蔬菜菌核病两种病原菌双重PCR检测方法的建立

Development of duplex PCR assay for the detection of two pathogens causing Sclerotinia rot on vegetables

核盘菌(Sclerotinia sclerotiorum)和小核盘菌(Sclerotinia minor)是引起蔬菜菌核病的两种主要病原菌。为了建立针对这两种病原菌的快速分子检测体系,本研究基于漆酶2编码基因(laccase 2)分别设计了针对S.sclerotiorum和S.minor的特异性引物对Sslcc2-F/R和Smlcc2-F/R,在此基础上建立了双重PCR检测体系。通过对该体系的灵敏度以及蔬菜菌核病模拟侵染样品进行检测,证实了双重PCR技术的可行性。研究结果表明:能通过扩增片段的大小区分S.sclerotiorum和S.minor,引物Sslcc2-F/R可扩增出大小为410 bp的S.sclerotiorum特异性条带,其检测灵敏度可达3.11×10^(-2) ng·μL^(-1);引物Smlcc2-F/R可扩增出大小为609 bp的S.minor特异性条带,其检测灵敏度为3.42×10^(-2) ng·μL^(-1)。在混合两对引物的体系中,检测灵敏度为3.0×10^(-1) ng·μL^(-1)。本研究建立的双重PCR体系能同时对引起蔬菜菌核病的两种主要病原菌进行准确、快速和灵敏的检测。

Sclerotinia sclerotiorum and Sclerotinia minor are the main pathogens causing Sclerotinia rot on vegetables.To establish a rapid and sensitive method for the detection of these two pathogens,Sslcc2-F/R and Smlcc2-F/R,S.sclerotiorum-and S.minor-specific primers,respectively,were designed based on the sequences of laccase 2 and used for duplex PCR assay in this study.The results of specificity and sensitivity tests showed that a 410 bp target band for S.sclerotiorum and a 609 bp target band for S.minor were amplified using the duplex PCR system we developed,with the sensitivities of 3.11×10^(-2) ng·μL^(-1) and 3.42×10^(-2) ng·μL^(-1),respectively.When mixed genomic DNA samples of S.sclerotiorum and S.minor were used as the template,both 410 bp and 609 bp bands could be amplified,with the sensitivity of 3.0×10^(-1) ng·μL^(-1).This duplex PCR system can also be used to detect S.sclerotiorum,S.minor or both the pathogens in one PCR reaction from diseased plant samples.In conclusion,the duplex PCR system we developed here provides an accurate and sensitive approach to simultaneously detect the two Sclerotinia species.