双重RT-PCR方法检测花生条纹病毒和黄瓜花叶病毒  

Detection of PStV and CMV by a Duplex RT-PCR Method

作　　者：谢宏峰[1] 吴菊香[1] 迟玉成[1] 许曼琳[1] 陈蕾[1] 许婷婷[1] 

机构地区：[1]山东省花生研究所,山东青岛266100

出　　处：《花生学报》2012年第1期16-20,共5页Journal of Peanut Science

基　　金：山东省优秀中青年科学家奖励基金项目(BS2009NY040)

摘　　要：为了建立花生条纹病毒(PStV)和黄瓜花叶病毒(CMV)的双重RT-PCR检测方法,根据Gen-Bank中登陆的PStV和CMV的核苷酸保守序列分别设计特异性引物,以感病叶片总RNA反转录物为模板,建立了双重RT-PCR反应体系,并对双重RT-PCR的特异性和灵敏性进行了验证。结果表明:此方法能够特异地从感染PStV和CMV的样品中扩增出PStV(300bp)和CMV(1054bp)2个条带,与实验设计相符;扩增产物测序结果表明,PStV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93%～99%,CMV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90%～99%,证明了检测结果的准确性;该方法特异性良好,灵敏性与单一扩增相同,能够特异、灵敏、准确地检测花生CMV和PStV。The aim of this study is to develop a duplex RT-PCR method which can simultaneously detect Peanut stripe virus （PStV） and Cucumber mosaic virus （CMV）. The reverse transcript of the total RNA extracted from diseased peanut leaves were used as templates for RT-PCR. The specific primers for duplex RT-PCR were designed according to conserved regions of the published sequences of PStV and CMV. The specificity and sensitivity of this duplex RT-PCR assay were tested. A duplex RT-PCR method for the detection of PStV and CMV was successfully established. There were two specific bands amplified in this system, one was 1054 bp refer to CMV, the other one was 300 bp refer to PStV. Sequence analysis of the amplified products showed that there were high homology of PStv subgroup （93%-99%）and CMV （90%-99%）with the sequences of PStV subgroup and CMV in GenBank. The results indicated that this duplex RT-PCR method can simultaneously detect PStV and CMV with high stability and specificity.

关 键 词：花生条纹病毒 黄瓜花叶病毒 双重RT-PCR 

分 类 号：S565.2[农业科学—作物学] S432.1