重组酶聚合酶扩增技术在流感病毒检测中的探索性研究  

Exploratory research on detection of influenza viruses by reverse transcriptase-recombinase polymerase amplification

作　　者：孙宁[1] 于娟[1,2] 严孝岭 高德玉[1] 陈波[3] 王卫萍 李晓军[1] Sun Ning;Yu Juan;Yan Xiaoling;Gao Deyu;Chen Bo;Wang Weiping;Li Xiaojun(Department of Clinical Laboratory Science,Nanjing General Hospital of People's Liberation Army,Nanjing 210002,China;Nanjing Lishui People′s Hospital,Zhongda Hospital Lishui Branch,Southeast University,Nanjing 210000,China;Ningbo Health Gene Technologies Co.Ltd.,Ningbo 315040,China)

机构地区：[1]东部战区总医院(原南京军区南京总医院)临床中心实验科,南京210002 [2]南京市溧水区人民医院、东南大学附属中大医院溧水分院,南京210000 [3]宁波海尔施基因科技有限公司,315040

出　　处：《中华实验和临床病毒学杂志》2019年第5期530-535,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金青年项目(81601857);江苏省科技计划项目(BL2014072);中国国家重点临床项目(2014ZDZK003);江苏省青年基金项目(BK20180293)。

摘　　要：目的建立和评估反转录酶-重组酶聚合酶扩增(reverse transriptase-recombinase polymerase amplification,RT-RPA)技术在流感病毒检测中的应用。方法分别以甲型流感病毒基质蛋白(matrix)基因、血凝素(HA)基因,以及乙型流感病毒非结构蛋白(NS)基因为靶标基因,设计引物,建立流感病毒检测和亚型(H1、H3)鉴定的RT-RPA方法,并分析该方法的特异性和灵敏性。结果所建立的流感病毒RPA检测方法仅对相应靶基因出现特异性扩增产物,对其他呼吸道病毒均无扩增,表明该方法特异性较高。利用上述方法检测流感病毒的检测限均为100拷贝/μl。SYBR Green I可与RT-RPA相结合检测乙型流感病毒,最低检测限为100拷贝/μl。结论初步证实了利用RPA方法检测流感病毒的可行性。Objective To establish and hevaluate a detection method for influenza virus using reverse transcriptase-recombinase polymerase amplification(RT-RPA).Methods RT-RPA was developed for detection of influenza viruses(type A and B)and subtyping of H1 and H3 using the primers targeted matrix and hemagglutinin(HA)genes of influenza A virus,and non-structural(NS)protein gene of influenza B virus.The specificity and sensitivity of RT-RPA were determined.Results The RT-RPA for the detection of influenza viruses showed specific amplification products of corresponding target gene,but no amplification products for other respiratory viruses,indicating that the method had good specificity.The detection limits of RT-RPA were 100 copies/μl.RT-RPA combined with SYBR Green I was used for the detection of influenza B virus with the detection limit of 100 copies/μl.Conclusions The feasibility of detecting influenza virus by RT-RPA was preliminarily confirmed.

关 键 词：流感病毒 检测 分型 重组酶聚合酶扩增 

分 类 号：R51[医药卫生—内科学]