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

摘要:

摘要: 【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

Abstract:

Abstract: 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．