Real-Time PCR optimization to identify environmental Vibrio spp. strains

Other titles Optimisation de PCR en temps réel pour l'identification de souches environnementales de Vibrio spp.
Type Article
Date 2012-08
Language English
Author(s) Tall Amadou1, Teillon Anna1, Boisset Claire2, Delesmont R.3, Touron-Bodilis A.4, Hervio-Heath Dominique1
Affiliation(s) 1 : IFREMER, Ctr Brest, Unite Environm Microbiol & Phycotoxines, Dept Ressources Biol & Environm,Lab Microbiol LNR, F-29280 Plouzane, France.
2 : IFREMER, Ctr Brest, Dept Ressources Biol & Environm, Lab Biotechnol & Mol Marines, F-29280 Plouzane, France.
3 : Eurofins, IPL Nord, Gravelines, France.
4 : EDF R&D, Lab Natl Hydraul & Environm, Chatou, France.
Source Journal Of Applied Microbiology (1364-5072) (Wiley-blackwell), 2012-08 , Vol. 113 , N. 2 , P. 361-372
DOI 10.1111/j.1365-2672.2012.05350.x
WOS© Times Cited 21
Keyword(s) dnaJ, identification, real-time PCR, Vibrio alginolyticus, Vibrio cholerae, Vibrio vulnificus
Abstract Aims: To identify Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus using standardized DNA extraction method and real-time PCR assays, among a large number of bacterial strains isolated from marine environment. Methods and Results: Methods for DNA extraction and real-time PCR were standardized to identify a large number of Vibrio spp. strains isolated through regular collection campaigns of environmental samples. Three real-time PCR assays were developed from a multiplex PCR, targeting V. vulnificus, V. cholerae and V. alginolyticus on the dnaJ gene. After testing their specificity, these systems were applied for the identification of 961 strains isolated at 22°C (446 strains) and 37°C (515 strains) in September 2009. The predominance of V. alginolyticus (82·6%) among the Vibrio spp. strains isolated at 37°C was shown. At 22°C, only 1·6% of the strains were identified by PCR and they were V. alginolyticus. Conclusions: Reproducible and specific real-time PCR assays combined to a DNA extraction method on microplates were used to constitute a large environmental Vibrio strains collection and to identify and detect potential human pathogenic Vibrio isolated at 37°C. For environmental strains isolated at 22°C, because of the higher species diversity, other approaches, like sequencing, should be chosen for identification. Significance and Impact of the Study: The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment in long-term studies.
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