三种甘薯病毒多重RT-PCR检测技术的建立

Establishment of multiplex RT-PCR system for detection of three viruses in sweet potato

本文根据GenBank中甘薯G病毒(SPVG)、甘薯卷叶病毒(SPLCV)和甘薯羽状斑驳病毒(SPFMV)外壳蛋白(CP)基因序列设计特异引物,对多重RT-PCR退火温度、延伸温度、模板浓度、引物浓度进行改良优化,建立能同时检测3种甘薯病毒的多重RT-PCR方法。该方法能同时扩增出SPVG、SPLCV和SPFMV特异片段,其大小分别是800、276和570bp。测序结果表明,扩增出的3种病毒序列与相应参考序列相似性达到98%以上。灵敏度分析结果表明,多重RT-PCR方法能够检测cDNA的量为0.1ng。应用建立的多重RT-PCR检测方法对田间样品进行检测,结果显示该方法可以特异、快速、灵敏地同时检测3种甘薯病毒。这些研究结果可为甘薯病毒检测提供参考。

Three pairs of specific primers for Sweet potato virus G,Sweet potato leaf curl virus and Sweet potato feathery mottle virus were designed based on the conserved region sequences of the coat protein（CP） gene pub- lished in GenBank. The crucial factors of multiplex RT-PCR including annealing temperature, extension tempera- ture, template concentration and primers concentration were optimized and a multiplex RT-PCR detection method for three sweet potato viruses was developed. Three specific fragments could be simultaneously amplified in uni- plex PCR reaction, with the lengths of 800, 276 and 570 bp, respectively. Sequence analysis indicated that the fragments of three viruses shared at least 98% homology with those of the other related viruses. Sensitivity analy- sis demonstrated that cDNA of 0.1 ng could be detected by multiplex RT-PCR. This multiplex RT-PCR protocol can simultaneously detect three sweet potato viruses from field samples with high specificity, rapidity and sensitivity.