A colorimetric Loop-mediated isothermal amplification (LAMP) assay based on HRP-mimicking molecular beacon for the rapid detection of Vibrio parahaemolyticus

Abstract

In the world wide, food poisoning accidents related to Vibrio spp. are on the rise, even numbers of food poisoning by other foodborne pathogens are decreasing. Therefore, the requirement of the rapid, sensitive and convenient detection method for V. parahaemolyticus has been grown. The objective of this study is to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay using a molecular beacon (HRPzyme connected with complementary oligonucleotides at the 5′ and 3′ ends) for the rapid, sensitive, and convenient detection of V. parahaemolyticus. The colorimetric LAMP assay optimized at 58.8°C showed a detection limit of 1 × 100 CFU/mL and was confirmed to be specific to V. parahaemolyticus. The colorimetric LAMP assay can be finished within 1 h including DNA extraction step. The method was successfully applied to flatfish samples artificially inoculated with known amount of V. parahaemolyticus, and its cut-off value for the flatfish samples was 1 × 101 CFU/g. In addition, the colorimetric LAMP assay developed in the study was found to be able to correct false-positive results, which are known to be a disadvantage of conventional LAMP assays. Therefore, these results indicated that the colorimetric LAMP method is a useful tool for the rapid, sensitive and convenient detection of V. parahaemolyticus in fishes and can also be used as a point-of-care molecular diagnostic technique since it does not require any expensive equipment such as a thermocycler and detectors.