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针对不同产地栽培的太子参(Pseudostellaria heterophylla)中主要存在芜菁花叶病毒(Turnip mosaic virus,Tu MV)和蚕豆萎蔫病毒(Broad bean wilt virus,BBWV)侵染且为害严重的情况,建立能同时检测这2种病毒的双重RT-PCR快速检测方法。根据Gen Bank数据库中的Tu MV、BBWV外壳蛋白(coat protein,CP)基因核苷酸序列的保守区域分别设计简并引物,在单一RT-PCR检测体系的基础上,建立和优化双重RT-PCR检测体系。双重RT-PCR的优化结果显示,最佳扩增循环数为35,最佳引物浓度为0.4μmol·L~(-1),最佳退火温度为51℃。其灵敏度测定结果显示,2种病毒在样品c DNA稀释至原液的10-4倍后仍能扩增出特异条带。应用该方法对7份栽培太子参样品和4份脱毒苗样品进行了检测,结果表明,建立的双重RT-PCR检测方法可稳定、准确、灵敏地同时检测Tu MV和BBWV。
In the case of Pseudostellaria heterophylla, which mainly infects Turnip mosaic virus (Tu MV) and Broad bean wilt virus (BBWV) and is severely damaged, Double RT-PCR rapid detection method for detecting these two viruses. Degenerate primers were designed according to the conserved regions of Tu MV and BBWV coat protein (CP) genes in Gen Bank database. Based on the single RT-PCR detection system, double RT-PCR Detection system. The optimized results of double RT-PCR showed that the optimal number of amplification cycles was 35, the optimal primer concentration was 0.4μmol·L -1, and the optimal annealing temperature was 51 ℃. The results of the sensitivity test showed that the two viruses could still amplify specific bands after the DNA of the two samples was diluted to 10-4 times of the original solution. The results showed that the established method of double RT-PCR could detect Tu MV and BBWV stably, accurately and sensitively with the method of 7 samples of Pseudostellaria heterophylla and 4 samples of virus-free seedlings.