基于颜色判定的逆转录环介导等温扩增技术检测GII型诺如病毒基因

Colorimetric Detection of Norovirus Genotype GII by Reverse Transcription Loop-Mediated Isothermal Amplification

建立了一种基于颜色判定的简单、快速和灵敏的逆转录环介导等温扩增(Reverse transcription loop-mediatedisothermal amplification,RT-LAMP)方法应用于GII型诺如病毒基因检测。针对诺如病毒RNA依赖的RNA聚合酶和衣壳蛋白基因序列(RNA-dependant RNA polymerase and capsid protein gene)设计出6条特异引物,在等温条件下(65℃)进行扩增反应60min。在扩增前加入染料羟基萘酚蓝(HNB)作为反应指示剂,以其颜色变化作为结果判断标准,并经琼脂糖凝胶电泳验证。本文利用此技术对不同腹泻病毒进行了特异性分析,对体外转录的GII型诺如病毒RNA的梯度稀释进行了灵敏度分析,同时与逆转录PCR(RT-PCR)方法进行比较,并对93份腹泻患者粪便中的病毒核酸进行了检测。结果显示,本研究建立的RT-LAMP方法特异性高,灵敏度达到1 000拷贝/μLRNA分子水平,与常规检测RT-PCR方法相当。对临床标本的阳性检出率也与常规RT-PCR相当。由于此方法特异性强,灵敏度较高,耗时短,结果直观,因此有望用于GII型诺如病毒的现场快速检测。

A simple,rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification（RT-LAMP） method was established to detect norovirus genotype GII.The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65℃ for 60 minutes.The amplification process of RT-LAMP was monitored by the addition of HNB（Hydroxy naphthol blue） dye prior to amplification.A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis.The specificity of the RT-LAMP was validated by detecting several different diarrhea viruses including norovirus genotype GII.The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection.The assay was further evaluated with 93 clinical specimens of diarrhea patients.The results showed that the sensitivity of RT-LAMP was 1 000 copies/μL with a high specificity and the relative sensitivity was at the same level as that of conventional RT-PCR.Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of conventional RT-PCR as well.This colorimetric RT-LAMP assay was potential for rapid detection of norovirus genotype GII on spot due to the observation of visual result with high specificity and sensitivity,time-saving and cost benefit.