三重PCR检测黄瓜炭疽病菌、菌核病菌和细菌性萎蔫病菌

Triplex PCR detection of Colletotrichum orbiculare,Sclerotinia sclerotiorum and Erwinia tracheiphila in infected cucumber tissues

本试验建立一种可同时检测黄瓜炭疽病(Colletotrichum orbiculare)、黄瓜菌核病(Sclerotinia sclerotiorum(Lib.)de Bary)和黄瓜细菌性萎蔫病(Erwinia tracheiphila)等黄瓜主要病害病原菌的三重PCR检测体系。采用正交试验设计方法,对三重PCR的影响因素分析研究,进行退火温度优化,并以3个引物组、Taq DNA聚合酶、dNTP和Mg2+共6因素3水平进行多重PCR体系优化,成功建立了适合黄瓜主要病害的三重PCR最佳检测体系,即25μL的反应体系中含有0.24μmol·L-1CY1/CY2;0.72μmol·L-1SSFWD/SSREV;0.336μmol·L-1ET-P1/ET-P2;1 U Taq聚合酶;0.15 mmol·L-1dNTP;1 mmol·L-1MgCl2,最适退火温度为63℃。该方法能够快速从田间黄瓜发病植株和根围土壤中将黄瓜炭疽病菌、黄瓜菌核病菌和黄瓜细菌性萎蔫病菌检测出来,灵敏度可以达到10 pg·μL-1。

A multiplex PCR-based method was designed for accurate and rapid identification of Colletotrichum orbiculare, Sclerotinia sclerotiorum （Lib.） de Bary and Erwinia tracheiphila simultaneously in the early stages of the diseases. Three-level designs for six factors （three primers, Taq DNA polymerase, dNTP and Mg2＋） were constructed to optimize the multiplex PCR system by using the orthogonal design method. The annealing temperature of the PCR reactions was also optimized. A triplex PCR system for simultaneous detection of these three pathogens of cucumber was successfully established. The reaction volume was 25 μL and the annealing temperature was 63oC. The reaction solution contained 0.24 μmol·L-1 CY1/CY2, 0.72 μmol·L-1 SSFWD/SSREV, 0.336 μmol·L-1 ET-P1/ ET-P2, 1 U Taq DNA polymerase, 0.15 mmol·L-1 dNTP and 1 mmol·L-1 Mg2＋. The triplex PCR system could detect C. orbiculare, S. sclerotiorum and E. tracheiphila in infected plant samples and soils rapidly with the sensitivity at 10 pg DNA·μL-1.