基于颜色判定的RT-LAMP技术检测HCoV-NL63冠状病毒基因  

Visual Detection of Human Coronavirus NL63 by Reverse Transcription LoopMediated Isothermal Amplification

作　　者：耿合员[1,2,3] 汪圣强[1] 谢晓谦[1] 肖雨[1] 张汀[1] 谭文杰[2] 苏川[3] 

机构地区：[1]无锡出入境检验检疫局中国检验检疫科学研究院(无锡)人体携带病原体研究室,无锡214101 [2]中国疾病预防控制中心病毒病预防控制所病毒病应急技术中心,北京102206 [3]南京医科大学基础医学院江苏省现代病原生物学重点实验室,南京210029

出　　处：《病毒学报》2016年第1期56-61,共6页Chinese Journal of Virology

基　　金：传染病重大专项(2014ZX10004001-002;2013ZX10004001)

摘　　要：建立了一种适合于国境口岸简便、快速、灵敏和特异检测人冠状病毒NL63(HCoV-NL63)的核酸检测方法。通过建立基于颜色判定的逆转录环介导等温扩增(Reverse transcription loop-mediated isothermal amplification,RT-LAMP)技术,针对HCoV-NL63核衣壳蛋白(Nucleocapsid,N)基因序列设计出6条特异性引物,在等温条件下(63℃)进行扩增反应60min,在扩增前加入钙黄绿素作为反应指示剂,并结合浊度仪进行实时监测,以最终反应颜色变化作为结果判断标准。利用该方法对同种属其他人冠状病毒、诺如病毒、流感病毒进行检测,以分析该方法的诊断特异性。应用该方法对梯度稀释的HCoV-NL63病毒RNA进行检测,以分析该方法的诊断灵敏性,并与常规real-time RT-PCR方法进行比较。结果显示,本研究建立的基于颜色判定的RT-LAMP方法在短时间内(40min)即可达到对HCoV-NL63的快速检测。该检测方法与加入其它病毒RNA模板溶液颜色均不产生改变,具有较高的检测特异性。检测灵敏性最低检测下限为1 600拷贝/反应。本研究建立的基于颜色判定的RT-LAMP方法可实现对HCoV-NL63冠状病毒现场快速检测,具有在国境口岸以及基层医疗机构和疫区现场推广应用的潜力。A simple and sensitive assay for rapid detection of human coronavirus NL63（HCoV-NL63）was developed by colorimetic reverse transcription loop-mediated isothermal amplification（RT-LAMP）.The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63℃for 1h Amplification of RT-LAMP was monitored by addition of calcein before amplification.A positive reaction was confirmed by change from light-brown to yellow-green under visual detection.Specificity of the RTLAMP assay was validated by cross-reaction with different human coronaviruses,norovirus,influenza A virus,and influenza B virus.Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6×109 to 1.6×101 per reaction.The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity.Hence,our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63.

关 键 词：HCOV-NL63 RT-LAMP 快速检测 

分 类 号：R373.9[医药卫生—病原生物学]