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为建立一种检测苹果茎沟病毒(Apple stem grooving virus,ASGV)的Taq Man探针实时荧光定量PCR(quantitative Real-time RT-PCR,q RT-PCR)方法,本研究根据ASGV外壳蛋白(coat protein,CP)基因保守序列设计了特异性引物和Taq Man探针,以构建的ASGV-cp重组质粒为阳性标准品绘制标准曲线,并对该方法的特异性、灵敏性和重复性进行检验。建立的标准曲线相关系数达0.999,扩增效率为96.8%;该方法特异性好,与苹果茎痘病毒(Apple stem pitting virus,ASPV)、苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)、苹果锈果类病毒(Apple scar skin viroid,ASSVd)均无交叉反应;灵敏度为10拷贝/μL,比常规RT-PCR高1 000倍;批内和批间变异系数均<1%。表明该方法具有特异性强、灵敏高和重复性好的优点,适用于实际样品中ASGV的快速准确检测。
In order to establish a TaqMan probe quantitative real-time RT-PCR (q RT-PCR) method for the detection of apple stem grooving virus (ASGV) protein, CP) gene was designed and a specific primer and Taq Man probe were designed to construct a standard curve of ASGV-cp recombinant plasmid as a positive standard. The specificity, sensitivity and repeatability of the method were tested. The correlation coefficient of the established standard curve was 0.999, and the amplification efficiency was 96.8%. The specificity of the method was good, and the specificity of the method was similar to that of apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV) , Apple scar skin viroid (ASSVd). The sensitivity was 10 copies / μL, which was 1 000 times higher than that of conventional RT-PCR. The coefficient of variation (CV) within and between batches was less than 1%. The results showed that this method has the advantages of high specificity, high sensitivity and good reproducibility. It is suitable for the rapid and accurate detection of ASGV in real samples.