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A strip test for the detection of Vibrio cholerae O1 was developed using two monoclonal antibodies (MAbs), VC-223 and VC-1226, specific to the lipopolysaccharides of Vibrio cholerae O1 Inaba and Ogawa serovars. The sensitivity of the test was 5 × 10(5)cfu/mL which was similar to that of dot blot test. The detection limit could be improved to 1cfu/mL of the original bacterial content after pre-incubation of the bacterium in alkaline peptone water (APW) for 12h. Detection of V. cholerae O1 in various fresh seafood samples such as shrimp, blood clam, mussel and oyster could be performed directly with sensitivities ranged from 5 × 10(5) to 10(6)cfu/mL. After pre-enrichment of the shrimp sample in APW, the detection sensitivities increased to 10(2) to 10CFU/mL of the original bacterial content after incubation for 12 and 24h. However, the detection sensitivities were also depending on the content of the other bacteria that might inhibit the growth of V. cholerae during pre-enrichment step. The V. cholerae O1 strip test has advantages in speed, and simplicity in not requiring sophisticated equipment or specialized skills and the sample could be directly examined without requirement for sample processing., (© 2013.)

The current study was aimed to develop a loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin ( tlh) gene followed by hybridization with an FITC-labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP-LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non- parahaemolyticus Vibrio isolates and 35 non- Vibrio bacterial isolates. The sensitivity of LAMP-LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 × 10 CFU g or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. The developed LAMP-LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample. [ABSTRACT FROM AUTHOR]

Aims: The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae. Methods and Results: A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65°C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non- cholerae Vibrio isolates and 37 non- Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2·2 × 103 CFU ml−1 or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2·2 × 104 CFU g−1 or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction. Conclusion: The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection. Significant and Impact of the study: The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae. This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample. [ABSTRACT FROM AUTHOR]