Specific detection of pathogenic Yersinia enterocolitica by two-step PCR using hot-start and DMSO

A pair of polymerase chain reaction (PCR) primers, YC1 and YC2, selected from the sequence of the invasin locus (inv) of Y. enterocolitica, has been evaluated for specific detection of pathogenic Y. enterocolitica by PCR. The primers were hybridized at high stringency conditions to DNA from 65 pathogenic Y. enterocolitica, 16 non-pathogenic Y. enterocolitica, 18 other Yersinia and 124 non-Yersinia strains. YC2 hybridized to the pathogenic Y. enterocolitica only, while YC1 hybridized weakly to nine non-Yersinia as well. In a PCR with annealing at 64 degrees C all Y. enterocolitica, pathogenic and non-pathogenic, were positive. However, DNA from 60 non-Y. enterocolitica was amplified. With annealing at 72 degrees C, 10 non-pathogenic Y. enterocolitica and 41 non-Y. enterocolitica were positive. When a two-step PCR assay with annealing at 72 degrees C, hot-start and 1% dimethylsulphoxide (DMSO) were used, only DNA from the pathogenic Y. enterocolitica were amplified. The limit of detection was shown for four different strains to be less than 10 cells per PCR tube.