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

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.