Real-time PCR optimization to identify environmental  Vibrio spp. strains.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Département Ressources Biologiques et Environnement, Unité Environnement Microbiologie et Phycotoxines, Ifremer, Centre de Brest, ZI de la Pointe du Diable, Plouzané, France.
A
A
Tall
A
A
Teillon
C
C
Boisset
R
R
Delesmont
A
A
Touron-Bodilis
D
D
Hervio-Heath
D004269:DNA, Bacterial / Q000302:isolation & purification; D004783:Environmental Microbiology; D005602:France; D019015:Geologic Sediments / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012623:Seawater / Q000382:microbiology; D012680:Sensitivity and Specificity; D013696:Temperature; D044149:Vibrio alginolyticus / Q000235:genetics / Q000302:isolation & purification*; D014734:Vibrio cholerae / Q000235:genetics / Q000302:isolation & purification*; D041261:Vibrio vulnificus / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial