地黄花叶病毒RT-LAMP快速检测方法的建立及应用

Establishment and application of RT-LAMP method for rapid detection of Rehmannia mosaic virus

基于反转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)技术建立了针对地黄花叶病毒(Rehmannia mosaic virus,ReMV)的快速、特异、灵敏的RT-LAMP检测方法。根据ReMV外壳蛋白(coat protein,CP)基因的核苷酸序列设计了3组LAMP引物,通过引物筛选确定最佳引物组合;利用电泳检测及可视化方法(加SYBR GreenⅠ显色剂)进行RT-LAMP温度优化、特异性检测、灵敏度比较。结果表明:本研究建立的RT-LAMP方法最适检测温度为60℃,优化后的LAMP方法能特异性检测ReMV,灵敏度是常规PCR的1000倍,可检测到1.2×10^(-1)拷贝/μL的模板浓度。对60份地黄样品的检测结果表明,RT-LAMP方法的阳性检出率为98.3%,高于常规RT-PCR方法(75%)。本研究建立的RT-LAMP检测技术为ReMV的准确检测提供了快速、高效的方法。

Based on reverse transcription loop-mediated isothermal amplification(RT-LAMP)technology,a rapid,specific and sensitive RT-LAMP detection method for Rehmannia mosaic virus(ReMV)was established.Three sets of LAMP primers were designed according to the nucleotide sequence of ReMV coat protein(CP),and the optimized primers were determined by primer screening.Temperature optimization,specificity detection and sensitivity comparison of RT-LAMP were carried out by using electrophoresis detection and visual(SYBR Green I chromogenic agent).The results showed that the optimum detection temperature of RT-LAMP method was 60℃.ReMV could be specifically detected by the optimized LAMP,and the sensitivity was 1000 times higher than that of conventional PCR.It could be detected in 1.2×10^(-1) copies/μL.The detection results of 60 samples of Rehmannia glutinosa showed that the positive detection rate of RT-LAMP was 98.3%,which was higher than that of conventional RT-PCR(75%).The RT-LAMP detection technology established in this study provides a fast and efficient method for the accurate detection of ReMV.