Direct Quantification of Campylobacter jejuni in Chicken Fecal Samples Using Real-Time PCR: Evaluation of Six Rapid DNA Extraction Methods

Direct and accurate quantification of Campylobacter in poultry is crucial for the assessment of public health risks and the evaluation of the effectiveness of control measures against Campylobacter in poultry. The aim of this study was to assess several rapid DNA extraction methods for their effectiveness for the direct quantification (without enrichment) of Campylobacter jejuni in chicken fecal samples using real-time PCR. The presence of inhibitory substances in chicken fecal samples may reduce or even completely impede the PCR amplification process making quantification very difficult. Six rapid DNA extraction methods were compared based on their limit of detection, efficiency, reproducibility, and precision. Standard curves were designed for all the methods tested in order to assess their performance on the direct quantification of C. jejuni in chicken fecal samples. As a result of this study, the Easy-DNA (Invitrogen) method generated lower Ct values, the best amplification efficiency (AE = 93.2 %) and good precision (R squared = 0.996). The method NucleoSpinA (R) Tissue was able to detect samples spiked with the lowest Campylobacter concentration level (10 CFU/ml) but the amplification efficiency was not optimal (AE = 139.5 %). DNA extraction methods Easy-DNA Invitrogen, MiniMAGA (R) and NucleoSpinA (R) Tissue produced good real-time PCR reproducibility generating standard deviations from 0.3 to 0.8 between replicates.