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Applied and Environmental Microbiology, September 2000, p. 3750-3755, Vol. 66, No. 9
0099-2240/00/$04.00+0

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

Alissa D. Jourdan,1,* Scott C. Johnson,2 and Irene V. Wesley1

USDA ARS National Animal Disease Center, Ames, Iowa 50010,1 and Molecular Biology Resources, Milwaukee, Wisconsin 532182

Received 13 March 2000/Accepted 2 June 2000

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 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 purified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strains 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 primer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica strains 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.

* Corresponding author. Mailing address: USDA ARS National Animal Disease Center, 2300 Dayton Ave., Ames, IA 50010. Phone: (515) 663-7291. Fax: (515) 663-7458. E-mail: ajourdan{at}nadc.ars.usda.gov.

Applied and Environmental Microbiology, September 2000, p. 3750-3755, Vol. 66, No. 9
0099-2240/00/$04.00+0


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