甘蔗花叶病和宿根矮化病多重PCR检测方法的建立

Development of a Multiplex PCR Protocol for the Detection of SCMV and Clavibacter xyli subsp. Xyli,lxx,the Causal Bacterium of Sugarcane RSD

【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。

[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 China and SCMV-B, D, E, SC strins and the region of 16s-23s ribosomal DNA of Lxx. Based upon the established systems for detecting SrMV/SCMV and Lxx respectively, the multiplex PCR for detecting of the SrMV/SCMV and Lxx was performed by using four specific primers simultaneously. [Result] The results show that all samples infected with SrMV/SCMV and Lxx can be detected simultaneously by the multiplex PCR. There were two specific bands amplified in this system. One is 400 bp refer to SrMV/SCMV, another one is 265 bp refer to Lxx of sugarcane. Sequence analysis of the amplified products showed that there were high homology of SCMV subgroup （81%-99%） and Lxx, （91%-99%）with the sequences of SCMV subgroup and Lxx, in GenBank. [Conclusion] This multiplex PCR protocol can simultaneously detect of SCMV and RSD of sugarcane with high stability and specificity.