大豆南方茎溃疡病菌PCR检测方法的建立  

Development of PCR methods for detection of Diaporthe aspalathi

作　　者：许忠祥 杨静 刘晓宇 钱茱希 许剑涛 李彬 吴翠萍 王振华 吴品珊[3] Xu Zhongxiang;Yang Jing;Liu Xiaoyu;Qian Zhuxi;Xu Jiantao;Li Bin;Wu Cuiping;Wang Zhenhua;Wu Pinshan(Animal,Plant and Food Inspection Center,Nanjing Customs District,Nanjing 210019,China;Technology Center of Wuhan Customs District;Chinese Academy of Inspection and Quarantine)

机构地区：[1]南京海关动植物与食品检测中心,江苏南京210019 [2]武汉海关技术中心 [3]中国检验检疫科学研究院

出　　处：《植物检疫》2022年第5期34-40,共7页Plant Quarantine

基　　金：2020年海关技术规范制(修)订计划(2020B088)。

摘　　要：大豆南方茎溃疡病是一种世界上危害严重的大豆病害,其病原菌(Diaporthe aspalathi)被列入我国进境植物检疫性有害生物名录。本文基于ITS序列差异设计引物和探针,建立了D. aspalathi常规PCR和荧光PCR检测方法。测试结果表明,常规PCR引物DM-F/DM-R3扩增33个供试菌株,6个D. aspalathi菌株出现394 bp的预期扩增条带,其余供试菌株均无目的条带,检测灵敏度为5 pg菌丝体DNA。探针DM-Pro仅对6个D. aspalathi菌株表现为阳性,其余供试菌株均为阴性,检测灵敏度为500 fg菌丝体DNA。同时,采用这两种方法对进境大豆种子及植株残体等样品进行了验证。以上结果表明,这两种检测方法具有快速、准确、灵敏等优点,可应用于口岸对大豆南方茎溃疡病菌的检疫鉴定及疫情监测。Southern stem canker is an important soybean disease that has been responsible for severe losses in the world. The pathogen Diaporthe aspalathi was listed in catalogue of quarantine pests for import plants to China. Based on the difference of ITS sequences,the primers and probe were designed and the methods of conventional PCR and real-time PCR of D. aspalathi detection were developed respectively.The test results showed that the primer pair DM-F/DM-R3 of conventional PCR could amplify 394 bp expected target band of 6 D. aspalathi strains among 33 tested strains,while the other strains showed no target PCR product,and the sensitivity of the method reached to 5 pg mycelium DNA amounts. The real-time PCR probe DM-Pro result of the 6 D. aspalathi strains appeared positive,while others were negative,and its sensitivity could get to 500 fg mycelium DNA amounts. Meanwhile the two PCR methods were verified by tests of imported soybean seed and plant debris samples. On the above results,the two fast,accurate and sensitive PCR methods showed good potential applications on both quarantine identification and monitoring of Diaporthe aspalathi.

关 键 词：大豆南方茎溃疡病菌 常规PCR 实时荧光PCR 

分 类 号：S41-32[农业科学—植物保护] S435.65