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Journal of Clinical Microbiology, September 2007, p. 3015-3021, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00256-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of a Real-Time Fluorescence Resonance Energy Transfer PCR To Detect Arcobacter Species

Université Victor Segalen Bordeaux 2, Centre National de Référence des Helicobacters et Campylobacters, F33076 Bordeaux, France,¹ INSERM U853, F33076 Bordeaux, France,² National Animal Disease Center, ARS, USDA, 2300 Dayton Road, Ames, Iowa³

Received 1 February 2007/ Returned for modification 26 March 2007/ Accepted 17 May 2007

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 done 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°C, 58.4°C, 60.6°C, and 51.8°C for the 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. The assay was then 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.

Published ahead of print on 25 July 2007.

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