Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation–pit‐stop semi‐nested PCR procedure

• Published in: Journal of applied microbiology 2000-09, Vol.89 (3), p.539-546
• Main Authors: Theron, J, Cilliers, J, Du Preez, M, Brözel, V.S, Venter, S.N
• Format: Article
• Language: English
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; cholera toxin; Cholera Toxin - genetics; culture media; detection limit; DNA Primers; DNA, Bacterial - analysis; drinking water; enrichment culture; Fresh Water - microbiology; Fundamental and applied biological sciences. Psychology; Microbiology; operon; polymerase chain reaction; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Sewage - microbiology; sewage treatment; Vibrio cholerae; Vibrio cholerae - isolation & purification; Water Microbiology
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1046/j.1365-2672.2000.01140.x

A pit‐stop semi‐nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml⁻¹ was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit‐stop semi‐nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.