Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Abstract

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. © 2009 The Society for Applied Microbiology.