猕猴桃溃疡病菌实时荧光定量PCR检测方法的建立和应用谢锦添俞.docVIP

猕猴桃溃疡病菌实时荧光定量PCR检测方法的建立和应用 谢锦添，俞超*，谭志文，吴月燕，马立孟 （浙江万里学院生物与环境学院，浙江 宁波 315100） 摘 要 为建立猕猴桃溃疡病PSA病原菌的实时荧光定量PCR方法，根据hrpW基因设计一对引物P3F/P5R，扩增片段大小为247bp，构建该片段的阳性质粒用于荧光定量 PCR标准曲线的制作；评价引物的灵敏度、特异性，并验证其实际应用效果。结果表明，该方法能特异性的检测出PSA病原菌，灵敏度为 100fg/μL。以采集的无病症和有病症猕猴桃枝条为样品，本方法可检测到无病症枝条中最低浓度为8.45×10‐5ng/μL的PSA病原菌。本实验建立的实时荧光定量PCR检测猕猴桃溃疡病菌方法可用于猕猴桃溃疡病感病样品的早期诊断，对该病原菌的预防诊治具有重要意义。 关键词 猕猴桃溃疡病菌；实时荧光定量PCR；检测方法；应用 Establishment and application of real－time PCR method for detection of Pseudomonas syringae pv. Actinidae Xie Jingtian，Yu Chao*，Tan Zhiwen，Wu Yueyan，Ma Limeng (College of Biological Environmental Sciences，Zhejiang Wanli University，Ningbo 315100， China) Abstract To establish a real-time PCR method of pathogenic bacteria （Pseudomonas．Syringae pv．Actinidae，PSA）detection , the pair of primers P3F/P5R were screened out according to hrpW gene sequence, with expected amplicons of 247bp in length. The recombinant plasmid of this amplification were used to build a standard curve real-time PCR . The sensitivity and specificity of these primers were analyzed and its real effect was validated. The results of sensitivity assays showed the sensitivity threshold of the real-time PCR was 100fg/μL. The results of actual effect validation experiments showed part of symptomless samples were positively detected by real-time PCR, with minimum DNA concentration 8.45×10‐5ng/μL. The methods in this study could be expected to greatly contribute to the early diagnosis and control of the bacterial canker disease on kiwifruit. Key words Pseudomonas syringae pv. Actinidae；real‐time PCR；detection method；application : 基金项目：浙江省分析测试科技计划项目（2015C37046）；宁波市农业攻关项目（2014C11005）；浙江省生物工程重中之重学科自设课题（ZS2016004） 作者简介：谢锦添，本科生，专业：生物工程。E-mail: 1031460871 @ *通信作者： 俞超，高级实验师，主要从事植物病害相关研究。 E‐mail: yuchao1997@163.com  猕猴桃(Actinidia chinensis)是一种经济价值很高的水果，国内外均有大量种植。然而，近年来随着猕猴桃栽培面积不断增加，细菌性溃疡病快速蔓延，自 2005 年以来，已在世界主要猕猴桃产区造成严重危害，成为猕猴桃栽培过程中最具毁灭性的病害，严重威胁世界猕猴桃的生产安全[1-2]。 2009－2012年，根据生物学特性、16S和16S-23S(ITS)rDNA序列比对分析，新西兰、法国、西班牙、中国等国家均将猕猴桃溃疡病的