环介导逆转录等温扩增技术检测中东呼吸综合征冠状病毒基因

Detection of Middle East Respiratory Syndrome Coronavirus by Reversetranscription Loop-Mediated Isothermal Amplification

建立了基于浊度仪和羟基萘酚蓝(Hydroxy naphthol blue,HNB)颜色变化的简单、快速和灵敏的环介导逆转录等温扩增技术(RT-LAMP)检测方法,应用于中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)的检测。针对MERS-CoV的核衣壳蛋白基因(Nucleocapsid,N)设计6条特异引物,在等温条件下(63℃)进行60min扩增反应。通过实时浊度仪记录扩增曲线和扩增前在反应体系中加入HNB观察颜色变化两种方式进行检测结果判定。本文对不同人类冠状病毒及常见呼吸道病毒进行了特异性验证,对梯度稀释的体外转录MERS-CoV全N基因RNA进行了定量和检测限分析,同时与已发表的实时荧光定量PCR(rRT-PCR)进行比较。结果显示,本研究建立的RT-LAMP方法特异性高,对于每反应管103至106的拷贝数RNA,出峰时间与每反应管N基因RNA拷贝数对数值有稳定的线性关系。基于浊度仪和颜色判定的RT-LAMP检测方法检测限分别为500拷贝和1 000拷贝。因此,该方法有望应用于MERS-CoV感染的快速筛选,具有在基层医疗卫生机构和现场推广和应用的潜力。

A simple,rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification（RT-LAMP）was developed for rapid detection of Middle East respiratory syndrome coronavirus（MERS-CoV）.The method employed six primers that recognized sequences of a nucleocapsid gene for amplification of nucleic acids under isothermal conditions at 63°C for 60 min.Products were detected through a LA-320 cLoopamp Turbidimeter（real-time RT-LAMP）or visual inspection of color change by pre-addition of Hydroxynaphthol Blue dye（visual RT-LAMP）.Specificity of RT-LAMP was validated by detection of several human coronaviruses and common respiratory viruses.MERS-CoV real-time RT-LAMP had a linear correlation（R2）of 0.995 at 103-106 copies.The limit of detection of real-time RT-LAMP,visual RT-LAMP and quantitative real-time PCR was 500,1 000 and 100copies/reaction,respectively.The established RT-LAMP assay was demonstrated to be a rapid screening tool for MERS-CoV infection,and could be suitable in resource-limited clinical sites and for field studies.