Development of Loop-mediated Isothermal Amplification (LAMP) Method for Detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus has been considered as one of the most important foodborne bacterial pathogens.The loop-mediated isothermal amplification(LAMP) that amplifies DNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of this pathogen for the first time.A set of four primers,two outer and two inner primers,was designed specifically to recognize the thermolabile hemolysin gene(tlh) of V.parahaemolyticus.The LAMP reaction mix was optimized.The most optimal reaction temperature and time of the LAMP assay for the tlh gene were 60℃ and 60min,respectively.Genomic DNAs from 28 bacterial strains including 14 V.parahaemolyticus strains were amplified using LAMP,and no amplicon was observed in other bacterial strains.The detection limit of this LAMP assay was around 90 fg of V.parahaemolyticus genomic DNA and 24 colonies forming units for pure cultures.In addition,this method was applied to detect artificially contaminated food samples,and the detection limit was 89 cfu/g for non-cultured artificially contaminated food samples.These results suggested that detection of V.parahaemolyticus by LAMP is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment.This assay is expected to become a valuable tool for rapid detection and identification of V.parahaemolyticus.