Evaluation of a Combined Culture and PCR Method (NMKL-163A) for Detection of Presumptive Pathogenic Yersinia enterocolitica in Pork Products

Abstract

A combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food (NMKL-163A) was evaluated by testing samples of artificially and naturally contaminated pork. The performance of the pre-PCR sample treatment, buoyant density centrifugation, was first compared with two commercially available methods (DNeasy tissue kit and PrepMan). We found that similar sensitivity was reached (i.e., 25 CFU/g of food was detected by single PCR) with the buoyant density centrifugation and the DNeasy Tissue kit when tested on overnight enrichments. However, the DNeasy tissue kit was superior when tested on nonenriched homogenates; the detection limit was 25 CFU/g in minced beef by single PCR and 25 CFU/g in sausage by nested PCR. We then analyzed 100 raw minced pork samples. Thirty-five tested positive for presumptive pathogenic Y. enterocolitica when analyzed by the NMKL-163A method, whereas none tested positive when analyzed in parallel by a standard culture method (ISO 10273). We also analyzed 97 samples of cold-smoked pork sausage, of which approximately 11% tested positive by the NMKL-163A method. This study showed that sensitivities such as those obtained by nested PCR were required for detection of the pathogen in naturally contaminated samples, and therefore the nested PCR primers, which are included in the NMKL-163A method only as an option, need to be validated and applied routinely.