辣椒三种病毒的多重RT-PCR同步检测

Multiplex RT-PCR Detection of Three Pepper Viruses

本研究根据辣椒轻斑驳病毒(PMMo V)、烟草花叶病毒(TMV)和黄瓜花叶病毒(CMV)的外壳蛋白(CP)编码序列设计了3对PCR检测引物,并初步建立了同步检测以上3种辣椒病毒的多重RT-PCR方法。以被感染了3种病毒的叶片的总RNA作为模板,可同时扩增到324 bp(TMV)、220 bp(PMMo V)和112 bp(CMV)的3个目标条带,在电泳图上清晰可辨。测序结果表明所扩增产物确为待检病毒靶序列。除阳性对照外,该多重RT-PCR体系不对ORSV、PVX和PVY等同属或不同属的其它病毒发生反应;该体系可检测到104倍稀释的病毒c DNA模板。同时,以建立的三重RT-PCR法对10份田间辣椒病样进行检测,其结果与单重RT-PCR结果一致,说明该法检测结果可靠,可用于PMMo V、TMV和CMV 3种辣椒病毒的同步检测。

This research designed 3 pairs of primer for multiplex RT-PCR detection through the coat protein（CP）coded sequences of Pepper mild mottle virus（PMMo V）, Tobacco mosaic virus（TMV） and Cucumber mosaic virus（CMV）, initial building the multiplex RT-PCR method for synchronous detection of the 3 pepper viruses above.We used total RNA of leaves which infected by 3 kinds of virus, three specific products（324 bp for TMV, 220 bp for PMMo V and 112 bp for CMV） can be amplified simultaneously and the corresponding bands were readily identified on agarose gel. The sequencing results showed that the amplified products are truly target sequences of the viruses. The detection system didn＇t produce any band when using total RNA of leaves infected with other viruses as the template. The viruses＇ c DNA diluted by 104 times can be detected with the system. Furthermore, the test results of 10 field pepper samples by the multiplex RT-PCR were consistent the same as that of single RT-PCR method, indicating that the multiplex RT-PCR results are reliable and the system can be used for the simultaneous detection of 3 pepper viruses of PMMo V, TMV and CMV.