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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Publications at this Location » Publication #114019
Title: DEVELOPMENT OF A FLUOROGENIC 5' NUCLEASE PCR ASSAY FOR THE DETECTION OF PATHOGENIC YERSINIA ENTEROCOLITICA IN SWINE AND SWINE PRODUCTS

Author
item Jourdan, Alissa
item JOHNSON, S - MOLECULAR BIOL RESOURCES
item WESLEY, IRENE

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 5/25/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: In this report we describe the development and evaluation of a 5' nuclease PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded invasion gene ail. Three different primer/probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. The TM1 set displayed the highest specificity, detecting each of the 26 Y. enterocolitica strains tested and none of the 21 non-enterocolitica strains, and was sensitive to approximately 0.5 pg of purified DNA. Similar sensitivities were achieved with the TM3 set and cross-reaction with non-enterocolitica strains was not observed. However, this set failed to positively identify all of the Y. enterocolitica strains tested. The TM2 set was the most sensitive, detecting in the range of 0.25 pg of purified DNA. However, it was not specific and failed to recognize 10 of the Y. enterocolitica strains. The TM1 set did not cross-react with any of 12 non-Yersinia pathogens commonly found in swine, indicating the assay is specific for Y. enterocolitica. The lower limit of bacterial detection with TM1 was 39 CFU/ml from pure culture and 1 CFU/ml after a 4 to 8 hour enrichment. Sensitivity levels decreased 10-fold when detecting Y. enterocolitica directly from artificially inoculated irradiated ground pork. In addition, higher ground pork concentrations (> 10%) reduced the sensitivity an additional 10-100 fold, indicating that ground pork inhibits the 5' nuclease assay.