Detection of Elm Yellows Phytoplasma in Elms and Insects Using Real-Time PCR
Detection of Elm Yellows Phytoplasma in Elms and Insects Using Real-Time PCR
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DOI：
10.1094/pdis-12-09-0783
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发表时间：
2010-11-01
期刊：
影响因子：
4.5
通讯作者：
Moorman, Gary W.
中科院分区：
文献类型：
--
作者：
Herath, Padmini;Hoover, Gregory A.;Moorman, Gary W.
建立了一种基于TaqMan微沟结合探针的榆树黄化植原体（EY）实时荧光定量PCR检测方法。根据EY植原体特异性易位蛋白secY基因DNA序列设计引物和探针。通过扩增叶绿体TRILL来评价DNA提取过程的成功。美洲榆（Ulmus americana）基因。实时PCR检测与EY和EY植原体株ULW DNA（一种发生在欧洲的分离物）反应呈阳性。它没有与伊利诺伊州EY或紫菀黄化植原体DNA发生交叉反应，这两种植原体DNA都已知发生在美国的榆树中，它也没有扩增属于16 SrV和其他系统发育组的其他几种植原体。实时PCR方案用于鉴定宾夕法尼亚州立大学，大学公园校园的30棵EY阳性榆树。从EY植原体感染榆树获得的阈值循环（C-T）值范围从15到37。EY植原体在几个叶蝉类群中检测到。该荧光定量PCR方法可用于榆树的诊断筛选和EY植原体可能的昆虫媒介的鉴定。
A rapid and accurate method to detect the common strain of elm yellows (EY) phytoplasma in elm and insect samples was developed using a real-time polymerase chain reaction (PCR) procedure based on the TaqMan minor-groove-binder probe. Primers and probe were designed based on the EY phytoplasma-specific translocation protein secY gene DNA sequence. Success of the DNA extraction procedure was evaluated by amplifying the chloroplast trill. gene of Ulmus americana. The real-time PCR assay reacted positively with EY and EY phytoplasma strain ULW DNA, an isolate which occurs in Europe. It did not cross-react with Illinois EY or aster yellows phytoplasma DNA, both of which are known to occur in elm trees in the United States, nor did it amplify several other phytoplasmas belonging to the 16SrV and other phylogenetic groups. The real-time PCR protocol was used to identify 30 EY-positive elm trees on The Pennsylvania State University, University Park campus. Threshold cycle (C-T) values obtained from the EY phytoplasma-infected elm trees ranged from 15 to 37. EY phytoplasma was detected in several leafhopper taxa. This real-time PCR method can be used for the diagnostic screening of elm trees and for the identification of possible insect vectors of EY phytoplasma.