Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias-IVIA, Carretera Moncada a Náquera km 5, 46113 Moncada, Valencia, Spain.
J Virol Methods. 2013 Mar;188(1-2):21-4. doi: 10.1016/j.jviromet.2012.11.034. Epub 2012 Dec 5.
A real-time multiplex RT-PCR has been developed for the simultaneous detection and identification of the major RNA viruses that infect grapevines (Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3 and Grapevine fleck virus). Serial dilutions of infected plant extracts were tested using the new method, and the results were compared with those obtained using a commercially available ELISA and real-time singleplex RT-PCR. The two real-time RT-PCR versions detected up to the same level of dilution and were at least 10,000 times more sensitive than the ELISA. In addition, 158 grapevine plants collected in a survey of the Protected Designation of Origin in Alicante, Spain were compared using the three methods. The results of the molecular methods were very similar, with only four discordant results, and both were able to detect many more infected plants than the ELISA. The high prevalence of Grapevine fleck virus, Grapevine leafroll-associated virus 3 and Grapevine fanleaf virus suggests that the main pathways of viral introduction are infected plant material that has escaped controls and/or uncontrolled traffic of propagating plant material. Real-time multiplex RT-PCR could be used to facilitate a better control of grapevine viruses.
一种实时多重 RT-PCR 方法已被开发出来，用于同时检测和鉴定感染葡萄的主要 RNA 病毒（葡萄扇叶病毒、阿拉伯 mosaic 病毒、葡萄卷叶伴随病毒 1、葡萄卷叶伴随病毒 3 和葡萄斑点病毒）。使用新方法对感染植物提取物的连续稀释液进行了测试，并将结果与商业上可用的 ELISA 和实时单重 RT-PCR 进行了比较。两种实时 RT-PCR 版本的检测稀释度相同，灵敏度至少比 ELISA 高 10,000 倍。此外，使用三种方法对西班牙阿利坎特原产地保护调查中收集的 158 株葡萄植物进行了比较。分子方法的结果非常相似，只有四个不一致的结果，而且这两种方法都比 ELISA 检测到了更多的感染植物。葡萄斑点病毒、葡萄卷叶伴随病毒 3 和葡萄扇叶病毒的高流行率表明，病毒传入的主要途径是逃脱控制的感染植物材料和/或繁殖植物材料的不受控制的运输。实时多重 RT-PCR 可用于促进葡萄病毒的更好控制。
