摘要
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。
To establish a Taq Man real-time RT-PCR assay for detecting Apple chlorotic leaf spot virus（ ACLSV）,a pair of primers and a Taq Man probe were designed based on the conserved nucleotide sequence of ACLSV coat protein（ cp） gene,and the recombinant plasmid of ACLSV-cp was constructed as positive standard to generate standard curve. The specificity,sensitivity and reproducibility of this method were evaluated. The results showed that the correlation coefficient of standard curve was 0. 999 and the amplification efficiency was 103. 7%. There was no crossing reaction with Apple stem grooving virus（ ASGV）,Apple stem pitting virus（ ASPV） and Apple scar skin viroid（ ASSVd）,indicating a strong specificity. The sensitivity of this method was 100 copies / μL,which was 100 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within0. 84%. This method could be used to detect ACLSV rapidly in practical samples with strong specificity,high sensitivity and reliable reproducibility.
出处
《植物保护学报》
CAS
CSCD
北大核心
2015年第4期551-556,共6页
Journal of Plant Protection
基金
国家现代农业产业技术体系建设专项(CARS-28)
