Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2007
Publication Date: 7/20/2007
Citation: Avdelbaqi, K., Buissoniere, A., Prouzet-Mauleon, V., Gesser, J., Wesley, I.V., Megraud, F., Menard, A. 2007. Development of a real-time fluorescence resonance energy transfer PCR to detect Arcobacter species. Journal of Clinical Microbiology. 45(9):3015-3021.
Interpretive Summary: Arcobacter butzleri is a newly described emerging human food-borne pathogen linked to consumption of contaminated poultry and water. It is isolated from livestock and poultry carcasses as well as from human cases of gastroenteritis. Isolation is time consuming and identification is based on PCR analysis. We developed a real-time PCR assay to detect, identify, and ultimate quantify this fastidious microbe. The assay detected Arcobacter in 1% of fecal samples (n=345) thus bypassing the need for cultural isolation and demonstrating the suitability of using this assay to screen field samples.
Technical Abstract: A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was made by probe hybridization and melting curve analysis, using Fluorescence Resonance Energy Transfer technology. Discrimination between Arcobacter species was straightforward, as the corresponding melting points showed significant differences with the characteristic melting temperatures of 63.5 deg C, 58.4 deg C, 60.6 deg C and 51.8 deg C, for Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter nitrofigilis type strains, respectively. The specificity of this assay was confirmed with pure cultures of 106 Arcobacter isolates from human clinical and veterinary specimens, identified by phenotypic methods and 16S rRNA gene sequencing. Next, the assay was used to screen 345 clinical stool samples obtained from patients with diarrhea. The assay detected A. butzleri in four of these clinical samples (1.2%). These results were confirmed by a conventional PCR method targeting the 16S rRNA gene with subsequent sequencing of the PCR product. In conclusion, this real-time assay detects and differentiates Arcobacter species in pure culture as well as in the competing microbiota of the stool matrix. The assay is economical since only one biprobe is used and multiple Arcobacter species are identified in a single test.