蔬菜中黄瓜花叶病毒的RT-LAMP快速检测

Development of a RT-LAMP Method for the Rapid Detection of Cucumber mosaic virus from Vegetables

为了建立黄瓜花叶病毒(Cucumber mosaic virus,CMV)可视化的反转录环介导等温扩增(Reverse transcription loop-mediated isothermal amplification,RT-LAMP)检测体系,根据CMV的外壳蛋白(coat protein,CP)基因核苷酸序列设计4条引物,优化了RT-LAMP反应体系中的d NTPs和Mg^(2+)浓度、反应温度和反应时间,同时对体系进行了特异性和灵敏性的测定。最终确定RT-LAMP的最优反应体系为d NTPs 1.2 mmol·L^(-1),Mg^(2+)6 mmol·L^(-1),在61℃的条件下反应40 min。该RT-LAMP体系比RT-PCR反应体系的灵敏度高100倍,并且反应产物可以通过将反应管快速离心后观察底部沉淀或通过加入SYBR GreenⅠ后的显色反应进行结果判定。该RT-LAMP检测体系可以对多种蔬菜的CMV进行高效快速的检测,操作简便,花费较小,十分适合生产上检测CMV使用。

In order to develop a reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay for rapid and sensitive detection of Cucumber mosaic virus（CMV）from different kind of vegetables,four primers were designed according to the coat protein（CP）gene of CMV. The reaction conditions of RT-LAMP,including the concentrations of d NTPs,Mg^2＋,reaction temperature and reaction time were optimized. The specificity and sensitivity of RT-LAMP were testified. The optimum conditions for RT-LAMP can carried out under the concentrations of 1.2 mmol · L-1 d NTPs and 6 mmol · L-1Mg^2＋. RT-LAMP reaction was carried out at 61 ℃ for 40 min. The sensitivity of the RT-LAMP assay was 100-folder than that of a standard RT-PCR method. Furthermore,the products amplified by RT-LAMP could be visibly detected by the precipitate of the tube after fast centrifugation or by reacting with SYBR GreenⅠ. The RT-LAMP is highly specific,sensitive and economical,which makes it a quick method for detecting CMV from several kinds of infected vegetables and a useful technique for CMV detection in field condition.