Molecular Methods for Campylobacter and Arcobacter Detection

Abstract

Twenty species and six subspecies of the genera Arcobacter and Campylobacter have been described to date. All are Gram-negative, microaerophilic, curved, spiral or S-shaped cells, and are members of the order Campylobacterales, class Epsilonproteobacteria phylum Proteobacteria. Though most members are pathogenic, C. jejuni, C. coli and A. butzleri are the most frequently isolated species from patients suffering from gastrointestinal illness. The current methods for their detection, identification, and differentiation are cumbersome, time consuming and lack specificity. DNA based molecular techniques including real-time Polymerase Chain Reaction (PCR) and Fingerprinting methods Terminal Restriction Fragments Length Polymorphism (T-RFLP) and Ligase Detection Reaction (LDR) have been used in this project to develop rapid detection and identification methods for Campylobacter and Arcobacter species. Five real-time PCR methods were developed which include: (a) rapid detection and identification of Campylobacter species using real-time PCR adjacent hybridisation probes, (b) rapid identification of C. jejuni using SYBR Green I, (c) rapid detection and differentiation of Arcobacter species using adjacent hybridisation probes, (d) rapid detection and differentiation of Arcobacter species and the Campylobacter group (C. coli, C. jejuni, C. lari, C. hyoilei, C. helviticus, C. hyointestinalis, C. insulaenigrae, C lanienae) using melting temperature (Tm) of adjacent hybridisation probes, and (e) a one tube real-time PCR multiplex for the rapid detection and identification of Campylobacter species, C. coli and C. jejuni using a TaqMan Probe, in an iCycler iQTM (BioRad, USA) and Light CyclerTM (Idaho Technology, USA). [Continued ...]