0.94, P < 0.05) and good consistency between our assay and a commercial HBV DNA qPCR kit were achieved.ConclusionsA novel two-probe real-time PCR method for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes was established. The assay was sensitive, specific and reproducible which can be applied to areas prevalent with HBV genotypes B and C, especially in China.