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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 #110713
Title: DEVELOPMENT OF A FLUOROGENIC 5' NUCLEASE PCR ASSAY FOR DETECTION OF THE AIL GENE OF PATHOGENIC YERSINIA ENTEROCOLITICA

Author
item Jourdan, Alissa
item JOHNSON, SCOTT - 3625-30-15
item WESLEY, IRENE

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Yersinia enterocolitica is a foodborne pathogens that causes ~3,000 to 20,000 cases of human disease annually in the United States. Healthy swine are the only animal known to harbor human pathogenic strains of Y. enterocolitica. This bacterium is frequently isolated from pig carcasses, ground pork, swine feces, chitterlings, tongue, and tonsils. Rapid, specific, and sensitive assays are needed to monitor the prevalence of Y. enterocolitica in animals and foods. The information generated would help identify on-farm management and processing practices leading to Y. enterocolitica contamination. Modification of such practices would ultimately decrease Y. enterocolitica transmission from pork products to humans. Polymerase chain reaction (PCR) is a powerful molecular biology tool with multiple applications. Fluorogenic PCR relies on cleavage of a fluorescently labeled probe and is more specific and sensitive than conventional PCR in detecting pathogens. A fluorogenic PCR assay was developed to specifically detect pathogenic strains of Y. enterocolitica in ground pork and feces. The assay was able to detect one Y. enterocolitica cell per gram of ground pork or feces. The assay is significantly more sensitive and efficient than current PCR protocols. The ability to identify pathogenic strains of Y. enterocolitica in diverse samples benefits not only hog producers and food processors, but also clinical microbiologists.

Technical 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 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. Utilizing the TM1 set, the fluorogenic PCR assay was able to detect less than or equal to 4 Y. enterocolitica CFU/ml in pure culture and 10 Y. enterocolitica CFU/ml independent of the presence of 10**8 CFU/ml contaminating bacteria. This set was also capable of detecting less than or equal to 1 CFU of Y. enterocolitica per gram ground pork or feces after a 24-hour enrichment in ITC broth.