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Title: SPECIES-SPECIFIC IDENTIFICATION OF CAMPYLOBACTERS BY PCR-RESTRICTION FRAGMENT LENGTH POLYMORPHISM AND PCR TARGETING OF THE GYRASE B GENE

Author
item KAWASAKI, SUSUMU - NATL FOOD RESEARCH INSTIT
item Fratamico, Pina
item Wesley, Irene
item KAWAMOTO, SHINICHI - NATL FOOD RESEARCH INSTIT

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/13/2008
Publication Date: 4/1/2008
Citation: Kawasaki, S., Fratamico, P.M., Wesley, I.V., Kawamoto, S. 2008. Species-specific identification of campylobacters by PCR-restriction fragment length polymorphism and PCR targeting of the gyrase b gene. Applied and Environmental Microbiology. 74(8):2529-2533.

Interpretive Summary: Campylobacter species are the most common cause of bacterial gastrointestinal infection in the United States and in many other countries. Because of technical limitations in methods currently employed for detection, isolation, typing, and differentiation of campylobacters, species other than Campylobacter jejuni are likely under-reported in clinical specimens, and it is difficult to determine sources of infection and the prevalence of these organisms in the environment. To address this problem, methods to detect, identify, and discriminate among Campylobacter species were developed based on the Campylobacter gyrase B gene. These include a DNA amplification method called the polymerase chain reaction (PCR), which yielded products of unique sizes for each of 12 Campylobacter species and a method based on the PCR followed by cutting of the resulting PCR product with specific enzymes yielding a unique fingerprint for each Campylobacter species. The latter method is referred to as PCR-restriction fragment length polymorphism (PCR-RFLP). These methods can be used to precisely discriminate among the different species of Campylobacter. In conclusion, PCR-RFLP analysis and the species-specific PCR assays provide valuable tools for clinical and veterinary diagnostics, the food industry, and regulatory agencies for rapid detection and unambiguous identification of the majority of Campylobacter species.

Technical Abstract: Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays and the existence of atypical strains. The phylogeny of 12 Campylobacter species was studied based on partial (1020-bp) gyrB gene (DNA topoisomerase beta-subunit gene) sequences. The topology of the phylogenic neighbor-joining tree based on the gyrB gene was similar to that of the tree based on the 16S rDNA gene previously reported. However, gyrB provides a better resolution for Campylobacter species with interspecies sequence similarities ranging from 58.3 to 89.2 %. A universal primer set designed to amplify a 900-bp fragment of the gyrB gene in Campylobacter spp. was developed and used for PCR-restriction fragment length polymorphism (PCR-RFLP) of 19 strains representing 12 Campylobacter species, including C. jejuni subsp. jejuni, C. coli, C. concisus, C. curvus; C. showae, C. mucosalis, C. fetus, C. hyointestinalis, C. sputorum biovar bubulus, C. helveticus, C. upsaliensis, and C. lari. Digestion of the 900-bp fragment with either DdeI or XspI restriction enzymes resulted in unique digest patterns for all 12 Campylobacter species; however, DdeI digestion could not distinguish between C. concisus and C. sputorum. In addition, PCR assays were developed using species-specific primer sets for amplification of regions of the gyrB gene specific for each Campylobacter species, yielding products ranging in size from 86 to 493 bp. Specificity testing using DNA from Campylobacter spp., Arcobacter spp., Helicobacter spp., and other bacterial genera, including Escherichia coli and Salmonella spp. showed that the Campylobacter species-specific primer sets were highly specific for the respective target species. In conclusion, PCR-RFLP analysis, and PCR using the species-specific identification primer sets based on the gyrB gene provide valuable tools for rapid detection and unambiguous identification of the majority of Campylobacter species.