Simple and Sensitive Quantification of Toxigenic Vibrio cholerae DNA by Real-Time Loop-Mediated Isothermal Amplification Based on ctxB Gene

Background: Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and specific method for the detection of bacterial pathogens within a sample. Methods: We designed and evaluated a new quantitative LAMP primer set specific to the ctxB gene for the rapid measurement of the load of toxigenic V. cholerae DNA in the reaction. Unlike the previously reported LAMP assays for V. cholerae detection, our LAMP primer set was able to detect and quantify toxigenic V. cholerae DNA with a detection limit of 2.8 pg of the ctxB gene equivalent to 8.3 copies of genomic DNA per reaction. Results: Our LAMP assay was found to be highly specific with no amplification detected in non-toxigenic V. cholerae bacterial strains. Thus, the ctxB-based LAMP assay developed in this study can serve as a simple, sensitive, specific, and quantitative molecular tool for the monitoring and epidemiological study of cholera. Conclusions: Overall, the low cost and simplicity of the LAMP assay can make it a preferable molecular method in the detection of pathogenic organisms.