Qualitative and Quantitative Detection of Potentially Virulent Vibrio parahaemolyticus in Drinking Water and Commonly Consumed Aquatic Products by Loop-Mediated Isothermal Amplification

Abstract

Vibrio parahaemolyticus can cause acute gastroenteritis, wound infection, and septicemia in humans. In this study, a simple, specific, and user-friendly diagnostic tool was developed for the first time for the qualitative and quantitative detection of toxins and infection process-associated genes opaR, vpadF, tlh, and ureC in V. parahaemolyticus using the loop-mediated isothermal amplification (LAMP) technique. Three pairs of specific inner, outer, and loop primers were designed for targeting each of these genes, and the results showed no cross-reaction with the other common Vibrios and non-Vibrios pathogenic bacteria. Positive results in the one-step LAMP reaction (at 65 °C for 45 min) were identified by a change to light green and the emission of bright green fluorescence under visible light and UV light (302 nm), respectively. The lowest limit of detection (LOD) for the target genes ranged from 1.46 × 10−5 to 1.85 × 10−3 ng/reaction (25 µL) for the genomic DNA, and from 1.03 × 10−2 to 1.73 × 100 CFU/reaction (25 µL) for the cell culture of V. parahaemolyticus. The usefulness of the developed method was demonstrated by the fact that the bacterium could be detected in water from various sources and commonly consumed aquatic product samples. The presence of opaR and tlh genes in the Parabramis pekinensis intestine indicated a risk of potentially virulent V. parahaemolyticus in the fish.