通用型人类呼吸道合胞病毒RT-RPA检测方法的建立  

Development of universal RT-RPA method for detecting human respiratory syncytial virus

作　　者：薛晓宁 徐翮飞 毛毛 王江凤 郑志坤 张娟 滕新栋 张瑾 XUE Xiaoning;XU Hefei;MAO Mao;WANG Jiangfeng;ZHENG Zhikun;ZHANG Juan;TENG Xindong;ZHANG Jin(Qingdao International Travel Healthcare Center,Qingdao,Shandong 266071,China;不详)

机构地区：[1]青岛国际旅行卫生保健中心,山东青岛266071 [2]上海国际旅行卫生保健中心 [3]青岛大学附属青岛市海慈医院 [4]青岛大学基础医学院

出　　处：《中国国境卫生检疫杂志》2023年第6期523-526,共4页Chinese Journal of Frontier Health and Quarantine

基　　金：山东省重点研发计划项目(2021CXGC011306)。

摘　　要：目的建立通用型人类呼吸道合胞病毒RT-RPA检测方法,完成呼吸道合胞病毒快速诊断。方法分析比较175株A型、B亚型呼吸道合胞病毒全基因组序列,针对保守的M基因设计合成符合重组酶聚合酶扩增(RPA)要求的通用型引物和探针,优化反应体系和条件,建立实时荧光RT-RPA检测方法,进行敏感性和特异性验证及临床样品的检测,评价方法的检测能力。结果成功建立了通用型呼吸道合胞病毒RT-RPA检测方法,最佳引物浓度210 nmol/L,探针浓度120 nmol/L,最佳反应温度41℃,20 min完成检测,最低检测限为10拷贝/μl,且与流感病毒、新冠病毒、鼻病毒、副流感病毒等无交叉反应;检测20份呼吸道合胞病毒阳性样本,与实时荧光RT-PCR检测结果的符合率为100%。结论建立的通用型呼吸道合胞病毒RT-RPA检测方法是一种敏感、特异的快速检测呼吸道合胞病毒的方法,为口岸现场快速检测呼吸道合胞病毒提供了新工具。Objective To develop a RT-RPA method for detecting human respiratory syncytial virus(HRSV),and provide a useful tool for rapid diagnosis of respiratory syncytial virus infection.Methods A total of 175 strains whole genome sequences of HRSV-A and HRSV-B were analyzed and compared.According to M gene of HRSV,RT-RPA primers and probes were designed to establish a RT-RPA method for the detection of HRSV.The specificity and sensitivity of the method were validated.Tests for clinical samples were carried out to evaluate the detection ability of the method.Results The universal RT-RPA method for the detection of HRSV was successfully established.The concentrations of optimized terminal RT-RPA detection primer and probe were 210 nmol/L and 120 nmol/L,respectively.The optimal reaction temperature was 41℃.The reaction time was 20 minutes.The sensitivity tests showed that the RT-RPA reaction system had a sensitivity of 10 copies/μl.It was not affected for other respiratory pathogens such as influenza virus,SARS-CoV-2,human rhinovirus and parainfluenza virus.20 HRSV positive samples were detected using this method and compared with Real time RT-PCR,the coincidence rate was 100%.Conclusion The universal RT-RPA assay for HRSV had strong specificity and high sensitivity.This method could be used for the rapid diagnosis of HRSV at port.

关 键 词：呼吸道合胞病毒 重组酶聚合酶扩增 通用型 

分 类 号：R183[医药卫生—流行病学] R373.13[医药卫生—公共卫生与预防医学]