Primer and probe conservation issue in the quantification of hepatitis B virus DNA

Current treatment strategies for chronic hepatitis B virus (HBV) infection aim at long-term suppression of the viral replication since a cure remains elusive. Its clinical management therefore relies greatly on routine monitoring of serum HBV DNA levels using quantitative polymerase chain reaction (qPCR) assays. Designing a highly conserved oligonucleotide set for the qPCR assay can be challenging due to the high genetic heterogeneity of the virus. The ever-increasing number of HBV genomes deposited in the GenBank nucleotide database warrants a revisit to previous primer and probe designs. We examined primer and probe sets from 53 qPCR assays published in the past 2 decades for their coverage in 9864 complete HBV genomes retrieved from GenBank. Of all the 53 qPCR assays, only 17% achieved >= 80% coverage. About 40% of the 53 assays covered less than 20% of the 9864 genomes.In silicoDNA thermodynamics analysis demonstrated reduced primer/probe binding affinity, which further increases the risk of viral load misdetection and underestimation for certain HBV variants. Taken together, there is a pressing need for improving available qPCR designs for the quantification of HBV DNA based on the updated genome data.

Automated summary

Current treatment strategies for chronic hepatitis B virus infection focus on long-term suppression of viral replication, with routine monitoring of serum HBV DNA levels being crucial. A study on 53 qPCR assays over the past two decades found that only 17% achieved a coverage of >= 80%, with about 40% covering less than 20% of the 9864 HBV genomes analyzed. The findings highlight the need for improving qPCR designs for accurate quantification of HBV DNA based on updated genome data.