Development and Evaluation of Real Time PCR for Detection and Identification of C. jejuni and C. coli from Human Food

Abstract

The current study aimed to develop, optimize, and evaluate the real-time polymerase chain reaction (qPCR) specificity and sensitivity on pure isolated strains of Campylobacter and on enriched broth for direct detection of these cells. To achieve this, HipO and CadF gene were purchased to target C. jejuni and C. coli, respectively. Using chessboard titration method, three critical reagents concentration of qPCR were optimized and described. After that, 130 of collected isolates from food samples were confirmed by the developed protocols. Lastly, 27 enriched broths culture-positive for Campylobacter spp. were subjected to direct qPCR. The earlier results indicated that the diluted DNA templates improved the amplification plot intensity (∆Rn ˃ 225.000) and the amendment of the amount of specific primers, probes, as well as the concentration of MgCl2 on the amplification curves affected positively the fluorescence signal for both C. jejuni and C. coli established protocols. The confirmation of 130 of C. jejuni and C. coli isolated from broiler chickens, turkeys, cattle, and handling surface swabs by qPCR optimized protocols demonstrated similar outcomes as phenotypic tests. From 27 broiler chickens’ samples, C. coli were the most isolated strain from culture method, biochemically confirmed, compared to C. jejuni. In contrast, the application of qPCR directly on enriched broth revealed higher percentage (74.07%) of co-existence of C. coli and C. jejuni from the same analyzed samples. The present study developed and validated reliable, rapid, robust, and specific qPCR method for screening, quantifying, and confirming Campylobacter in food safety monitoring.