甘薯羽状斑驳病毒实时荧光定量PCR检测方法的建立

Development of Real-time Fluorescent Quantitative PCR Assay for Detection of Sweet potato feathery mottle virus

依据甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)外壳蛋白(CP)基因的保守区设计引物和TaqMan探针,经过对反应体系和反应条件的优化,建立了特异、灵敏、高效的SPFMV实时荧光定量PCR检测方法。试验结果表明:该方法能检测到目的病毒,标准曲线的斜率和相关系数分别为-3.307和0.998,扩增效率为100.6%。最低可检测到约5.46个拷贝的阳性质粒,灵敏度比常规PCR高2个数量级。建立的实时荧光定量PCR方法可用于田间样品的检测,为SPFMV的早期预警和流行学研究提供了技术手段。

A rapid, sensitive and stable real-time RT-PCR assay was established for detecting Sweet potato feather mottle virus （SPFMV）based on primers and TaqMan probe derived from coat protein gene sequences of SPFMV. The results showed that the correlation coefficient and the slope value of the standard curves with plasmid DNA were 0.998 and -3.307, respectively. The efficiency of the PCR was 100.6%.The assay was specific allowing detection only SPFMV, the detection limit of the real-time PCR assay was 5.46 copies. Compared with conventional PCR method, the real-time fluorescent RT-PCR assay showed 100 fold higher in detection sensitivity. This real-time PCR assay reported here is useful for rapid and quantitative detection of SPFMV and epidemiological investigation.