Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems

doi:10.1046/j.1462-2920.2003.00443.x

If you are seeing this message, you may be experiencing temporary network problems. Please wait a few minutes and refresh the page. If the problem persists, you may wish to report it to your local Network Manager.

It is also possible that your web browser is not configured or not able to display style sheets. In this case, although the visual presentation will be degraded, the site should continue to be functional. We recommend using the latest version of Microsoft or Mozilla web browser to help minimise these problems.

Wiley InterScience

Environmental Microbiology

See Also:

Environmental Microbiology Reports

Volume 5 Issue 7, Pages 599 - 606

Published Online: 19 Jun 2003

Published jointly with the Society for Applied Microbiology

Go to Society Site

< Previous Abstract | Next Abstract >

Save Article to My Profile Download Citation Request Permissions

Abstract | References | Full Text: HTML, PDF (Size: 100K) | Related Articles | Citation Tracking

Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems

Irma N. G. Rivera ^1,2 *^†, Erin K. Lipp ^1,3 , Ana Gil ⁴ , Nipa Choopun ¹ , Anwar Huq ^1,5 and Rita R. Colwell ^1,5

¹ Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA.
² Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, CEP 05508-900, SP, Brazil.
³ Department of Environmental Health Science, University of Georgia, Athens, GA 30602, USA.
⁴ Instituto de Investigación Nutricional (IIN), La Molina Lima 18, Peru.
⁵ Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.

*For correspondence. E-mail igrivera@usp.br; Tel. (+55) 11 3091 7204; Fax (+55) 11 3091 7354.

^† Present address: Departamento de Microbiologia, ICB-USP, 1374 Lineu Prestes Av. – Edif. ICB II, USP-S.P., S. Paulo, Brasil – CEP 05508-900.

Summary

Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 µg µl⁻¹. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.