Development of a fluorogenic 5 ' nuclease PCR assay for detection of the ail gene of pathogenic Yersinia enterocolitica

In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assa y targets the chromosomally encoded attachment and invasion gene, ail. Thre e primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlappi ng, regions of ail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of pur ified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strain s examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the Y. enterocolitica strains tested. None of the pr imer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica stra ins examined. When the TR II set was utilized, the fluorogenic PCR assay wa s able to detect less than or equal to 4 Y. enterocolitica CFU/ml in pure c ulture and 10 Y enterocolitica CFU/ml independent of the presence of 10(8) CFU of contaminating bacteria per mi. This set was also capable of detectin g less than or equal to CFU of Y. enterocolitica per g of ground pork or fe ces after a 24-h enrichment in a Yersinia selective broth.