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本研究建立了一种用于沙地葡萄茎痘相关病毒(Grapevine rupestris stem pitting-associated virus,GRSPaV)的RT-LAMP检测方法。以GRSPaV的RdRp基因序列(GenBank登录号:GQ478314)为靶序列,设计3组RT-LAMP引物,从中筛选出1组有效引物,并确定了适宜的反应温度和反应时间。对RT-LAMP产物进行HhaⅠ酶切,酶切片段与理论片段大小一致,证明了RT-LAMP产物的特异性。RT-LAMP方法能够检测出GRSPaV的RNA最大稀释倍数为10-4,与RT-PCR方法相比更为灵敏。田间葡萄样品RT-LAMP检测结果与已知样品带毒情况相同,表明RT-LAMP检测GRSPaV具有较好的可靠性。在RT-LAMP反应产物中加入染料SYBR GreenⅠ(×1000)可直接观察反应结果。建立的GRSPaV RT-LAMP检测方法具有简便、快速、灵敏、可视化等特点,尤其适合基层使用,具有良好的应用前景。
In this study, we established a RT-LAMP method for Grapevine rupestris stem pitting-associated virus (GRSPaV). Three RT-LAMP primers were designed based on the RdRp gene sequence of GRSPaV (GenBank accession number: GQ478314). One effective primer was screened out and the suitable reaction temperature and reaction time were determined. The RT-LAMP product was digested with Hha I, and the size of the fragment was the same as that of the theoretical fragment, which proved the specificity of RT-LAMP product. The RT-LAMP method was able to detect the maximum dilution of GRSPaV RNA of 10-4, which is more sensitive than the RT-PCR method. The RT-LAMP test results of field grape samples were the same as those of known samples, indicating that RT-LAMP has good reliability in detecting GRSPaV. The reaction results can be directly observed by adding the dye SYBR Green I (× 1000) to the RT-LAMP reaction product. The established GRSPaV RT-LAMP detection method is simple, rapid, sensitive and visualized. It is especially suitable for primary use and has good application prospect.