Polymerase chain reaction-gene probe detection system specific for pathogenic strains of Yersinia enterocolitica

Abstract

The polymerase chain reaction technique was used to develop a rapid diagnostic assay for detection of pathogenic Yersinia enterocolitica strains. The assay targeted a stretch of 163 bp of the yst gene and could be applied to both pure cultures and crude DNA extracted from feces. The defined primer pair amplified the targeted sequence from only pathogenic strains and fecal samples seeded with the serotype O:3 strain of Y. enterocolitica, whereas neither nonpathogenic strains nor normal stools yielded any amplified fragments. Of the other Yersinia species and non-Yersinia species tested, only two strains of Y. kristensenii yielded the same amplified product. A 20-mer oligonucleotide probe specifically hybridized within the amplified yst fragment of Y. enterocolitica but did not hybridize with the amplified yst fragment of Y. kristensenii by Southern and dot blot hybridizations. This confirms the reliability of this diagnostic assay in both clinical and epidemiological studies. The availability of the extracted DNA for the polymerase chain reaction was checked by simultaneous amplification of a part of the 16S rDNA and the yst gene. The entire diagnostic assay, including a simplified technique for DNA extraction, the amplification process, and gel electrophoresis, could be completed within 1 working day, which is better than the time required for the time-consuming traditional techniques used in clinical laboratories.