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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。
In this study, we established a triplex PCR system for the simultaneous detection of pathogen of cucumber major diseases such as Colletotrichum orbiculare, Sclerotinia sclerotiorum (Lib.) De Bary and cucumber bacterial wilt disease (Erwinia tracheiphila) . The orthogonal test design was used to analyze the influence factors of triplex PCR. The annealing temperature was optimized and the multiplex PCR system was optimized by three primer sets, Taq DNA polymerase, dNTP and Mg2 +. The multiplex PCR system was successfully established The optimal triple-PCR detection system for cucumber diseases was 25μL containing 0.24μmol·L-1CY1 / CY2, 0.72μmol·L-1SSFWD / SSREV, 0.336μmol·L-1ET-P1 / ET- 1 U Taq polymerase; 0.15 mmol·L-1dNTP; 1 mmol·L-1 MgCl2. The optimum annealing temperature was 63 ℃. The method can rapidly detect cucumber anthracnose, cucumber sclerotium and cucumber bacterial wilt disease from field cucumber plants and rhizosphere soil, and the sensitivity can reach 10 pg · μL-1.