Detection of Campylobacter spp. in chicken fecal samples by real-time PCR

Abstract

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.

FIG. 1.

Log-linear plot of the amplification profile for fecal samples spiked with serial 10-fold dilutions of C. jejuni in eight replicates (A) and C. coli in six replicates (B). The quantity of Campylobacter in the samples from which the DNA is isolated is plotted against the threshold cycle.