Method for hepatitis B virus DNA detecting in biological material at low viral load based on nested PCR with detection on three viral targets in real-time mode.

Abstract

A method has been developed for HBV DNA finding in biological material at low viral load based on nested PCR with real-time detection of three viral targets. When developing the method, blood plasma samples were used from 128 CHB patients living in the regions of the Russian Federation and countries of Central Asia and 173 hemodialysis center patients living in the North-West Federal District. Analytical sensitivity was tested using the stepwise dilution method. HBV was detected by nested PCR. According to the method developed by us, at the first stage, the HBV DNA is amplified using at the first stage oligonucleotides complementary to the greatest similarity regions of the various HBV isolates genomes flanking the entire virus genome. At the second stage, when using the amplification product of the first stage as a template, PCR was performed using three pairs of oligonucleotides and the corresponding oligonucleotide fluorescently labeled probes to three virus genome regions (Core gene, S gene and X gene), as well as one pair of primers and the corresponding probe complementary to a human HPRT gene region. The method sensitivity for DNA extraction from plasma with a 100 μl volume was 10 IU/ml. Obtaining a threshold Ct cycle for only one fluorophore may indicate the presence of HBV DNA in the 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 the disease in various HBV subgenotypes and can be used to diagnose CHB in the population and risk groups, including those with the HBsAg-negative form of the disease.