草莓6种病毒多重PCR快速检测方法的建立

A multiplex PCR assay for simultaneous detection and identification of six viruses in strawberry

为快速鉴定草莓病毒病种类以预防病毒病扩散,根据草莓镶脉病毒(strawberry vein banding virus,SVBV)、草莓斑驳病毒(strawberry mottle virus,SMoV)、草莓轻型黄边病毒(strawberry mild yellow edge virus,SMYEV)、草莓白化病毒(strawberry pallidosis-associated virus,SPaV)、草莓病毒1(strawberry virus 1,StrV-1)和芸薹黄化病毒(brassica yellows virus,BrYV)的基因保守区域设计特异性引物,建立了能够同时检测这6种病毒的多重PCR方法。该多重PCR反应体系中,2×SanTaq PCR Master Mix用量为10μL,上下游引物(10μmol/L)用量为4μL,其中SVBV为0.25μL、SMoV为0.50μL、SMYEV为0.10μL、SPaV为0.15μL、StrV-1为0.50μL和BrYV为0.50μL,模板1μL,ddH_(2)O为5μL,总体积20μL。多重PCR反应程序设定为:94℃预变性5 min,94℃变性30 s,53℃退火30 s,72℃延伸120 s,最后72℃延伸10 min,循环数为35。该检测体系的灵敏度可以达到107copies/μL。该多重PCR方法可以同时实现6种草莓病毒的高效快速检测,为草莓病毒病的早期发现和针对性防治提供了技术支持。

In order to rapidly identify strawberry virus disease species and prevent the spread of virus disease,a multiplex reverse transcription polymerase chain reaction system was established for the simultaneous detection and identification of strawberry vein banding virus(SVBV),strawberry mottle virus(SMoV),strawberry mild yellow edge virus(SMYEV),strawberry pallidosis-associated virus(SPaV),strawberry virus 1(StrV-1)and brassica yellows virus(BrYV).Six pairs of specific primers designed on the conserved genomic regions of these viruses.In this multiplex PCR reaction system,the dosage of 2×SanTaq PCR Master Mix was 10μL,the dosage of upstream and downstream primer(10μmol/L)was 4μL,among which SVBV was 0.25μL,SMoV was 0.50μL,SMYEV was 0.10μL,SPaV was 0.15μL,StrV-1was 0.50μL,BrYV was 0.50μL,template volume was 1μL,ddH_(2)O was 5μL,and the total volume was 20μL.The multiple PCR procedure was pre-denaturation at 94℃for 5min,denaturation at 94℃for 30s,annealing at 53℃for 30s,extension at 72℃for 2min,and final extension at 72℃for 10min,with a cycle number of 35,and the sensitivity of this system achieved 107 copies/μL.In conclusion,the proposed multiplex PCR can realize the efficient detection of six strawberry viruses at the same time,which provides technical support for the early prevention and treatment of strawberry virus diseases.