Development of a PCR-based method for detection of pathogenic Yersinia enterocolitica in pork

Thisted Lambertz, S. 2005. Development of a PCR-based method for detection of pathogenic Yersinia enterocolitica in pork. Doctor ́s dissertation. ISSN 1652-6880, ISBN 91-576-6922-8 During the last decade, Yersinia enterocolitica has been reported to cause between 500 and 800 cases of human gastroenteritis per year in Sweden. As pigs are the only animals in human food production that regularly harbour the pathogen, pork is probably an important source of infection. Earlier it has only rarely been possible to recover the bacterium from pork, but in the last few years this was made possible by DNA-based technology. In this project, a PCR-based method for the detection of pathogenic Y. enterocolitica in pork was developed. The chromosome-located gene attachment invasion locus (ail) was chosen as the PCRtarget. The ail PCR assay was evaluated according to criteria for a standardised PCR-based method set by the European research project FOOD-PCR. In a trial involving 14 European laboratories, the ail PCR assay showed high repeatability and robustness. The complete PCR-based method comprises a sample treatment step prior to the ail PCR assay. The assay consists of either one (single) or two (nested) PCR analyses and an internal amplification control for monitoring false-negative results. The detection limit of the complete (single) PCR method, using inoculated enriched homogenates, was established to 10 cfu or less per gram. An increased sensitivity in the form of a nested PCR was required to enable detection of the bacterium in naturally contaminated pork. This is in practice very important. Finally, for characterisation of isolated strains, a multiplex PCR assay was developed, directed towards four different virulence-associated genes (yst, rfbC, ail and virF). As presence or absence of the four PCR targets was established, the following groups were identified: pathogenic Y. enterocolitica 4/O:3 strains, pathogenic Y. enterocolitica serotypes other than 4/O:3, Y. pseudotuberculosis strains and nonpathogenic strains. The method does not allow for confirmation of the viability of the pathogen, the reason being that the bacterium cannot be isolated by traditional culture. The method can therefore preferably be used where information about viability is not important, for example in studies to identify the critical points during slaughter, important to limit contamination by the bacterium.