Specific Detection of Viable Vibrio parahaemolyticus Using Loop-mediated Isothermal Amplification Combined with Propidium Monoazide

Introduction: Vibrio parahaemolyticus, a Gram-negative marine bacterium, is a major foodborne pathogen predominantly in raw or undercooked seafood. The viable but non-culturable (VBNC) state of V. parahaemolyticus cannot be detected by traditional culture methods while remains alive for long time and maintains the potential virulence.

Purpose: The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with loop-mediated isothermal amplification (LAMP) to detect only viable cells of V. parahaemolyticus.

Methods: The viable or dead cells suspensions were treated with PMA in the dark for 10 min and were subsequently exposed to a 650 W halogen lamp for 5 min. The bacterial cells were harvested and DNA was extracted and amplified by LAMP. The primers targeted six distinct regions in the tlh gene of V. parahaemolyticus were designed for the PMA-LAMP method.

Results: The treatment with 3 μg/ml PMA and a 5min light exposure was suitable for PMA-LAMP to distinguish the viable cells from dead cells of V. parahaemolyticus. The optimized assay was specific and sensitive, it could detect as low as 12 CFU/ml viable V. parahaemolyticus in pure culture and artificially contaminated seafood samples (pomfret, shrimp, scallop and salted fish). The assay can detect VBNC state of V. parahaemolyticus without any interference of dead cells and other bacteria.

Significance: The results indicate that PMA-LAMP could effectively detect viable V. parahaemolyticus without being disturbed by other cells, it is a suitable technique for the detection of VBNC cells of foodborne pathogens in contaminated food.