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依据Gen Bank中登录的甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)西非株系(WA)的核苷酸序列,分别设计两对特异性引物和一条Taq Man探针。以SPCSV-WA外壳蛋白(cp)基因的重组质粒为阳性标准质粒绘制标准曲线,通过优化反应体系和反应条件,建立了SPCSV-WA的实时荧光定量PCR检测方法。试验结果表明,该方法只能检测到目的病毒,标准曲线的斜率和相关系数分别为-3.239和1,扩增效率为103.568%。最低可检测到约3.31 copies/μL的阳性质粒,灵敏度比常规PCR高1 000倍。本研究建立的SPCSV实时荧光定量PCR方法可用于田间样品的检测,为SPCSV的早期预警和流行学研究提供了技术手段。
Two pairs of specific primers and one Taq Man probe were designed based on the nucleotide sequence of the West African strain (WA) of Sweet potato chlorotic stunt virus (SPCSV) registered in GenBank. The SPCSV-WA coat protein (cp) gene recombinant plasmid was used as a positive standard plasmid to draw a standard curve. By optimizing the reaction system and the reaction conditions, a real-time fluorescence quantitative PCR detection method of SPCSV-WA was established. The experimental results show that this method can only detect the target virus. The slope and the correlation coefficient of the standard curve are -3.239 and 1 respectively, and the amplification efficiency is 103.568%. A minimum of about 3.31 copies / μL positive plasmids were detected with a sensitivity of 1 × 10-fold higher than that of conventional PCR. The SPCSV real-time fluorescence quantitative PCR method established in this study can be used for the field sample detection, providing a technical measure for the early warning and epidemiological study of SPCSV.