1 Division of Microbiology and Risk Assessment, National Food Institute, Technical University of Denmark2 National Food Institute, Technical University of Denmark3 Section of Poultry Diseases, Division of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark4 Division of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark5 National Veterinary Institute, Technical University of Denmark6 Section for Veterinary Epidemiology and public sector consultancy, Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark7 Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark8 Division of Food Microbiology, National Food Institute, Technical University of Denmark9 unknown10 Department of Applied Mathematics and Computer Science, Technical University of Denmark
Certified Campylobacter-free poultry products have been produced in Denmark since 2002, the first example of fresh (unprocessed and nonfrozen) chickens labeled "Campylobacter free." This success occurred partly through use of a 4-hour gel-based PCR testing scheme on fecal swabs. In this study, a faster, real-time PCR approach was validated in comparative and collaborative trials, based on recommendations from the Nordic system for validation of alternative microbiological methods (NordVal). The comparative real-time PCR trial was performed in comparison to two reference culture protocols on naturally contaminated samples (99 shoe covers, 101 cloacal swabs, 102 neck skins from abattoirs, and 100 retail neck skins). Culturing included enrichment in both Bolton and Preston broths followed by isolation on Preston agar and mCCDA. In one or both culture protocols, 169 samples were identified as positive. The comparative trial resulted in relative accuracy, sensitivity, and specificity of 98%, 95%, and 97%, respectively. The collaborative trial included nine laboratories testing neck skin, cloacal swab, and shoe cover samples, spiked with low, medium, and high concentrations of Campylobacter jejuni. Valid results were obtained from six of the participating laboratories. Accuracy for high levels was 100% for neck skin and cloacal swab samples. For low levels, accuracy was 100% and 92% for neck skin and cloacal swab samples, respectively; however, detection in shoe cover samples failed. A second collaborative trial, with an optimized DNA extraction procedure, gave 100% accuracy results for all three spiking levels. Finally, on-site validation at the abattoir on a flock basis was performed on 400 samples. Real-time PCR correctly identified 10 of 20 flocks as positive; thus, the method fulfilled the NordVal validation criteria and has since been implemented at a major abattoir.
Applied and Environmental Microbiology, 2006, Vol 72, Issue 8, p. 5463-8