DEVELOPMENT OF A HIGHLY SPECIFIC ASSAY FOR RAPID IDENTIFICATION OF PATHOGENIC STRAINS OF YERSINIA-ENTEROCOLITICA BASED ON PCR AMPLIFICATIONOF THE YERSINIA HEAT-STABLE ENTEROTOXIN GENE (YST)

The chromosomal gene yst, which encodes a heat-stable enterotoxin of Y ersinia enterocolitica, is a useful diagnostic marker because it occur s only in invasive strains of this species. A homologous gene also occ urs in some strains of Yersinia kristensenii. Sequence analysis of the yst genes from two different strains of Y. enterocolitica and from Y. kristensenii revealed a substantial number of mismatches at the 3' en ds of the yst genes of the so-called American and European biotypes of Y. enterocolitica. Moreover, several mismatches and a deletion of 5 c odons were found in the yst of Y. kristensenii. These findings were us ed to develop a PCR-based assay for yst of Y. enterocolitica which yie lded a detectable product in as little as 50 min. The assay was 100% s pecific in terms of its ability to identify potentially pathogenic str ains of Y. enterocolitica regardless of biotype or serotype. The PCR y ielded an amplicon that was visible on agarose gel electrophoresis fro m as few as 100 CFU, or 10 CFU when the PCR was combined with dot blot hybridization with a digoxigenin-labeled oligonucleotide probe that c orresponded to an internal sequence of yst. These results establish th e value of the yst gene as a target for the identification of pathogen ic bioserotypes of Y. enterocolitica and the usefulness of PCR for thi s purpose.