中东呼吸综合征冠状病毒RT-RAA快速检测方法的建立及应用

Development and Application of a RT-RAA Assay to Detect Middle East Respiratory Syndrome Coronavirus

建立基于重组酶介导的中东呼吸综合征冠状病毒（MERS-CoV）核酸检测方法。本研究设计、合成特异性中东呼吸综合征冠状病毒基因的重组酶介导检测（RAA）引物及探针,制备MERS-CoV假病毒颗粒阳性对照品,通过一系列条件优化建立了MERS-CoV的快速、灵敏、特异的RT-RAA检测方法,分别与荧光定量RT-PCR检测方法做比较,并且采用首例中国MERS-CoV韩国地区输入病例的咽拭子样品、其它类似呼吸道病毒样本以及英国QCMD的MERS-CoV室间质评灭活样本做临床验证。结果显示：建立的RT-RAA方法检测中东呼吸综合征冠状病毒灵敏度为10拷贝,高于本实验室建立的荧光RT-PCR方法灵敏度（100拷贝）,且检测时间（4.8~13.6min）大大低于荧光RT-PCR检测时间（90min）;用该方法检测中东呼吸综合征冠状假病毒颗粒为阳性,而检测其他8种对照呼吸道病毒均呈阴性;检测临床阳性样本也与实际结果相符。本研究建立的中东呼吸综合征冠状病毒RT-RAA检测方法灵敏、特异、快速,可用于中东呼吸综合征冠状病毒感染的现场快速诊断和流行病学调查。

We wished to establish a rapid,sensitive and specific assay to detect the Middle East Respiratory Syndrome Coronavirus（MERS-CoV）using a reverse-transcription recombinase-aid assay.Several primers and probes were designed in according to the conserved sequence of MERS-CoV.RT-RAA was performed using optimized parameters and positive RNA prepared by serial tenfold dilution of virus-like particles.In addition,the specificity and sensitivity of the RT-RAA assay wereverified,and clinical samples were tested using this assay.The RT-RAA assay could detect as few as 10 copies,and was more sensitive than the realtime polymerase chain reaction（PCR）developed by our research team（100 copies）.Positive signals were observed in MERS-CoV virus-like particles.No signals were observed with eight control respiratory disease pathogens and coronavirus were detected using the RT-RAA assay,and its detection time（4.8~13.6 min）was far faster than that of real-time PCR（90 min）.the result of using the RT-RAA assay on clinical samples was consistent with the actual result.In summary,we developed a RT-RAA assay was.that was highly sensitive,specific and rapid,This RT-RAA assay lays the foundation for onsite diagnosis and the epidemiologic study of MERS-CoV infection.