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Page No. : 32-39

ABSTRACT

Chicken and pork are considered as main sources of Campylobacter jejuni and Campylobacter coli and culture-based methods for their detection are time-consuming. In the current study, SYBR Green-based real-time (RTi) PCR assays were developed for culture-independent/dependent detection of C. jejuni and C. coli in chicken and pork, respectively. Specificity was demonstrated with genomic DNA extracted from the C. jejuni, C. coli and non-Campylobacter bacterial genera. Ten fold serial dilution of bacterial DNA was prepared and used for determining the analytical sensitivity. Artificial spiking experiments were carried out in chicken and pork. The standardized real-time assay did not reveal any cross-reactivity with other bacterial genera revealing its high specificity for C. jejuni and C. coli. The analytical sensitivity of the RTi PCR and PCR assays was 500 fg and 50 pg, respectively. In artificial spiking experiment, the limit of detection of 1000 CFU/g and 100 CFU were achieved without enrichment and 12 h enrichment, respectively. The RTi PCR assay displayed significant enhancement (10-fold) in the sensitivity and limit of detection as compared to conventional endpoint PCR. The specificity, sensitivity, NPV, PPV and accuracy of the developed RTi PCR assays for detection of C. jejuni and C. coli were calculated to be 100%.

Keywords: Campylobacter jejuni, Campylobacter coli, chicken, pork, enrichment, real-time PCR