New Approaches on Quantification of Campylobacter jejuni in Poultry Samples: The Use of Digital PCR and Real-time PCR against the ISO Standard Plate Count Method

Abstract

Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

Detection and Quantification of Viable but Non-culturable Campylobacter jejuni

“…In the preliminary spiking experiment, the recovery rate of culturable C. jejuni cells on chicken samples was 84.14%. The LOD of PMA-qPCR in the current study was improved in comparison to a previous study, in which qPCR exhibited a LOD of 1.2 × 10 4 CFU/g for the quantification of viable C. jejuni in poultry neck-skin samples (Papic et al, 2017). Others employing PMA-qPCR to quantify Campylobacter in chicken carcasses reported LOD values as low as 10 CFU/g (Rantsiou et al, 2010) and 2 log CFU/ml (Josefsen et al, 2010).…”

Comparison among the Quantification of Bacterial Pathogens by qPCR, dPCR, and Cultural Methods

“…In this regard, two recent papers reported similar results: one was addressed to the quantification poultry pathogens (Rothrock et al, 2013) and the other to the quantification of Campylobacter jejuni (Papic et al, 2017). A table summarizing these observations is reported in Data Sheet 2 (Supplementary Material 2).…”

Knowledge gaps in control ofCampylobacterfor prevention of campylobacteriosis

“…Propidium monoazide intercalates into the double‐helical DNA from dead cells with compromised membranes exposure to visible light cross‐links the two strands of DNA, preventing PCR amplification. Optimization of molecular‐based methods to enable reliable Campylobacter quantification reduces time and expense and facilitates Campylobacter surveillance throughout the food production chain (Papić et al., ).…”