Research Institute of Influenza, Ministry of Healthcare of Russian Federation, Laboratory of Molecular Virology, Prof. Popova 15/17, Saint Petersburg, 197376, Russia; Saint Petersburg University, Faculty of Biology, Department of Biochemistry, 7/9 Universitetskaya emb., Saint Petersburg, 199034, Russia; ITMO University, Department of Laser Systems and Technologies, Kronverkskiy Ave, 49, Saint Petersburg, 197101, Russia.
Research Institute of Influenza, Ministry of Healthcare of Russian Federation, Laboratory of Molecular Virology, Prof. Popova 15/17, Saint Petersburg, 197376, Russia.
Mol Cell Probes. 2017 Oct;35:57-63. doi: 10.1016/j.mcp.2017.06.005. Epub 2017 Jun 23.
Influenza and other acute respiratory infections are of great concern for public health, causing excessive morbidity and mortality throughout the world. Influenza virus A(H2N2), which caused a pandemic of so called "Asian flu" in 1957 was expelled from the human population by the new pandemic virus subtype H3N2 in 1968, however, influenza A(H2) viruses continue to circulate in wild birds and poultry. The lack of immunity in human population and the continued circulation of influenza A(H2) among animals makes emergence of a new pandemic virus possible. One of the basic techniques of molecular diagnostics of infectious diseases is the realtime polymerase chain reaction (PCR). The aim of this work was to design oligonucleotide primers and probes for the rapid detection of influenza A virus subtype H2 by realtime reverse transcription - polymerase chain reaction (rRT-PCR). Analysis of 539 sequences of influenza A(H2N2) virus hemagglutinin gene from GISAID EpiFlu database revealed conservative regions suitable for use as binding sites for primers and probes. 191 probes were designed and 2 sets of primers and probes (H2-1 and H2-2) were selected for further experimental evaluation. Detection limit of RT-PCR system was 50 copies of DNA per 25 μl reaction when 10-fold dilutions of pCI-neo-H2 plasmid used as template. Analytical specificity of selected sets of primers and probes were tested on wide range of influenza strains and non-influenza respiratory viruses. H2-2 set found to have insufficient specificity detecting seasonal influenza A(H1N1) viruses and was excluded from further analysis. Analytical sensitivity was further tested on vaccine strain A/17/California/66/395 (H2N2) and A/Japan/305/1957 (H2N2), limit of detection for primers-probe set H2-1 was 3.2 (CI95%: 3.07-3.48) lg EID/ml. Designed primers and probes for the realtime RT-PCR universal detection of influenza A(H2) viruses could be used in clinical trials of vaccines against influenza A(H2) and screening for H2 in cases of unsubtypeable influenza A in humans.
流感和其他急性呼吸道感染对公共卫生极为关注，在全球范围内导致过高的发病率和死亡率。1957 年导致所谓“亚洲流感”大流行的甲型 H2N2 流感病毒于 1968 年被新型的 H3N2 大流行病毒亚型所取代，但甲型 H2 病毒仍在野禽和家禽中传播。人类缺乏免疫力，以及甲型 H2 病毒在动物中的持续传播，使得新的大流行病毒有可能出现。传染病分子诊断的基本技术之一是实时聚合酶链反应（PCR）。本工作旨在设计用于通过实时逆转录-聚合酶链反应（rRT-PCR）快速检测甲型 H2 流感病毒的寡核苷酸引物和探针。对 GISAID EpiFlu 数据库中 539 个甲型 H2N2 病毒血凝素基因序列的分析显示了适合用作引物和探针结合位点的保守区域。设计了 191 个探针，并选择了 2 组引物和探针（H2-1 和 H2-2）进行进一步的实验评估。当使用 pCI-neo-H2 质粒作为模板进行 10 倍稀释时，RT-PCR 系统的检测限为 25 μl 反应中的 50 个拷贝 DNA。选择的引物和探针组的分析特异性在广泛的流感株和非流感呼吸道病毒上进行了测试。发现 H2-2 组对季节性甲型流感 A（H1N1）病毒的特异性不足，因此被排除在进一步分析之外。进一步在疫苗株 A/17/加利福尼亚/66/395（H2N2）和 A/日本/305/1957（H2N2）上测试了分析敏感性，引物-探针组 H2-1 的检测限为 3.2（CI95%：3.07-3.48）lg EID/ml。设计的用于实时 RT-PCR 通用检测甲型 H2 病毒的引物和探针可用于针对甲型 H2 流感的疫苗临床试验和人类未分型流感 A 的筛查。
