论文部分内容阅读
为建立一种检测苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)的Taq Man探针实时荧光定量RT-PCR方法,根据ACLSV外壳蛋白基因(coat protein,cp)保守序列设计了特异性引物和Taq Man探针,以构建的ACLSV-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、重复性进行检验。结果显示,以ACLSV-cp重组质粒为标准品建立的标准曲线相关系数达0.999,扩增效率为103.7%;建立的Taq Man探针实时荧光定量RT-PCR方法特异性好,与苹果茎沟病毒(Apple stem grooving virus,ASGV)、苹果茎痘病毒(Apple stem pitting virus,ASPV)、苹果锈果类病毒(Apple scar skin viroid,ASSVd)均无交叉反应;灵敏度为100拷贝/μL,比常规RT-PCR高100倍;批内和批间变异系数均小于0.84%。表明Taq Man探针实时荧光定量RT-PCR方法具有特异性强、灵敏性高、重复性好的优点,适用于实际样品中ACLSV的快速准确检测。
In order to establish a TaqMan probe real-time fluorescence quantitative RT-PCR method for detecting apple chlorotic leaf spot virus (ACLSV), a specific primer was designed according to the conserved sequence of ACLSV coat protein (cp) And Taq Man probe to construct a standard curve of ACLSV-cp recombinant plasmid as a positive standard, and test the specificity, sensitivity and repeatability of the method. The results showed that the correlation coefficient of standard curve established by ACLSV-cp recombinant plasmid was 0.999 and the amplification efficiency was 103.7%. The real-time fluorescent quantitative RT-PCR method of TaqMan probe was established, (ASGV), Apple stem pitting virus (ASPV) and Apple scar skin viroid (ASSVd). The sensitivity was 100 copies / μL, which was lower than that of conventional RT -PCR was 100 times higher than that of the control. The coefficient of variation (CV) within and between batches was less than 0.84%. Taq Man probe real-time fluorescence quantitative RT-PCR method has the advantages of strong specificity, high sensitivity and good repeatability, and is suitable for rapid and accurate detection of ACLSV in real samples.