甘薯羽状斑驳病毒(SPFMV)和矮化退绿病毒(SPCSV)的双重RT-PCR检测技术体系构建

Establishment of Duplex RT-PCR Detection Method for SPFMV and SPCSV Infecting Sweet Potato

在大田生产中,甘薯极易受到甘薯羽状斑驳病毒(sweet potato feathery mottle virus, SPFMV)和甘薯矮化退绿病毒(sweet potato chlorotic stunt virus,SPCSV)的混合侵染,导致甘薯产量和品质严重下降,因此构建能同时检测SPFMV和SPCSV两种病毒的检测技术体系对甘薯病毒病(sweet potato virus disease,SPVD)的预防具有重要意义。RT-PCR病毒检测技术具有灵敏度高、特异性强的优点。针对SPFMV和SPCSV病毒分别设计4对和3对特异性引物,通过单一RT-PCR检测技术筛选出特异性强的引物和最佳退火温度;在双重RT-PCR检测体系中,针对SPFMV和SPCSV两种病毒的引物浓度组合设置5个处理,优化双重RT-PCR病毒检测技术体系。单一RT-PCR检测结果表明,SPFMVS2/A2和SPCSVS2/A2引物的特异性最强,目的片段大小为461bp,对于SPCSV病毒来说,SPCSVS2/A2引物的特异性最强,目的片段大小为304bp,最佳退火温度为56℃;在双重RT-PCR检测技术体系中,SPFMV/SPCSV的引物组合浓度比为1∶3时,目标片段扩增效果最好。对两种病毒最低检测浓度的测试结果表明,SPFMV和SPCSV两种病毒RNA的最低检测浓度相当于模板RNA浓度的10-4μL。双重RT-PCR检测技术体系的构建,为大田生产中受SPFMV和SPCSV共侵染的甘薯病毒病的预防提供技术支持。

Sweetpotatoes can be easily infected by the combination of Sweet Potato Feathery Mottle Virus(SPFMV)and Sweet Potato Chlorotic Stunt Virus(SPCSV),which usually bring about decline in their production and quality.Therefore,developing a detection system which can detect both SPFMV and SPCSV at the same time is quite important.RT-PCR(Reverse Transcription-Polymerase Chain Reaction)virus detecting techniques has its advantages for its high sensitivity and specificity.This research designed 4 and 3 pairs of specific primers which aims at SPFMV and SPCSV virus,by using single RT-PCR detection technique,specificprimersand optimal annealing temperature were determined.Subsequently,in duplex RT-PCR detecting techniques,5 concentration combination of SPFMV and SPCSVwereset to optimize the detection ofduplex RT-PCR.Single RT-PCR detection techniqueshowed that the primers of SPFMVS2/A2 and SPCSVS2/A2 have strongest specificity andtarget fragment is 461 bp.For SPCSV virus,SPCSVS2/A2 have strongest specificity and the size oftarget fragment is 304bp.Optimal annealing temperature in this experiment is 56℃.When duplex RT-PCR detecting techniques wasused,target fragment has the best amplificationeffect when primer concentrationcombinationof SPFMV/SPCSV wasgiven by 1∶3.The sensitivityexperiment of both virusesindicatedthat detection limit of SPFMV and SPCSV is equal to 10^-4μL concentration of template concentration.Therefore duplex RT-PCR detecting technique will provide technique support in farmlands.