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Background   Vibrio parahaemolyticus  is a leading cause of seafood-related bacterial gastroenteritis and outbreaks worldwide. Sensitive and specific detection methods are needed to better control  V. parahaemolyticus  infections. This study aimed at developing a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting  V. parahaemolyticus  in oysters. A set of five LAMP primers, two outer, two inner, and one loop were designed based on the published  V. parahaemolyticus toxR  sequence. Specificity of the assay was evaluated using a panel of 36  V. parahaemolyticus  and 39 other strains. The assay sensitivity was determined using serial dilutions of  V. parahaemolyticus  ATCC 27969 culture ranging from 10 8  CFU/ml to extinction. The assay was also tested in experimentally inoculated oyster samples.    Results  The  toxR -based LAMP assay was able to specifically detect all of the 36  V. parahaemolyticus  strains without amplification from 39 other strains. The detection limit was 47-470 cells per reaction in pure culture, up to 100-fold more sensitive than that of  toxR -PCR. When applied in spiked oysters, the assay was able to detect 1.1 × 10 5   V. parahaemolyticus  cells per gram of oyster without enrichment, up to 100-fold more sensitive than that of  toxR -PCR. Standard curves generated for detecting  V. parahaemolyticus  in both pure culture and spiked oyster samples showed good linear relationship between cell numbers and the fluorescence or turbidity signals.    Conclusions  The  toxR -based LAMP assay developed in this study was sensitive, specific, and quantitative, holding great potential for future field detection of  V. parahaemolyticus  in raw oysters.

Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus