Development of a Fluorogenic 5′ Nuclease PCR Assay for Detection of the ail Gene of Pathogenic Yersinia enterocolitica

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Abstract
In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions ofail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of theY. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocoliticastrains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect ≤4 Y. enterocolitica CFU/ml in pure culture and 10 Y. enterocolitica CFU/ml independent of the presence of 108 CFU of contaminating bacteria per ml. This set was also capable of detecting ≤1 CFU of Y. enterocolitica per g of ground pork or feces after a 24-h enrichment in a Yersinia selective broth.