Digital Droplet-PCR for Quantification of Viable Campylobacter jejuni and Campylobacter coli in Chicken Meat Rinses

Abstract

The EU commission established Regulation (2017/1495) in 2017 to reduce Campylobacter on chicken skin and to decrease the number of human cases of campylobacteriosis attributable to the consumption of poultry meat. A Process Hygiene Criterion based on colony-forming unit data was set to a maximum of 1000 CFU Campylobacter spp. per gram chicken neck skin at slaughterhouses. Confronted with stressors, including cold, oxidative stress or antibiotic treatment, live cells may enter into a viable but non-cultivable state (VBNC) and lose the ability to grow, in reference to the plate count ISO 10272-2:2017 method, but still possess the potential to recover and cause infections under favorable conditions. In this study, a droplet digital PCR combined with the intercalating dye propidium monoazide (PMA) was established for quantification of C. coli and C. jejuni in chicken meat rinses. The PMA was used to inactivate DNA from dead cells in this technique. This method was successfully validated against the reference method according to ISO 16140-2:2016 for accuracy and relative trueness. Additionally, it presented a 100% selectivity for Campylobacter jejuni and C. coli. Moreover, the technical measurement uncertainty was determined according to ISO 19036:2019, and the applicability of ddPCR for quantifying C. coli and C. jejuni in chicken meat rinses was investigated on naturally contaminated samples from slaughterhouses and supermarkets. Results obtained from this study demonstrated a strong correlation to qPCR as well as the classical microbiological reference method.

Molecular detection and characterization of foodborne bacteria: Recent progresses and remaining challenges

“…Shortcomings of the ddPCR compared to qPCR remain the high cost of instrumentations and consumables, and the inaccuracy in quantifying target DNA molecules present at high concentrations in the sample, or present in samples containing a high level of contaminants (Govindaswamy et al., 2022; Peruzy et al., 2020; Porcellato et al., 2016; Wang et al., 2018). ddPCR technique warrants further exploration and technical optimization before it can be routinely used in food laboratory settings.…”

Multiplex Real-Time PCR for the Detection of Tetracycline, Ciprofloxacin, and Erythromycin Resistance Determinants from Human and Foodborne Campylobacter jejuni and Campylobacter coli

“…The DNA copy number was adjusted to 5000 copies/µL DNA in ddPCR [41] based on an absolute quantification of DNA copy number. All DNAs were diluted to five dilution levels (5000, 1000, 500, 100, and 50 copies/µL DNA).…”