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利用逆转录重组酶介导的等温扩增技术（Reverse transcription-recombinase aided amplification, RT-RAA）与RT-RAA单链荧光探针结合，建立甲型H1N1流感病毒（Influenza A virus H1N1, FLU A H1N1）的快速分子检测方法。设计RT-RAA引物和荧光探针，用于FLU A H1N1模板核酸的等温扩增和荧光检测，以评价优化方法的灵敏度与特异性；将实时荧光定量PCR(real-time PCR)作为对照方法，测试RT-RAA荧光方法对临床样本的检测效果。FLU A H1N1的RT-RAA荧光检测方法能在30 min内完成检测，灵敏度达到101拷贝/μL；特异性试验表明，其与十六种常见呼吸道传染性病毒均无交叉反应。49份临床样本检测结果和real-time PCR检测结果一致性高（Kappa=0.958），临床灵敏度为100%，临床特异性为95.2%。建立的RT-RAA荧光检测方法检测时间短，灵敏度高，特异性强，适用于FLU A H1N1的快速检测。
Abstract：To establish a rapid and sensitive molecular detection method for Influenza A virus H1N1(FLU A H1N1) via reverse transcription recombinase-aided amplification(RT-RAA) and RT-RAA fluorescent probe approaches. We designed RT-RAA amplification primers and fluorescent probe for FLU A H1N1. Nucleic acids were amplified by RT-RAA and amplification products were evaluated for sensitivity and specificity using isothermal fluorescence equipment. Forty-nine suspected clinical specimens were analyzed by RT-RAA fluorescence assay and real-time PCR. The established RT-RAA fluorescent assay completed detection within 30 min. The detection limit for FLU A H1N1 was 101 copies/μL, and this assay showed no cross-reaction with another 16 common respiratory infectious viruses. Detection results for 49 clinical specimens were highly consistent with real-time PCR(Kappa = 0.958), with a clinical sensitivity of 100% and a clinical specificity of 95.2%. The established RT-RAA fluorescent assay has the advantages of short detection duration, high sensitivity, and strong specificity. It can quickly detect FLU A H1N1 and provides a more convenient detection method for the diagnosis of undeveloped areas.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004266
中图分类号:R373.13
引用信息:
[1]安柏霖,郭悦,刘丹丹,等.逆转录重组酶介导的等温扩增技术快速检测甲型H1N1流感病毒[J].病毒学报,2023,39(01):45-51.DOI:10.13242/j.cnki.bingduxuebao.004266.
基金信息:
江苏省社会发展面上项目（项目号：BE2019761）,题目：基于离心力微流控芯片的重要境外输入性传染病核酸即时检测技术及应用; 江苏省社会发展重点项目（项目号：BE2020682）,题目：重大传染病特异性诊断试剂开发与标准化示范~~
2022-05-29
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