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

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．

Key words:Keywords: Salmonella，Target mining，EvaGreen real-time PCR，Internal amplification control (IAC)