杂交诱捕实时荧光PCR检测番茄环斑病毒.pdfVIP

植物病理学报 ACTA PHYTOPATHOLOGICA SINICA 37(6)：666-669(2007) 杂交诱捕实时荧光PCR检测番茄环斑病毒 赵文军 一，陈红运 ，朱水芳 ，谭天伟 ( 中国检验检疫科学研究院，北京100029； 北京化工大学，北京100029) Tomato ringspot nepovirus detection using hybridization capture real—time PCR zHAO Wen-jun ，CHEN Hong—yun ，ZHU Shui—fang ，TAN Tian—wei ( Chinese Academy of Inspection and Quarantine，Beijing 100029，China； Beijing University of Chemical Technology，Beijing 100029，China) Abstract： A new virus detection method was developed by combining hybridization capture and real—time PCR technology with the sample of Tomato ringspot nepovirus．High purified DNA or RNA was crucial for the rea1．-time PCR detection as the inhibitors of PC R reaction in template and might cause the false．．negative ca- ses．A probe was covalent bound to the surface of PC R tube for the DNA Capture followed by real—time PCR detection．The steps of nucleic acids preparation and detection were both done in one the same tube．ThiS method reduced the possibility of contamination as well increased the detection efficiency．It Can be applied in plant virus detection especially for the sam ple with many inhibitors of PCR reaction． Key words：hybridization capture；real—time PCR；Tomato ringspot nepovirus；detection 文章编号：04124)914(2007)06-0666-04 番茄环斑病毒(Tomato ringspot nepovirus， 实时荧光PCR技术因结果准确和不需要电泳 ToRSV)广泛分布于欧美及大洋洲，能侵染葡萄、 等优点得到了广泛关注，也成为最近几年研究的热 桃、苹果、樱桃、番茄及烟草等150多种植物，引起 点 J。但它对核酸纯度要求更严格，含有聚合酶 多种病害，严重时导致绝产ll J。ToRSV被我国列 抑制物质的核酸很容易导致检测结果的假阴性，这 为对外检疫性有害生物，实施严格检疫。传统的 限制了该技术在某些病害鉴定中的应用。本研究 PCR电泳技术是最经典的分子检测方法，但存在 利用共价结合于PCR管壁的寡核苷酸定向诱捕核 如下3个方面问题：1)样品前处理复杂；对于某些 酸粗提液中的目标核酸，去除杂质及聚合酶抑制物 植物材料，尤其是果树苗木，含有大量酚类及单宁 质，然后在同一管内利用实时荧光PCR进行检测， 等物质，难以获得高纯度核酸，有时甚至影响PCR 有效避免了PCR的漏检现象，并降低污染可能，提 成败；2)传统PCR扩增后需要电泳检验，操作繁 高了检测效率。 琐，需接触