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Open Access Methodology article

PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

Maria Hoffmann12*, Eric W Brown1, Peter CH Feng1, Christine E Keys1, Markus Fischer2 and Steven R Monday1

Author Affiliations

1 Division of Microbiology, Office of Regulatory Science Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Paint Branch Parkway, College Park, MD 20740, USA

2 Institute of Food Chemistry, University of Hamburg, Grindelallee 117, D-20146 Hamburg, Germany

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BMC Microbiology 2010, 10:90  doi:10.1186/1471-2180-10-90

Published: 23 March 2010

Abstract

Background

The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species.

Results

In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well.

Conclusion

This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations providing information more quickly than other time-honoured methods traditionally used in these types of analyses.