双重PCR检测马铃薯晚疫病和环腐病方法的建立

Development of duplex PCR assay for detection of Phytophthora infestans and Clavibacter michiganensis subsp.sepedonicum

通过克隆马铃薯环腐病菌和晚疫病菌转录间隔区(ITS)序列,并对测序结果进行同源性比较,选取差异位点分别设计了两对引物P.IN1/P.IN2和C.IN1/C.IN2,并检测了引物的特异性及方法的灵敏度。引物P.IN1/P.IN2可扩增出1条363bp马铃薯晚疫病菌的特异性条带,在DNA水平上其灵敏度达18fg/μL;引物C.IN1/C.IN2可扩增出1条218bp马铃薯环腐病菌的特异性条带,在细菌数上检测灵敏度为104 cfu/mL。混合这两对引物构建双重PCR反应体系,能从马铃薯环腐病菌和晚疫病菌的混合DNA及感染这两种菌的马铃薯植株中同时扩增到363bp和218bp的特异片段。实现了同时对马铃薯晚疫病菌和环腐病菌的快速可靠检测。

Based on cloning and analysis of the internal transcribed spacer（ITS）sequences of Phytophthora infestans and Clavibacter michiganensis subsp.sepedonicum,two specific pairs of primer,P.IN1/P.IN2 and C.IN1/C.IN2,were designed to establish a duplex PCR system,with the optimized PCR parameters to ensure the sensitivity and specificity.The system could simultaneously detect the two pathogens of potato.Using P.IN1/P.IN2,a single unique PCR band of 363 bp was amplified fromP.infestans and the sensitivity was 18 fg/μL of DNA.Using C.IN1/C.IN2,a single unique PCR band of 218 bp was amplified fromC.michiganensis subsp.sepedonicum and the sensitivity was 104cfu/mL of bacteria.With the duplex PCR system,the 363 bp and 218 bp PCR bands from P.infestans and C.michiganensis subsp.sepedonicum could be specifically amplified.So a duplex PCR system has been developed for simultaneously and rapidly detecting P.infestans and C.michiganensis subsp.sepedonicum.