半巢式RT-Realtime PCR方法检测藜草花叶病毒

Detection of Sowbane mosaic virus by Semi-nested Real-time PCR Method

[目的]根据藜草花叶病毒(SoMV)全基因组中多聚蛋白质基因序列保守区,分别设计2套TaqMAN探针和引物,建立半巢式实时荧光PCR检测SoMV的新方法。[方法]提取病毒核酸进行反转录操作合成cDNA,依据设计好的2套TaqMAN探针和引物,分别进行实时荧光PCR特异性与灵敏度检测,并进行2套引物探针的扩增效率对比试验。[结果]方法特异性强,灵敏度高达0.4 fg/μL植物总RNA,2套引物探针的扩增效率试验对比结果显示扩增效率几乎完全一样。[结论]该方法适用于藜草花叶病毒的检疫鉴定。

[Objective]Two sets of TaqMAN probes and primers were designed according to the conserved region of the polyprotein gene sequence in the whole genome of Sowbane mosaic virus (SoMV),and a new semi-nested real-time fluorescent PCR method for detecting SoMV was established.[Method]The virus nucleic acid was extracted for reverse transcription to synthesize cDNA.The specificity and sensitivity of real-time fluorescent PCR were detected by two sets of TaqMAN probes and primers.The amplification efficiency of two sets of primer-probes was compared.[Result]The method was highly specific and sensitive,reaching 0.4 fg/μL of plant total RNA.The efficiency of the two sets of primer probes was almost the same.[Conclusion]This method is suitable for quarantine and identification of Sowbane mosaic virus.