Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus.
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 x 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.
Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, USA.
Siyi
S
Chen
Beilei
B
Ge
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D006801:Humans; D016014:Linear Models; D008856:Microscopy, Fluorescence; D009391:Nephelometry and Turbidimetry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010125:Ostreidae / Q000382:microbiology*; D017747:Seafood / Q000382:microbiology*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics*; D014735:Vibrio Infections / Q000382:microbiology*; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C066719:toxR protein, bacteria