Study on Simultaneous Detection of Vibrio Parahaemolyticus and Vibrio Cholerae in Aquatic Products by Loop-Mediated Isothermal Amplification Method
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Abstract:
A loop-mediated isothermal amplification (LAMP) method was developed for simultaneous and rapid detection of Vibrio parahaemolyticus and Vibrio cholerae in aquatic products. Based on the nucleic acid sequences of toxR of V. parahaemolyticus and ompW of V. cholerae, a pair of primers was designed for LAMP. After optimizing the reaction conditions, the levels of V. parahaemolyticus and V. cholerae in aquatic products were estimated using LAMP and PCR, and the detection rate and efficiency of the two methods were compared. The optimal reaction temperature was found to be 61 ℃; at this temperature, the detection limit for the DNA templates of V. parahaemolyticus and V. cholerae was 3.1 fg, and no cross-reaction with other common bacteria was found, yielding a detection specificity of 100%. For direct detection of V. parahaemolyticus and V. cholerae in artificially contaminated food samples, the detection limit was found to be 50 cfu/mL. In addition, when 50 seafood samples were tested by LAMP and PCR, 6 of them were found to be positive, but only 4 were detected to be positive by conventional microbiological methods. Thus, the LAMP method had high specificity and sensitivity for detecting V. parahaemolyticus and V. cholerae in aquatic products, and would be suitable for rapidly detecting V. parahaemolyticus and V. cholerae.
