METHOD FOR DETECTING THE DNA OF THE HEPATITIS B VIRUS IN BIOLOGICAL MATERIAL WITH A LOW VIRAL LOAD BASED ON TWO-STAGE PCR WITH DETECTION BY THREE TARGETS IN REAL TIME

摘要

FIELD: biotechnology.;SUBSTANCE: described is a method for detecting the hepatitis B virus with a low viral load by determining the DNA of the virus in a blood sample by means of PCR with detection by three targets in real time. Detection is executed in blood plasma and/or serum, a blood clot, hepatic tissues. Diagnostics is executed in two stages. At the first stage, amplification of the DNA of the virus is performed using oligonucleotide primers flanking the full genome of the virus, and at the second stage - using three pairs of oligonucleotide primers for the three regions (S gene, X gene, Core gene) of the genome of the virus, as well as one pair for a fragment of the human HPRT gene and the corresponding fluorescently labeled oligonucleotide probes complementary to the sections of the amplified fragments carrying fluorophores at the 5' end and non-fluorescent quenching agents at the 3' end. The obtained results are recorded by means of hybridisation fluorescence detection in real time. At the second stage, a positive control sample is introduced into the study, constituting a plasmid containing the nucleotide sequences of the three target regions of the HBV genome.;EFFECT: method provides an increase in the sensitivity and accuracy of diagnostics.;1 cl, 2 dwg, 2 tbl, 4 ex