Specific detection of plasmid bearing Yersinia isolates by PCR

A total of 210 isolates belonging to 9 different species of the genus Yersinia (Y.) was investigated with three different PCR assays targeting two plasmoidal genes, the Yersinia adhesin gene (yadA) and the V-antigen gene. The yadA PCR assay described in 1995 by Blais and Phillipe, targeting a Y. enterocolitica specific gene region and a newly designed assay targeting the gene region functionally responsible for autoagglutination, were compared. Both assays identified the same Y. enterocolitica strains. To exclude the possibility that false negative results were obtained due to mutations that had occurred in parallel in both gene regions, a third PCR assay by Neubauer et al. (2000) targeting a conserved region of the V-antigen gene was used as control. Again, DNA of the same Y. enterocolitica strains was amplified. In contrast to the yadA PCR assay described by Blais and Phillipe, the newly established yadA and the V-antigen PCR assays amplified DNA from Y. pseudotuberculosis strains. Therefore, by using the PCR technique as a molecular tool spontaneous mutations could be excluded as the cause of anomalous reactions in PCR assays targeting genes of the Yersinia virulence plasmid. Based on these results, it can be assumed that all presumptive pathogenic Yersinia isolates can be identified on the basis of PCR analysis. These molecular assays may also produce fewer false positive reactions in comparison to phenotypic tests such as the autoagglutination test which depend heavily on the handler's experience. It has to be stressed that the PCR assays used in this study have not been evaluated for routine use. Therefore, standardization of the PCR methodology including sample preparation, primer target sequences and PCR reagents is needed for the reliable and safe diagnosis of pathogenic Yersinia spp. in future.