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| Construction of Standard Template and Development of Real-Time Fluorescence Quantitative PCR Assay for Detection of Vibrio parahemolyticus |
| CHEN Gang;SHAO Biao;XU Bei-bei;JI Ge-zhen |
| Nantong Products Quality Supervision and Inspection Institute, Nantong 226011, China |
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Abstract Objective: To construct recombinant plasmids for use as standard positive template and establish a real-time
(RT) fluorescent quantitative PCR assay for determining Vibrio parahemolyticus in meat products. Methods: Primers and
Taqman probes were designed and synthesized using the specific fragment of toxRS gene from Vibrio Parahemolyticus as
target sequence. Recombinant plasmids were constructed by inserting the target gene into PGM-T vector. A RT fluorescent
quantitative PCR assay was established and its sensitivity, specificity, repeatability and accuracy were investigated. Results:
Recombinant plasmids were successfully constructed for use as standard positive template for fluorescent quantitative PCR.
The following calibration curve was established: Y =－3.151 lgX + 42.86. The sensitivity of the PCR assay was 40 copies
per reaction system, and good specificity for Vibrio parahemolyticus and high repeatability were observed. The intra-batch and
inter-batch coefficients of variation for five replicate assays were both below 5%. Conclusion: The fluorescent quantitative PCR
based on Taqman probes allows rapid, simple, accurate and efficient detection of Vibrio parahemolyticus in foods.
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2012, Vol. 26 
