马铃薯病毒PVY,PVS和PLRV多重RT-PCR检测

Detection of PVY,PVS and PLRV by Multiplex RT-PCR

病毒是马铃薯生产过程中危害较大的一类病原,为快速检测出云南省马铃薯主要病毒种类,依据病毒外壳蛋白基因序列设计合成了马铃薯Y病毒(PVY)、马铃薯S病毒(PVS)、马铃薯卷叶病毒(PLRV)的特异性引物,从退火温度、最低检测浓度、热反应循环条件、病毒间的模板及引物间的最佳组合浓度进行优化,建立了三重RT-PCR反应体系。结果表明:该优化体系可同步扩增出上述3种病毒,分别得到480 bp(PVY)、187 bp(PVS)、336 bp(PLRV)大小的扩增片段。优化的三重RT-PCR反应体系可快速、灵敏、简便的检测到单一或复合侵染的马铃薯病毒,有利于马铃薯病毒病的早期检测和防治,将为云南省马铃薯种苗脱毒、继代、田间植株的高产栽培等有重要实践作用。

Virus is the one of the plant pathogen which is harmful to the potatoes production. In order to rapid detect the main potatoes virus species of Yunnan Province, the PCR primers for potato virus Y （PVY）, potato virus S （PVS）, and potato leaf roll virus （PLRV） were designed and synthesized based on their coat protein gene sequences. Then the optimal system of triple RT-PCR detection system was estab- lished from annealing temperature, minimum detectable concentration, thermal cycling condition and best combination concentration of virus template with primers. The result showed that this optimized multiplex RT-PCR could amplify three viruses simultaneously, and the fragments were 480 bp （PVY）, 187 bp （PVS） and 336 bp （PLRV）, respectively. This optimized multiplex RT-PCR reaction system was proved to be rap- id, sensitive and simple to detect single or compound infection of the above potato virus and to early detec- tion and control of potato virus diseases. This result will have an important practical effect on potato seed- lings detoxification, trans-generational and high yield cultivation in the field in Yunnan Province.