沙地葡萄茎痘相关病毒的RT-LAMP检测方法

 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Hha Ⅰ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 Green Ⅰ （×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。

RT-LAMP assay for detection of Grapevine rupestris stem pitting-associated virus

The RT-LAMP assay was established to detect Grapevine rupestris stem pitting-associated virus（GRSPaV）. Three sets of specific primers were designed based on GRSPaV RdRp gene（GenBank: GQ478314）for RT-LAMP assay. One feasible set of primers was selected to establish the suitable temperature and times for the RT-LAMP reaction. The products of RT-LAMP were cut by restriction enzyme Hha Ⅰ， and the obtained fragments had identical sizes according to RT-LAMP theory. The maximal diluted limit of RNA detected by RT-LAMP was 10^-4, so the RT-LAMP assay has more sensitivity than the RT-PCR assay. Consistent results for GRSPaV detection of grapevine samples from the field were obtained by the RT-LAMP and RT-PCR assay, suggesting the reliability of the RT-LAMP in GRSPaV detection. Furthermore, Detection results were also observed by naked-eyes after adding SYBR Green Ⅰ (×1 000) into RT-LAMP products and omitted electrophoresis． The RT- LAMP technique appeared characteristic of simple, quick，sensitivity and visual. This method is fit for rapid diagnosis to viruses, especially in the laboratory which lacking of special equipments, and showing good deve-lopment prospects．