Optimization of Sample Processing for Droplet Digital PCR Quantification of Campylobacter coli and Campylobacter...

Accurate detection of Campylobacter in chicken liver is hindered by strong matrix inhibition. This study evaluated sample-processing strategies to improve droplet digital PCR (ddPCR) quantification of Campylobacter coli and Campylobacter jejuni in chicken liver. Mechanical homogenization (Stomacher) and enzymatic/mechanical dissociation (gentleMACS), with and without 8 μm filtration, were compared. Particle-size analysis showed that filtration, especially following gentleMACS treatment, produced smaller, more uniform particles and reduced variability. Percent-degradation assays confirmed that gentleMACS achieved substantially greater tissue disruption than Stomacher homogenization. The multiplex ddPCR assay, which simultaneously targets C. coli and C. jejuni, produced droplet counts comparable to single-target reactions, indicating minimal interference between targets under the conditions tested. In inoculated liver samples, gentleMACS processing yielded droplet counts similar to those obtained from pure cultures, whereas unprocessed liver caused severe matrix interference and inconsistent quantification. Furthermore, gentleMACS-treated samples exhibited strong log-to-log linearity for quantifying C. coli and C. jejuni, enabling detection near 1 genome copy equivalent per reaction. Overall, the results indicate that enzymatic/mechanical dissociation combined with fine-pore filtration improves ddPCR detection of Campylobacter species in chicken liver.