Isothermal amplification and colorimetric detection of Vibrio cholerae in environmental matrices

Vibrio cholerae (V. cholerae), causing deadly cholera, which is responsible for significant public health problems in endemic regions, should be monitored in contaminated environments. Here, we report an integrated protocol incorporating alkaline lysis DNA extraction and loop-mediated isothermal amplification (LAMP) targeting the V. cholerae rpoB gene and producing a colorimetric reading within 1 h. This protocol yielded 100% specificity for V. cholerae after testing three non-cholerae Vibrio species and four Enterobacteriaceae as negative controls. A positive signal was detected across a range of 3 × 10⁸ to 3 × 10³ cells/mL for four V. cholerae isolates within 30-60 min. Following the inoculation of 3 × 10⁴ V. cholerae cells into five simulated environmental matrices (soil, sand, seawater, tap water, and anthropogenic effluents), positive results were similarly obtained between 3 × 10⁴ and 3 × 10³ cells/mL over the same time interval. In contrast, at the same conditions, Vibrio alginolyticus (V. alginolyticus) and Escherichia coli mock-infected matrices, which were used as negative controls, remained negative. These data suggested that this LAMP protocol could be implemented in the field to monitor V. cholerae in environmental matrices at the point of interest in countries affected by this deadly pathogen.IMPORTANCECholera is an intestinal infection caused by the bacterium Vibrio cholerae, resulting in vomiting and acute watery diarrhea. If left untreated, it can cause severe dehydration and death. Although various detection tests have been developed for different types of samples, cholera remains endemic in several countries, particularly where access to safe drinking water is limited and sanitation infrastructure remains inadequate. The objective of our research is to explore different environmental matrices to design a screening system for V. cholerae in environmental samples, thus contributing to the development of a system that can be used in the environment.