Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets

Area of Science:

• Molecular Biology
• Virology
• Infectious Diseases

Background:

• Hepatitis B virus (HBV) DNA quantification is vital for managing chronic infections.
• Genetic variations in HBV and drug-resistant mutations can lead to inaccurate viral load measurements by commercial kits.
• Primer or probe mismatches in commercial DNA quantification kits can cause underestimation of HBV DNA levels.

Purpose of the Study:

• To evaluate the accuracy of commercial kits for quantifying HBV DNA.
• To develop an improved duplex real-time PCR assay for more accurate HBV DNA quantification.

Main Methods:

• A novel duplex real-time PCR assay was developed using two primer pairs and two probes targeting conserved S and C regions of the HBV genome.
• HBV DNA quantitative detection was performed on HBV samples.
• Results were compared against the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays.

Main Results:

• The duplex real-time PCR assay showed good agreement with commercial assays.
• Two Chinese HBV infection samples exhibited underestimated viral loads with the Roche kit due to primer sequence mismatches.
• Six samples showed undervalued HBV DNA levels with duplex C region assays because of mutations in the C region primer.

Conclusions:

• A new duplex real-time PCR assay was developed with comparable results to commercial kits.
• The COBAS TaqMan HBV Test version 2 can underestimate HBV DNA levels in Chinese HBV infections due to primer/probe mismatches.
• A duplex real-time PCR assay targeting S and C regions offers a solution to improve HBV DNA quantification accuracy.