Development of Loop-mediated Isothermal Amplification (LAMP) Assay Based on Propidium Monoazide (PMA) for Detecting Vibrio parahaemolyticus
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Abstract:
Vibrio parahaemolyticus is a common foodborne pathogen frequently detected in seafood, and consumption of raw, pickled, or uncooked food contaminated with V. parahaemolyticus can cause food poisoning. Nearly all current methods for rapidly detecting V. parahaemolyticus can not distinguish live cells from dead cells, resulting in false-positive results. In this study, a real-time visual loop-mediated isothermal amplification (LAMP) method was developed based on the characteristics of the DNA binding dye propidium monoazide (PMA) and fluorescent dye calcein to detect viable V. parahaemolyticus strains. This method was compared to quantitative polymerase chain reaction (qPCR). The results of real-time visual PMA-LAMP assay could be observed by the naked eye and monitored in real-time using fluorescence analysis instruments. A preliminary semi-quantitative analysis was conducted, providing a basis for in-depth quantitative analysis. The results indicated that the sensitivities of the real-time visual PMA-LAMP assay in the sensitivity experiment and artificial contamination experiment were 5.0 × 102 CFU/mL and 1.9 × 102 CFU/mL, respectively, which were consistent with the values from PMA-qPCR. The entire detection process required only 2 h, excluding the pre-enrichment step. Furthermore, the method showed high accuracy and may be an effective technical method for rapid detection of V. parahaemolyticus.
