Bio-Protection Research Centre, Lincoln University, Lincoln, Canterbury, New Zealand.
J Virol Methods. 2011 Jan;171(1):46-52. doi: 10.1016/j.jviromet.2010.09.028. Epub 2010 Oct 7.
This study investigated the suitability of a two step real-time RT-PCR melting curve analysis as a tool for the detection and discrimination of nine species in the plant virus family Luteoviridae, being Soybean dwarf virus [SbDV], Bean leafroll virus [BLRV], Beet chlorosis virus [BChV], Beet mild yellowing virus [BMYV], Beet western yellows virus [BWYV], Cereal yellow dwarf virus-RPV [CYDV-RPV], Cucurbit aphid-borne yellows virus [CABYV], Potato leafroll virus [PLRV] and Turnip yellows virus [TuYV]. Melting temperature and shape of the melting peak were analysed for 68 bp and 148 bp coat protein gene amplicons using SYBR® GreenER™ fluorescent dye. Specific melting peaks with unique melting temperature were observed for the various species of the family Luteoviridae using the 68 bp amplicon, but not with the 148 bp amplicon. Due to the high variability of sequences for some members of this family, different melting temperatures were also observed between different isolates of the species CYDV-RPV and TuYV. Nevertheless, discrimination between species was achieved for SbDV, BLRV, BChV, BMYV, CABYV and either PLRV or BWYV. Melting curve analysis, in this study, is a faster and more discriminatory alternative to gel electrophoresis of end-point PCR products for the detection of Luteoviridae infection.
本研究探讨了两步实时 RT-PCR 熔解曲线分析作为一种工具，用于检测和区分植物病毒科 Luteoviridae 中的九种病毒，包括大豆矮化病毒（SbDV）、菜豆卷叶病毒（BLRV）、甜菜黄化病毒（BChV）、甜菜黄脉病毒（BMYV）、甜菜西部黄化病毒（BWYV）、小麦黄花叶病毒-RPV（CYDV-RPV）、葫芦科蚜虫传播的黄化病毒（CABYV）、马铃薯卷叶病毒（PLRV）和芜菁黄花叶病毒（TuYV）。使用 SYBR®GreenER™荧光染料，对 68bp 和 148bp 外壳蛋白基因扩增子的熔解温度和熔解峰形状进行了分析。使用 68bp 扩增子观察到了 Luteoviridae 家族各种病毒的特异性熔解峰，且具有独特的熔解温度，但使用 148bp 扩增子则没有。由于该科的一些成员序列高度变异，因此在不同的 CYDV-RPV 和 TuYV 分离株之间也观察到了不同的熔解温度。然而，仍实现了 SbDV、BLRV、BChV、BMYV、CABYV 以及 PLRV 或 BWYV 之间的物种区分。在本研究中，熔解曲线分析是一种比终点 PCR 产物凝胶电泳更快、更具区分度的方法，可用于检测 Luteoviridae 感染。
