Herath Padmini, Hoover Gregory A, Angelini Elisa, Moorman Gary W
Department of Plant Pathology.
Department of Entomology, The Pennsylvania State University, University Park 16802.
Plant Dis. 2010 Nov;94(11):1355-1360. doi: 10.1094/PDIS-12-09-0783.
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 trnL 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) 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.
基于TaqMan小沟结合探针的实时聚合酶链反应（PCR）程序，开发了一种快速准确地检测榆树和昆虫样本中榆树黄化病（EY）常见植原体菌株的方法。引物和探针是根据EY植原体特异性转运蛋白secY基因的DNA序列设计的。通过扩增美国榆的叶绿体trnL基因来评估DNA提取程序的成功率。实时PCR检测与EY和欧洲出现的EY植原体菌株ULW DNA呈阳性反应。它与伊利诺伊州EY或紫菀黄化病植原体DNA均无交叉反应，这两种植原体在美国的榆树中均有发现，它也没有扩增属于16SrV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帕克校区的30棵EY阳性榆树。从感染EY植原体的榆树获得的阈值循环（C）值范围为15至37。在几种叶蝉类群中检测到了EY植原体。这种实时PCR方法可用于榆树的诊断筛查以及鉴定EY植原体可能的昆虫传播媒介。
