甘蔗高粱花叶病毒RT-LAMP快速检测方法的建立及评价

Establishment and Evaluation of RT-LAMP for the Rapid Detection of Sorghum Mosaic Virus

为加强SrMV在蔗区的流行动态监控,本研究建立了反转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)方法。实验中针对SrMV外壳蛋白(coat protein,CP)保守序列设计特异性引物,构建了含CP基因的标准质粒用于RT-LAMP反应体系的优化,并采用实时浊度仪和优化的荧光可视化检测方法进行了特异性、敏感性及符合性实验。结果显示,RT-LAMP方法特异性强,可检测到的标准质粒的最低量为2.02 fg,其灵敏度是RT-PCR的100倍,对田间样品的检测结果与RT-PCR相符。建立的RT-LAMP方法特异性强、灵敏性高、操作简便,可用于甘蔗健康种苗中SrMV的检测及蔗区中SrMV流行动态监控。

In order to manage and control the sugarcane disease caused by SrMV,the detection method of reverse transcription loop-mediated isothermal amplification(RT-LAMP)was developed and evaluated.In the experiment,the primers were designed according to the conservative region of coat protein(CP)gene sequences,and the standard vector contained the CP gene sequence was constructed,which was used as template of RT-LAMP reaction to optimized the reaction system.Furthermore,real-time turbidity instrument and visual fluorescence detection assay were both used to evaluate the specificity,sensitivity and consistency of RT-LAMP assay.The results indicated that RT-LAMP has a great specificity,and the lowest detected concentration is 2.02 fg when the standard vector was used as the template,which sensitivity is 100 times higher than that of RT-PCR method.Besides,the RT-LAMP achieved excellent agreement with the RT-PCR for the field detection of SrMV.Taken altogether,our findings indicate that RT-LAMP is a specific,sensitive,and simplemethod,which can be used to detect SrMV in healthy sugarcane seedlings and monitor the prevalence of SrMV in sugarcane field.