甘蔗花叶病和宿根矮化病多重PCR检测方法的建立.pdfVIP

中国农业科学 2008,41(12):4321-4327 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2008.12.051 甘蔗花叶病和宿根矮化病多重PCR检测方法的建立 张显勇，蔡文伟，杨本鹏，张树珍，王业桥 （中国热带农业科学院热带生物技术研究所，海口 571101 ） 摘要：【目的】建立能同时检测甘蔗花叶病（sugarcane mosaic virus，SCMV）和宿根矮化病（ratoon stunting disease，RSD）的多重PCR 检测方法。【方法】以病株带鞘叶组织总核酸反转录物为模板，根据中国甘蔗花叶病的 主要致病病原高梁花叶病毒SrMV和甘蔗花叶病毒中国大陆优势株系SCMV-A，结合B、D、E和SC株系的外壳蛋白 核苷酸序列的保守区设计 PCR 引物对和已有的检测甘蔗宿根矮化病病原木质部赖氏杆菌木质部亚种（Leifsonia xyli subsp. Xyli ，Lxx）的PCR引物对，在SCMV RT-PCR和 RSD单一PCR体系的基础上，建立并优化可同时检测 SCMV和RSD的多重PCR检测方法。【结果】此检测方法可特异地从感染SCMV和RSD的样品中扩增出SCMV（400 bp） 和 RSD（265 bp）2 个条带，扩增产物测序结果表明，SCMV 扩增产物与 GenBank 中其它株系或分离物的核苷酸序 列同源性为 81%～99%，RSD 扩增产物与 GenBank 中其它株系或分离物的核苷酸序列同源性为 91%～99%。【结论】 应用此检测方法可稳定、特异地检测出蔗株中是否有导致甘蔗花叶病和宿根矮化病的单一或混合的病原。 关键词：甘蔗花叶病毒；甘蔗宿根矮化病；RT-PCR；病原检测；多重PCR Development of a Multiplex PCR Protocol for the Detection of SCMV and Clavibacter xyli subsp. Xyli, lxx, the Causal Bacterium of Sugarcane RSD ZHANG Xian-yong, CAI Wen-wei, YANG Ben-peng, ZHANG Shu-zhen, WANG Ye-qiao (Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (CATAS), Haikou 571101) Abstract: 【Objective 】 The aim of this study is to develop a multiplex PCR protocol which can simultaneously detect of Sugarcane mosaic virus (SCMV) and Clavibacter xyli subsp. Xyli , lxx, the causal bacterium of sugarcane ratoon stunting disease (RSD). 【Method 】The reverse transcripts of the total nucleic acid extracted from diseased sugarcane leaves with sheath was used as templates for PCR. The specific primers for detecting of the SCMV and RSD of the sugarcane were designed according to conserved regions of the coat protein gene of the dominating pathogeny SrMV-H and dominant strain SCMV-A in C