Purpose: The purpose of this study was to design and then validate a new detection protocol using current media and molecular techniques against the reference FDA method.
Methods: Pure cultures of ten different strains of Campylobacter (jejuni & coli) and five cultures of non-Campylobacter strains were serially diluted and inoculated at different levels (5, 50,125 CFU/25g) into separate 25g samples of raw silo milk. Each sample was then enriched in 100 ml of Bolton Broth without blood and incubated in microaerophilic conditions at 41-42°C for 24 hrs. For each sample, 10 μl was streaked in duplicate onto R & F® Campylobacter Chromogenic Plating Medium (CCPM) and modified Cefoperazone Charcoal Deoxycholate Agar (mCCDA) which was then incubated in microaerophilic conditions at 41-42°C for 48 hrs. Confirmation was conducted by real-time PCR (qPCR) with a Cepheid Smartcycler utilizing primers and probes (FAM-cj and TxR-cc) within the ceuE gene of Campylobacter.
Results: At the lowest spiking level (5 cfu/25g), 100% cells were recovered with CCPM compared to 0% with mCCDA. At the low spiking level (50 cfu/25g), the results showed 100% recovery with CCPM compared to 20% with mCCDA. At the high spiking level (125 cfu/25g), both CCPM and mCCDA recovered 100% cells. Confirmation, using a qPCR, verified all the positive isolates as either C. jejuni or coli.
Significance: In conclusion, CCPM is a more viable choice for detection at low levels and our qPCR protocol is a demonstrated confirmation step for C. jejuni and coli. More research with a larger sample size is needed to determine the significant differences for recovering low levels of Campylobacter from complex food matrices.