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

<jats:title>Summary</jats:title><jats:p>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. <jats:italic>In silico</jats:italic> DNA 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.</jats:p>