半巢式RT-Realtime PCR检测马铃薯Y病毒脉坏死株系  

Dectection of Potato virus Y vein necrosis strain by semi-Nested RT-Realtime PCR.

作　　者：耿金培 粟智平 张京萱 方绍庆 王真 

机构地区：[1]烟台出入境检验检疫局,264000

出　　处：《植物检疫》2009年第3期17-20,共4页Plant Quarantine

基　　金：山东检验检疫局课题:马铃薯重要病毒类病害实时荧光PCR检测技术研究(SK13-2004)

摘　　要：马铃薯Y病毒脉坏死株系(Potato virusY,PVYn)是马铃薯中一个非常重要的病毒病害。本文根据PVYn基因组中RNA依赖的RNA聚合酶基因序列保守区域,设计合成了3条巢式PCR引物和1条TaqMAN荧光探针,建立了半巢式RT-Realtime 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和TaqMAN探针检测技术。第二步半巢式RT-Realtime PCR既是对第一步信号的进一步放大,也是对第一步PCR产物的确认,因此,检测的准确性、灵敏度比巢式PCR、Real time PCR等方法高。实验结果表明,该方法检测灵敏度可达4fg/μL植物总RNA。Potato virus Y vein necrosis strain （PVYn） is one of the most serious desease of the potato. Three nested primers and one TaqMAN probe were designed according to the RNA dependent RNA polymerase （RDRP） gene of the PVY, with which we set up a new method of semi - Nested RT Real time PCR to detect the PVY^n. The E. Z. N. ATM was used to extract the total RNA of the plant. The nest - PCR and the probe - detection technique were conbined maneuverably in this study. The semi - nested RT - Realtime PCR in the second step can both magnify the sigal and validate the result of the first step ,which can provide a more sensitive and specific detection of the PVY^n. The sensitivity of the method can reach 4fg/μL of total plant RNA.

关 键 词：马铃薯Y病毒脉坏死株系(PVYN) 半巢式RT-Realtime PCR 检测 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治] S41-3[农业科学—植物保护]