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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 establish a visualization system of cucumber mosaic virus (CMV) reverse transcription loop-mediated isothermal amplification (RT-LAMP), according to the CMV coat protein (CP ) Nucleotide sequence of the gene was designed to optimize the concentration of dNTPs and Mg 2+ in the RT-LAMP reaction system, the reaction temperature and the reaction time, and the specificity and sensitivity of the system were determined. The optimal reaction system for final determination of RT-LAMP was 1.2 mmol·L -1 dNTPs and 6 mmol·L -1 Mg 2+, and the reaction was carried out at 61 ° C for 40 min. The RT-LAMP system is 100 times more sensitive than the RT-PCR reaction system, and the reaction product can be determined by observing the bottom sediment by rapid centrifugation of the reaction tube or by the color reaction after the addition of SYBR Green I. The RT-LAMP detection system for a variety of vegetables, CMV efficient and rapid detection, easy to operate, less costly, very suitable for the production of CMV testing.