添加有扩增内标的沙门氏菌荧光定量PCR检测体系的建立与评价

Establishment and evaluation of a real-time IAC-PCR for the detection of Salmonella

【目的】建立添加有扩增内标(IAC,Internal amplification control)的沙门氏菌EvaGreen荧光定量PCR检测体系,提高PCR检测可靠性。【方法】通过比较已有沙门氏菌属细菌的基因组序列,筛选沙门氏菌属特异检测靶点,设计特异引物;再用复合引物法构建扩增内标,优化参数,建立沙门氏菌内标PCR检测体系,利用特异性和灵敏度实验评价体系的检测性能。【结果】筛选得到的新特异靶点基因编码III型分泌系统蛋白(ssaQ)。针对该基因设计特异引物(SsaQ6),建立了添加有扩增内标的常规PCR和EvaGreen荧光定量PCR检测体系;二者对151株沙门氏菌和34株非沙门氏菌的检测符合率均达100%,对基因组DNA的检测下限达14.9拷贝/PCR和2.76拷贝/PCR;人工污染牛奶样品(初始染菌量:4-6 cfu/10 mL),増菌10 h和8 h后分别可检出沙门氏菌。【结论】本研究发掘的新靶点基因ssaQ特异性强,基于这一新靶点建立的添加有扩增内标的EvaGreen荧光定量PCR比常规内标PCR的检测限更低,重复性更好,快速方便,在12 h内即可得出检测结果,并且定量准确,有利于推进沙门氏菌PCR检测方法的标准化应用。

[Objective] The aim of this study was to establish a new EvaGreen real-time IAC-PCR for the rapid detection of Salmonella.[Methods] We used Salmonella genomic comparison analysis to mine Salmonella-specific targets,and Primer Premier 5.0 to design primers which were evaluated by specificity and sensitivity tests.[Results] We obtained a Salmonella-specific gene that encodes putative type Ⅲ secretion protein（ssaQ）,and specific primers（SsaQ6L/SsaQ6R） were designed based on this gene.Then we established IAC-PCR and EvaGreen real-time IAC-PCR assays,which showed 100% inclusivity and 100% exclusivity on all strains tested.Their detection limits of purified Salmonella genomic DNA were 14.9 copies/PCR and 2.76 copies/PCR respectively.Artificial contamination assays showed that Salmonella could be detected after 10 hours and 8 hours enrichment when the original bacterial concentration was 4.2 cfu/10 mL.[Conclusion] A new EvaGreen real-time IAC-PCR with high specificity and sensitivity was successfully developed for the rapid detection of Salmonella.