Simultaneous Detection and Differentiation of Campylobacter jejuni, C. coli, and C. lari in Chickens Using a Multiplex Real-Time PCR Assay

Abstract

A multiplex real-time PCR (qPCR) assay was developed for simultaneous detection and differentiation of the three most important Campylobacter species in chickens. Three novel sets of PCR primers and TaqMan probes were designed to amplify the unique DNA sequences within the hipO, cdtA, and pepT genes which are specific to Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, respectively. To avoid competition in the multiple target amplifications, the concentrations of primers and probes were optimized. By using the optimized qPCR conditions together with a minor-groove binding probe of pepT, amplification efficiency greater than 92% and detection sensitivity of 38 genome copies/reaction have been achieved for all three targets. The assay was highly specific for C. jejuni, C. coli, and C. lari with testing of 33 Campylobacter strains and 20 non-Campylobacter strains. In chicken samples spiked with known quantities of Campylobacter cells, the assay was able to detect 1 CFU/g after a 24-h enrichment. Application of the assay in food was further evaluated using 21 fresh chicken samples obtained from local supermarkets. The results revealed that, after a 24-h or 48-h enrichment, 14 samples (66.7%) were positive for C. jejuni, five samples (23.8%) were positive for C. coli, and none of the samples was contaminated by C. lari. Taken together, the multiplex qPCR assay combined with an enrichment step is a sensitive, species-specific, and non-labor-intensive method suitable for rapid detection of C. jejuni, C. coli, and C. lari in chicken samples.