巢式-多重RT-Realtime PCR检测马铃薯Y病毒脉坏死株系的方法

Dectection of Potato Virus Y Vein Necrosis By Nested-Multiplex Rt-Realtime PCR

马铃薯Y病毒脉坏死株系(Potato virus Yvein necrosis strain,PVYn)是马铃薯中一个非常重要的病毒病害。本研究根据PVYn基因组中RNA依赖的RNA聚合酶(RNA dependentRNA polymerase,RDRP)基因序列保守区域,设计合成了两对巢式PCR引物和一条TaqMAN荧光探针,建立了巢式-多重RT-Real-time PCR检测PVYn的新方法。该方法采用E.Z.N.ATM快速提取植物总RNA,并有机地结合了巢式PCR、多重PCR和探针检测技术。实验结果表明,本方法检测的准确性、灵敏度比巢式PCR、单重Real time PCR等方法高。该方法检测灵敏度可达3 fg/μL植物总RNA。利用四对引物和一条TaqMAN探针对PCR阳性产物进行确认,检测的准确性比其它方法高。

Potato virus Y vein necrosis（PVYn） is one of the most serious desease of the potato.In this study,we set up a new method of Nest-multiplex-RT Real time PCR for detection of the ArMV.E.Z.N.ATM was used to extract total RNA of the plant.Two-pair of primers and one TaqMAN probe were designed according to the RNA dependent RNA polymerase（RDRP） gene of the PVYn to detect the PVYn.The nested-PCR and multiplex-PCR and probe-detection technique were conbined maneuverably in this study.The method provides a more sensitive and specific detection of PVY^n. The sensitivity of the method can reach 3fg/μL of total plant RNA.