Assay for Evaluating the Abundance of Vibrio cholerae and Its O1 Serogroup Subpopulation from Water without DNA Extraction

Abstract

Cholera is a severe diarrheal disease caused by Vibrio cholerae, a natural inhabitant of brackish water. Effective control of cholera outbreaks depends on prompt detection of the pathogen from clinical specimens and tracking its source in the environment. Although the epidemiology of cholera is well studied, rapid detection of V. cholerae remains a challenge, and data on its abundance in environmental sources are limited. Here, we describe a sensitive molecular quantification assay by qPCR, which can be used on-site in low-resource settings on water without the need for DNA extraction. This newly optimized method exhibited 100% specificity for total V. cholerae as well as V. cholerae O1 and allowed detection of as few as three target CFU per reaction. The limit of detection is as low as 5 × 103 CFU/L of water after concentrating biomass from the sample. The ability to perform qPCR on water samples without DNA extraction, portable features of the equipment, stability of the reagents at 4◦ C and user-friendly online software facilitate fast quantitative analysis of V. cholerae. These characteristics make this assay extremely useful for field research in resource-poor settings and could support continuous monitoring in cholera-endemic areas.