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

• Main Authors: Teh, Chye Phing, Chook, Jack Bee, Ngeow, Yun Fong, Tong, Tommy Yuh Koon, Tee, Kok Keng, Bong, Jan Jin, Mohamed, Rosmawati
• Format: Article
• Published: Wiley 2021
• Subjects: R Medicine (General)
• Online Access: http://eprints.um.edu.my/28865/
• Institution: Universiti Malaya

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.