Development of a Sampling and Real-time PCR Method for the Quantitative Detection of Campylobacter spp. in Retail Chicken Meat Without DNA Extraction

Abstract

Campylobacter food poisoning is caused by consumption of the contaminated foods, especially poultry meat. Continuous quantitative measurement of Campylobacter spp. in contaminated foods is crucial to develop preventive measures. We developed a direct-qPCR method for determining the viable cell counts of Campylobacter spp. using qPCR without DNA extraction from enriched food samples and a sampling method (the wrap procedure) in which the sample is wrapped in a sheet, different from the conventional homogenization procedure.The viable cell counts of Campylobacter spp. before and after enrichment of the samples sampled using the wrap and homogenization procedures from chicken samples inoculated with Campylobacter jejuni were determined using the culture method, and the cycle threshold (CT) values after enrichment were determined using the direct-qPCR. An enrichment regression equation was generated from the viable cell counts obtained before and after enrichment, and a direct-qPCR regression equation was generated from the CT values and viable cell counts obtained after enrichment, enabling the viable cell counts before enrichment to be estimated from the CT values.Estimated viable cell counts were similar for the culture method when sampled by the homogenization procedure, but lower for the wrap procedure. However, the detection rate of direct-qPCR was 37.5% for liver and 89.7% for breast fillet using the homogenization procedure, whereas using the wrap procedure, it was 100% for both samples.The detection rate of direct-qPCR for retail chicken was 30.4–35.7% for the homogenization procedure, and 85.7–100% for the wrap procedure. Colonies were observed using the culture method, but their quantification was difficult due to swarming or their low number. However, estimating viable cell counts using the combination of wrap procedure and direct-qPCR methods is possible.The developed method can provide baseline data for the risk assessment Campylobacter food poisoning.