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Applied and Environmental Microbiology, August 2004, p. 4468-4477, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4468-4477.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR

Cinta Rachman,1 Petia Kabadjova,1,2 Rosica Valcheva,1 Hervé Prévost,1* and Xavier Dousset1

Unité de Recherche Qualité Microbiologique et Aromatique des Aliments, Laboratoire de Microbiologie Alimentaire et Industrielle, ENITIAA, 44322 Nantes Cedex 3, France,1 Department of Microbiology, Faculty of Biology, University of Sofia "St. Kliment Ohridski," 1421 Sofia, Bulgaria2

Received 12 November 2003/ Accepted 29 March 2004

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNAAla and tRNAIle, which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.

* Corresponding author. Mailing address: QM2A, LMAI, ENITIAA, rue de la Géraudière, BP 82225, 44322 Nantes Cedex 3, France. Phone: 33-2-51785524. Fax: 33-2-51785520. E-mail: prevost{at}enitiaa-nantes.fr.

Applied and Environmental Microbiology, August 2004, p. 4468-4477, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4468-4477.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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