兰花两种主要病毒双重RT-PCR检测方法的建立  

Development of Duplex RT-PCR Protocol for Detection of Two Viruses in Orchid

作　　者：曾燕君[1,2] 莫饶[1] 刘志昕[2] 王健华[2] 

机构地区：[1]海南大学农学院,海南儋州571737 [2]中国热带农业科学院热带生物技术研究所,海南海口571101

出　　处：《江西农业学报》2009年第7期12-14,共3页Acta Agriculturae Jiangxi

基　　金：国家科技支撑课题(2007BAD45B07-02;2007BDA48B01)

摘　　要：根据建兰花叶病毒（Cymbidium mosflic virus，CymMV）和齿兰环斑病毒（Odontoglossumringspotvirus，ORSV）这两种主要兰花病毒的保守序列设计特异性引物，在CymMV和ORSV单一RT～PCR优化体系的基础上。建立了同时检测CymMV和ORSV的双重RT—PCR检测方法。此方法可特异性地从感染CymMV和ORSV的样品中扩增出2条目的带，即CymMV（781bp）和ORSV（588bp）。扩增产物测序表明，CymMV扩增产物与GenBank中登陆的分离物核苷酸同源性为84％～97％，ORSV扩增产物与GenBank中其它分离物的核苷酸序列同源性为98％-99％。运用该方法对随机抽取的田间样品及组培苗样品进行检测，都得到了特异性扩增条带，其灵敏度达到了10^-5。A duplex reverse transcription- polymerase chain reaction （RT- PCR） technique was successfully adopted to detect cymbidium mosaic virus （CymMV） and odontoglossum ring spot virus （ORSV） at the same time. Based upon the establishment of the optimized RT - PCR detection of CymMV and ORSV, the duplex RT - PCR which can simultaneously detect the two viruses in orchid was developed. The results showed that all samples infected by CymMV and ORSV could be amplified simultaneously by duplex RT - PCR, and yield two specific bands of CymMV （781 bp） and ORSV （588 bp） visualized by agarose gel electrophoresis. Sequence analysis of the amplified products showed high homology of CymMV （84% -97% ） and ORSV （98% -99% ） with the sequences of Cym-MV and ORSV in GenBank. The random samples collected from field and tissue culture were researched and amplified specifically by this method and its sensitivity was up to 10^-5 .

关 键 词：双重RT-PCR 建兰花叶病毒 齿兰环斑病毒 检测 

分 类 号：S436.8[农业科学—农业昆虫与害虫防治]