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  被引量:6

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

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作  者:关丽[1] 聂凯[1] 张丹[1,2] 李鑫娜[1] 王延群[1] 谭文杰[1] 马学军[1] 

机构地区:[1]中国疾病预防控制中心病毒病预防控制所,北京102206 [2]广州医科大学基础医学院,广州511436

出  处:《病毒学报》2015年第3期269-275,共7页Chinese Journal of Virology

基  金:传染病重大专项(2013ZX10004-001;2012ZX10004-215;2013ZX10004-202和2013ZX10004804-007)

摘  要: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.

关 键 词:环介导逆转录等温扩增 中东呼吸综合征冠状病毒 

分 类 号:R373.9[医药卫生—病原生物学]

 

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