Development of colorimetric multiplex loop mediated isothermal amplification combined with lateral flow dipstick (mLAMP-LFD) assay for the identification of TDH toxin-producing Vibrio parahaemolyticus

Vibrio parahaemolyticus in seafood is associated with gastroenteritis diseases worldwide. Rapid and sensitive detection methods of the pathogenic strain are essential for monitoring and preventing the diseases. Colorimetric multiplex loop-mediated isothermal amplification combined with lateral flow dipstick assay (mLAMP-LFD) was developed to differentiate pathogenic strains from non-pathogenic V. parahaeholyticus. Thermolabile hemolysin (TLH) and thermo-stable direct hemolysin (TDH) genes were designed and used for mLAMP-LFD assay. The assay specificity was validated using strains belonging to 11 species of other foodborne pathogens, while the sensitivity was evaluated by using a DNA template ranging from 0.005 to 0.4 ng/reaction. Shrimp samples inoculated at 10¹, 10², and 10³ CFU/ 10 g, and enrichment times of 0, 2, 4, 6, and 8 h were used for assay validation. The standardized assay accurately identified 20 V. parahaemolyticus strains and showed no cross-amplification. The assay’s limit of detection was 0.02 ng and took 30 min to complete. Shrimp samples inoculated at 10 CFU of V. parahaemolyticus could be detected following a four-hour enrichment. In conclusion, the high sensitivity and specificity of the mLAMP-LFD assay developed in the present study could be used to identify pathogenic strains of V. parahaemolyticus.