Rapid identification of Campylobacter species by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA

Abstract

Restriction fragment length polymorphism analysis of a PCR-amplified DNA fragment of the gene coding for 16S rRNA was performed on 148 previously characterized strains of Campylobacter, Helicobacter, Arcobacter, and Wolinella succinogenes and 13 Campylobacter-like isolates. These strains included clinical, animal, and environmental isolates. PCR amplification generated a 283-bp fragment from all species. The amplicon from each strain was digested with six restriction endonucleases (AccI, AvaI, DdeI, HaeIII, HpaII, XhoI). DdeI was useful for the initial grouping of the strains. Additional discrimination within the different DdeI groups was obtained with AccI, HaeIII, HpaII, and XhoI digestions. The PCR-restriction fragment length polymorphism analysis allowed for the discrimination of members of the genus Campylobacter from members of closely related genera and discrimination between Campylobacter species. The proposed method is simple and rapid and can be useful for the routine identification of Campylobacter-like organisms in clinical or epidemiologic studies.

Applicability of a rapid duplex real-time PCR assay for speciation ofCampylobacter jejuniandCampylobacter colidirectly from culture plates

“…This lack of specificity in the Taqman assay is being addressed, in order to determine whether it is failure of the primers or probes, or the absence or variation in the genes, which is responsible for this small proportion of negative results. Eleven strains (0.2%) were identified as non‐ C. jejuni / coli but as other Campylobacter species (two C. upsaliensis , one C. fetus and eight C. lari ), using 16S rRNA RFLP [16].…”

“…Following the initial trial of 60 isolates, all subsequent isolates were serotyped, but only isolates with contradictory serotype (or non‐serotypeable) and Taqman speciation were validated with the other methods above. Further validation was carried out where required, using a campylobacter 16S rRNA restriction fragment length polymorphism (RFLP) strategy based on amplification of a region of the 16S rRNA gene, followed by digestion with a combination of the restriction endonucleases Dde I, Taq I and Bsr I to identify C. lari , C. upsaliensis or C. fetus [16]. Where required, sequencing of the mapA and ceuE genes was carried out by standard protocols using a Beckman CEQ8000 sequencer.…”

Rapid identification of diverse Campylobacter lari strains isolated from mussels and oysters using a reverse hybridization line probe assay

“…Several DNA-based methods have been developed for the detection and identification of Campylobacter species in food, clinical and environmental samples (Oyofo et al 1992;Oyofo and Rollins 1993;Eyers et al 1993;Uyttendaele et al 1994;Korolik et al 1995;Stucki et al 1995;Cardarelli-Leite et al 1996;Jackson et al 1996;Giesendorf et al 1992Giesendorf et al , 1993. Although these methods are faster than conventional methods, the efficacy is often limited, since they require multiple PCR reactions per sample or digestions with multiple restriction enzymes.…”

Molecular detection identified a type six secretion system in Campylobacter jejuni from various sources but not from human cases

“…PCR confirmation of Camp. jejuni was performed on 16S rRNA gene and hipO (hippurate hydrolysis gene) (Cardarelli-Leite et al 1996;Atanassova and Ring 2000).…”