Method for detecting hepatitis B virus in blood plasma at low viral load using real-time PCR

Abstract

A method for detecting HBV DNA in peripheral blood at low viral load using real-time PCR was developed and its significance in identifying HBsAg-negative viral hepatitis B was evaluated. When developing the method, blood plasma samples and liver tissue biopsy material were used from 128 patients living in St. Petersburg, in various regions of the Russian Federation, as well as in the Central Asia countries. We also used blood plasma samples from 96 pregnant women and 37 hemodialysis center patients living in Northwestern Federal District, 199 foreign citizens undergoing medical examination to obtain work permits at the Directorate for Migration in the Northwestern Federal District, 397 conditionally healthy people living in the Socialist Republic of Vietnam. HBV was detected by nested PCR. Analytical sensitivity was tested using the stepwise dilution method. According to the method developed by us, at the first stage, the HBV DNA is amplified using at the first stage oligonucleotides flanking the genome region 2932-3182 ... 1-1846 nt., and at the second stage two oligonucleotides pairs to the genome virus regions (gene S and gene X) and corresponding oligonucleotide fluorescently labeled probes complementary to the amplified fragments regions carrying fluorophores at the 5’-end, and non-fluorescent quenchers at the 3’-end. The channel corresponding to the FAM fluorophore detects the HBV DNA S-region amplification product, and the channel corresponding to the ROX fluorophore detects the HBV DNA X-region amplification product. The method sensitivity for DNA extraction from plasma with a 100 μl volume was 10 IU/ml. Obtaining a threshold cycle Ct for only one FAM or ROX fluorophore may indicate the HBV DNA presence in a sample at a load of less than 10 IU / ml, HBV detection in this case is possible with a repeated PCR study of the corresponding sample with HBV DNA extraction from an increased plasma volume (200-1000 μl). The developed method makes it possible to identify various HBV genovariants, both characteristic and rare in the Russian Federation, circulating in other world regions. The method can be used to detect HBV in risk groups, in the population, as well as in screening blood donors in order to ensure the blood transfusions safety.