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| Construction of Standard Positive Template and Development of a Real-Time Fluorescence Quantitative Polymerase Chain Reaction (FQ-PCR) Assay for PathogenicSalmonella spp. |
| CHEN Chen;SHAO Biao;CHEN Gang;HUANG Weidong |
| 1. Nantong Products Quality Supervision and Inspection Institute, Nantong 226011, China;
2. School of Life Science and Technology, China Pharmaceutical University, Nanjing 211198, China |
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Abstract 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 pathogenicSalmonellaspp. in foods. Methods: Primers and Taqman probe were designed and synthesized with the specific fragment of InvA 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 pathogenicSalmonella spp. could be used as a standard positive template for fluorescent quantitative PCR. The standard curve wasY=-3.151 lgX+42.86 (R2=0.999), and the sensitivity of the method was 80 copies per reaction. It was specific to detect Salmonellaspp. 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 pathogenicSalmonellaspp.
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2014, Vol. 28 
