Optimization of Loop-Mediated Isothermal Amplification Methods for the Detection of Vibrio parahaemolyticus in Aquatic Products
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Abstract:
The heat-sensitive hemolysin (tlh) gene was used as the target in this study, and loop-mediated isothermal amplification (LAMP) conditions were optimized to develop a method for the detection of Vibrio parahaemolyticus in aquatic products. The LAMP reaction system was catalyzed using Bst DNA polymerase and the reaction proceeded at a constant temperature for 60 min. The amplified products were identified by 2% agarose gel electrophoresis and SYBR Green I staining, and all reaction parameters were optimized. LAMP and polymerase chain reaction (PCR) amplification were performed after ten-fold serial dilution of the fresh bacterial culture, and the sensitivities of the two methods were compared. LAMP was performed on 32 foodborne pathogens (including 25 strains of Vibrio parahaemolyticus isolated from aquatic products, Vibrio parahaemolyticus strain ATCC 17802, Escherichia coli strain D5, Staphylococcus aureus strain CMCC26003, Shigella boydii strain B4, Bacillus cereus strain 63302, Listeria monocytogenes strain 54001, and Salmonella Enteritidis strain 50041), and the specificity of LAMP was verified. The reliability of LAMP was evaluated by measuring shrimp samples contaminated with V. parahaemolyticus culture. The optimum conditions were as follows: 3.6 mmol/L magnesium ion, 0.96 mmol/L dNTPs, 4.8 U Bst DNA polymerase, 8:1 ratio of inner and outer primers, reaction temperature of 63 ℃, and reaction time of 60 min. The detection limit of the LAMP assay was 1 CFU/mL, which was lower than that of the PCR method (1×105 CFU/mL). In specificity tests, all 26 strains of V. parahaemolyticus were positive, and the other six strains were negative. In the detection of artificially contaminated samples, the detection limits were 1 CFU/mL and there were no false positive results. In conclusion, the established LAMP assay is a simple, rapid, specific, and sensitive detection method that is suitable for the rapid on-site detection of V. parahaemolyticus in aquatic products.
