Saint-Petersburg Pasteur Institute.
Saint-Petersburg State Medical University n.a. acad. I.P. Pavlov.
Klin Lab Diagn. 2021 Feb 10;66(1):59-64. doi: 10.18821/0869-2084-2021-66-1-59-64.
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.
建立了一种使用实时 PCR 检测低病毒载量外周血 HBV DNA 的方法，并评估了其在鉴定 HBsAg 阴性乙型肝炎病毒中的意义。在方法开发过程中，使用了来自圣彼得堡的 128 名居住在俄罗斯联邦不同地区以及中亚国家的患者的血浆样本和肝组织活检材料。我们还使用了来自西北联邦区 96 名孕妇和 37 名血液透析中心患者、西北联邦区移民局 199 名接受体检以获得工作许可的外国公民以及越南社会主义共和国 397 名条件健康人的血浆样本。使用巢式 PCR 检测 HBV。使用逐步稀释法测试分析灵敏度。根据我们开发的方法，在第一阶段，使用侧翼基因组区域 2932-3182…1-1846 nt 的寡核苷酸扩增 HBV DNA，在第二阶段，使用两对基因组病毒区域（基因 S 和基因 X）的寡核苷酸对以及与扩增片段区域互补的相应荧光标记探针，该区域在 5'端携带荧光团，在 3'端携带非荧光淬灭剂。与 FAM 荧光团对应的通道检测 HBV DNA S 区扩增产物，与 ROX 荧光团对应的通道检测 HBV DNA X 区扩增产物。用 100μl 体积提取的血浆进行 DNA 提取的方法灵敏度为 10IU/ml。仅获得一个 FAM 或 ROX 荧光团的阈值循环 Ct 可能表明样品中 HBV DNA 的载量低于 10IU/ml，在这种情况下，可以通过重复 PCR 研究相应的样品并用 HBV DNA 提取增加的血浆体积（200-1000μl）来检测 HBV。该方法可用于鉴定俄罗斯联邦内常见和罕见的各种 HBV 基因变体，以及其他世界区域内的循环变体。该方法可用于在风险人群、人群中以及在筛选献血者中检测 HBV，以确保输血安全。
