Combining enrichment with multiplex real-time PCR leads to faster detection and identification of Campylobacter spp....

Conventional protocols for the detection of Campylobacter from foods are laborious and time-consuming. This research describes an alternative procedure (EMRT-PCR) for the detection of Campylobacter from food by combining ISO 10272-1:2017 enrichment in Bolton broth (BB) with a multiplex real-time (MRT-) PCR assay. Species differentiation was done by targeting C. jejuni (mapA), C. coli (ceuE), and both species (cje). The detection limit of the MRT-PCR assay was 4.5 and 5.5 log₁₀ cfu/ml in BB and BB containing chicken skin, respectively. A Monte Carlo simulation was conducted to predict the probability that campylobacters reach the MRT-PCR detection threshold throughout enrichment in BB, and results suggested that cold-stressed campylobacters could reach the detection limit after 40 h of enrichment (p = 0.99). As a proof of principle, 23 naturally contaminated meat products were enriched according to ISO 10272-1:2017 procedure A, and the EMRT-PCR in parallel. After 24 h, 12 and 11 samples already tested positive for Campylobacter with the ISO method and EMRT-PCR, respectively. After 40 h, the 24-h-negative sample was also positive with EMRT-PCR. The EMRT-PCR takes about 2 days to produce reliable results, while results using ISO 10272-1:2017 can take up to 8 days, which demonstrate the potential of the EMRT-PCR method.