Abstract
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 1 hour at 63°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 107 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.
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