Evaluation of a loop-mediated isothermal amplification assay for detecting Vibrio vulnificus in raw oysters

Human consumption of raw or undercooked seafood, particularly oysters, may lead to severe infections due to the presence of Vibrio vulnificus. In this study, a sensitive and specific loop-mediated isothermal amplification (LAMP) assay was developed to detect this pathogen in raw oysters. Two outer and two inner primers were designed to specifically recognize the V. vulnificus cytolysin/hemolysin gene (vvhA), and the reaction could be completed in I hour at 63 degrees C. Direct visual observation of the LAMP amplicons was achieved with the aid of SYBR Green I fluorescent dye. The assay specificity was determined using 50 bacterial strains, including multiple Vibrio spp. and bacteria of other genera. No false-positive or false-negative results were observed. The detection limit of the LAMP assay was approximately 20 colony-forming units (CFU) in pure cultures, 10-fold more sensitive than a conventional polymerase chain reaction (PCR). When directly applied in oyster homogenate, the LAMP assay had a detection limit of approximately 10(7) CFU/g. After 5-hour enrichment, LAMP was capable of detecting 7 CFU of V. vulnificus per gram of oyster tissue without lengthy DNA extraction steps. This level of detection was 1000-fold more sensitive than PCRs included for comparison. Because of its isothermal format and unique amplicon detection technique, this rapid and sensitive LAMP assay holds potential for future field applications.