Smith Donna S, De Boer Solke H, Gourley Jane
Charlottetown Laboratory, Canadian Food Inspection Agency, Charlottetown, PE, C1A 5T1, Canada.
Plant Dis. 2008 May;92(5):684-693. doi: 10.1094/PDIS-92-5-0684.
An internal reaction control was integrated into a TaqMan polymerase chain reaction (PCR) assay for the detection of Clavibacter michiganensis subsp. sepedonicus, the causal organism of bacterial ring rot of potato. The reaction control, cloned into plasmid pCmsC4, consisted of a sequence unrelated to C. michiganensis subsp. sepedonicus flanked by the primer sequences used in the TaqMan PCR, thus eliminating the need for multiplexing. Inclusion of the reaction control plasmid in the TaqMan assay had no effect on either the limit of detection or the specificity of the method. Addition of SYBR Green permitted melt analysis of PCR products. The 242-bp reaction control amplicon, with a melt temperature of approximately 94.5°C, could easily be distinguished from the 152-bp primary diagnostic target amplicon, which had a melt temperature of about 85.5°C. Electrophoretic analysis showed that appearance of either melt peak correlated well with the presence of the appropriate amplicon. Two different substances, guanidine-HCl and humic acid, inhibited the amplification of the reaction control at concentrations lower than those that inhibited the primary diagnostic target, demonstrating the reaction control's effectiveness in detecting inhibition or reaction failure. Using the reaction control plasmid, a quantitative threshold for inhibitor detection was established. This permitted the validation of negative results, and thus facilitated the use of TaqMan real-time PCR in the routine testing of diagnostic samples for C. michiganensis subsp. sepedonicus.
将一种内部反应对照整合到TaqMan聚合酶链反应（PCR）检测方法中，用于检测密执安棒杆菌马铃薯环腐亚种，即马铃薯细菌性环腐病的致病生物。克隆到质粒pCmsC4中的反应对照，由一段与密执安棒杆菌马铃薯环腐亚种无关的序列组成，其两侧为TaqMan PCR中使用的引物序列，从而无需进行多重检测。在TaqMan检测中加入反应对照质粒对检测限或方法的特异性均无影响。添加SYBR Green可对PCR产物进行熔解分析。242 bp的反应对照扩增子熔解温度约为94.5°C，可轻松与152 bp的主要诊断靶标扩增子区分开来，后者的熔解温度约为85.5°C。电泳分析表明，任一熔解峰的出现与相应扩增子的存在密切相关。两种不同物质，盐酸胍和腐殖酸，在低于抑制主要诊断靶标的浓度下就能抑制反应对照的扩增，证明了反应对照在检测抑制或反应失败方面的有效性。使用反应对照质粒建立了抑制剂检测的定量阈值。这使得阴性结果得以验证，从而便于在密执安棒杆菌马铃薯环腐亚种诊断样品的常规检测中使用TaqMan实时PCR。
