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Title: A PCR ELISA METHOD FOR THE DETECTION OF YERSINIA ENTEROCOLITICA

Author
item UYEDA, J - CALIFORNIA STATE UNIV
item Harmon, Karen
item Wesley, Irene

Submitted to: Swine Research Report
Publication Type: Proceedings
Publication Acceptance Date: 1/31/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Yersinia enterocolitica is a potential human foodborne pathogen which is present in pigs. Because of its public health significance, rapid methods are needed to determine its prevalence in pork products and market-weight hogs. The ail gene is only found in pathogenic strains of Y. enterocolitica. We have developed a PCR ELISA, which utilizes 5'-Biotin (5'-BIOT-TTAATGTGTACGCTGCGAGTG-3') and a 3' (3'-CTGCGAAGTATACTTATGAGG-5') PCR primers. For ELISA detection, a fluorescein-tagged internal probe (5'FLCTCCCCAGTTATCATCGAGTTCFL-3') was designed to hybridize with a portion of the ail gene. PCR products are visualized colorimetrically, thus bypassing the need for gel electrophoretic detection. PCR ELISA was as specific as gel electrophoresis in detecting the ail gene (425 bp) product of 6 of the 7 ATCC reference strains. Failure to amplify strain ATCC 9610 may be due to the loss of this virulence attribute since its acquisition in n1939. Seven of the nine NADC field strains, for which no mouse virulence tests were conducted, also gave a positive reaction. No amplification occurred with strains of Yersinia ruckeri, Y. pseudotuberculosis, Y. fredricksenii, Y. kristensenii, or L. monocytogenes. For pure cultures, PCR ELISA was as sensitive as gel electrophoresis in detecting as few as 6 CFU per reaction mixture (50 ul). The PCR ELISA was at least as sensitive as gel electrophoresis in detecting Y. enterocolitica in experimentally contaminated ground pork. The PCR ELISA is simple to perform, eliminates the need for electrophoresis and thus is suitable for the rapid processing of large sample numbers encountered in surveys of hogs and pork products.