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Applied and Environmental Microbiology, September 2000, p. 4131-4135, Vol. 66, No. 9
Department of Animal Sciences and
Industry,1 Food Animal Health and
Management Center,2 and Department of
Diagnostic Medicine and Pathobiology,3 Kansas
State University, Manhattan, Kansas 66506, and Applied
Biosystems Division, Perkin-Elmer Corporation, Foster City, California
944044
Received 9 December 1999/Accepted 19 June 2000
We have developed a rapid procedure for the detection of virulent
Yersinia enterocolitica in ground pork by combining a
previously described PCR with fluorescent dye technologies. The
detection method, known as the fluorogenic 5' nuclease assay (TaqMan),
produces results by measuring the fluorescence produced during PCR
amplification, requiring no post-PCR processing. The specificity of the
chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic
serotypes of Y. enterocolitica, other species of
Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri,
Y. frederiksenii, and Y. kristensenii), and
other enteric bacteria (Escherichia, Salmonella, Citrobacter, and
Flavobacterium). The assay was 100% specific in
identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Rapid 5' Nuclease (TaqMan) Assay for Detection of
Virulent Strains of Yersinia enterocolitica
102 CFU/ml
in pure cultures and 103 CFU/g in spiked ground pork
samples. Results of the assay with food enrichments prespiked with
Y. enterocolitica serotypes O:3 and O:9 were comparable to
standard culture results. Of the 100 field samples (ground pork)
tested, 35 were positive for virulent Y. enterocolitica
with both 5' nuclease assay and conventional virulence tests. After
overnight enrichment the entire assay, including DNA extraction,
amplification, and detection, could be completed within 5 h.
*
Corresponding author. Mailing address: Department of
Animal Sciences and Industry, College of Agriculture, Kansas State
University, 225 Call Hall, Manhattan, KS 66502. Phone: (785) 532-5654. Fax: (785) 532-5681. E-mail: DFUNG{at}oz.ozNET.KSU.edu.
Contribution no. 00-171-J from the Kansas Agricultural Experiment Station.
Applied and Environmental Microbiology, September 2000, p. 4131-4135, Vol. 66, No. 9
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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