title: Development of a real-time fluorescence resonance energy transfer PCR to detect arcobacter species.
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 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 degrees C, 58.4 degrees C, 60.6 degrees C, and 51.8 degrees 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.
authors.affiliation: Université Victor Segalen Bordeaux 2, Centre National de Référence des Helicobacters et Campylobacters, F33076 Bordeaux, France.
authors.forename: Khalil
authors.initials: K
authors.lastname: Abdelbaqi
authors.forename: Alice
authors.initials: A
authors.lastname: Buissonnière
authors.forename: Valérie
authors.initials: V
authors.lastname: Prouzet-Mauleon
authors.forename: Jessica
authors.initials: J
authors.lastname: Gresser
authors.forename: Irene
authors.initials: I
authors.lastname: Wesley
authors.forename: Francis
authors.initials: F
authors.lastname: Mégraud
authors.forename: Armelle
authors.initials: A
authors.lastname: Ménard
mesh_terms: D000328:Adult; D000368:Aged; D020640:Arcobacter / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics; D029779:Cupressaceae; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D003967:Diarrhea / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D027081:DNA Gyrase