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为建立一种检测苹果茎痘病毒(Apple stem pitting virus,ASPV)的Taq Man探针实时荧光定量RT-PCR方法,根据ASPV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,以体外转录的RNA为标准品构建标准曲线,并对该方法的特异性、灵敏性、重复性进行检验。建立的标准曲线决定系数达0.996,扩增效率为99%;该方法特异性好,与苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)、苹果锈果类病毒(ASSVd)均无交叉反应;其最低检测限为1.31×102 copies·μL-1,灵敏度比常规RT-PCR高100倍;批内和批间变异系数均小于1.85%。该方法具有特异性强、灵敏度高、重复性好等优点,适用于田间样品中ASPV的快速定量检测。
In order to establish a TaqMan probe real-time fluorescence quantitative RT-PCR method for the detection of apple stem pitting virus (ASPV), specific primers and Taq primers were designed according to the conserved sequence of ASPV coat protein (cp) Man probe was used to construct a standard curve using in vitro transcribed RNA as a standard. The specificity, sensitivity and repeatability of the method were tested. The established standard curve had a coefficient of determination of 0.996 and an amplification efficiency of 99%. The method was of good specificity and had no significant difference with ASGV, ACLSV and ASSVd The detection limit was 1.31 × 102 copies · μL-1. The sensitivity was 100-fold higher than that of conventional RT-PCR. The coefficient of variation (CV) was less than 1.85%. The method has the advantages of strong specificity, high sensitivity and good repeatability, and is suitable for rapid and quantitative detection of ASPV in a field sample.