Construction of Standard Positive Template and Development of a Real-Time Fluorescence Quantitative Polymerase Chain Reaction(FQ-PCR) Assay for Pathogenic Salmonella spp.

Objective: To construct recombinant plasmids for use as standard positive template and establish a real-time fluorescent quantitative polymerase chain reaction(FQ-PCR) assay for determining pathogenic Salmonella spp. in foods. Methods: Primers and Taqman probe were designed and synthesized with the specific fragment of Inv A gene as the target sequence. Recombinant plasmids were constructed by inserting the target gene into PGM-T carriers. Real-time fluorescent quantitative PCR method was established and its sensitivity, specificity, repeatability and accuracy were investigated. Results: The recombinant plasmids constructed using the specific sequence of pathogenic Salmonella spp. could be used as a standard positive template for fluorescent quantitative PCR. The standard curve was Y=-3.151 lg X + 42.86(R2=0.999), and the sensitivity of the method was 80 copies per reaction. It was specific to detect Salmonella spp. with good repeatability(the inter-batch and intra-batch coefficients of variation were both less than 5%). Conclusion: The FQ-PCR method allows qualitative and quantitative detection of pathogenic Salmonella spp.