AMB Volume 40, Issue 4, 2024 Pages 485-493 https://doi.org/10.59393/amb24400408
Optimization and Evaluation of Real-Time PCR for Detection and Identification of Campylobacter jejuni and Campylobacter coli from Meat and Surface Swabs Samples
El Baaboua A., El Maadoudi M., Belmehdi O., Kounnoun A., Bouyahya A., Skali-Senhaji N., Abrini J.
The current study aimed to optimize the real-time polymerase chain reaction (qPCR) specificity and sensitivity on isolated strains of Campylobacter and on enriched broth for direct detection of these cells. To achieve this, the HipO and CadF gene were purchased to target Campylobacter jejuni and Campylobacter coli, respectively. Using the chessboard titration method, three critical reagent concentrations of qPCR were described and optimized. After that, 130 of the collected isolates from meat and swab samples were confirmed by the optimized 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 (ΔRn ˃ 225.000) and the amendment of the amount of specific primers, and 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 surface swabs by qPCR optimized protocols demonstrated similar outcomes as phenotypic tests. Of 27 enriched broiler chicken broths, C. coli was the most isolated strain by culture method, compared to C. jejuni. In contrast, the application of qPCR directly on enriched broth revealed a higher percentage (74.07 %) of co-existence of C. coli and C. jejuni from the same analyzed samples. The present study optimized reliable, rapid, robust, and specific qPCR methods for screening, quantifying, and confirming Campylobacter.
Keywords: Campylobacter, CadF gene, HipO gene, food safety, real-time PCR.
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