Development of loop-mediated isothermal amplification (LAMP) method for rapid detection of Vibrio parahaemolyticus.

摘要

A technique for detecting Vibrio parahaemolyticus using a novel DNA amplification procedure designated loop-mediated isothermal amplification (LAMP) has been developed 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 was 600 degrees C and 60 min. Genomic DNAs from 28 bacterial strains including 14 V. parahaemolyticus strains were amplified using LAMP, and LAMP product was observed in other bacterial strains. The detection limit of this LAMP assay was approximately 90 fg. testtube-1 of V. parahaemolyticus genomic DNA and 24 cfu.mL-1 for pure cultures. In addition, this method was applied to detect noncultured artificially contaminated food samples. These results suggest that detection of V. parahaemolyticus by the LAMP assay 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.