AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE RAPID DETECTION OF VIBRIO-PARAHAEMOLYTICUS

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of Vibrio parahaemolyticus in foods. The assay was based on the detection of two outer-membrane proteins (molecular weights 36,000 and 34,000) of V. parahaemolyticus. Following an 18-h incubation in alkaline peptone salt broth containing 0.1% Teepol, the culture supernatant was added to the microtiter plate coated with antibodies against the two outer-membrane proteins. After washing, the same antibodies absorbed with V. alginolyticus and labeled with horseradish peroxidase were used as secondary antibodies. The detection limit of the assay for total outer-membrane proteins was 10 ng/ml. For 29 strains of V. parahaemolyticus and 73 strains (including 27 isolates of vibrios) of other bacteria tested, the sensitivity and specificity of the ELISA were 100 and 96%, respectively. Strains producing false positives were V. tubiashii, V. campbellii, and V. vulnificus. Of 23 seafood samples tested, V. parahaemolyticus was detected in 17 and 15 samples, respectively, by the ELISA and by a conventional culture method. V. parahaemolyticus was also detected in samples artificially inoculated with the microorganism at levels less than 10 colony-forming units (CFU) per g. Compared to the conventional culture methods, which may take 4 to 6 days to complete, the ELISA can detect low numbers of V. parahaemolyticus in foods with a total analytical time of only 24 h. The ELISA is recommended as a rapid screening method.