Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica
- 1 September 2000
- journal article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 66 (9), 4131-5
- https://doi.org/10.1128/aem.66.9.4131-4135.2000
Abstract
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 ≥10 2 CFU/ml in pure cultures and ≥10 3 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.Keywords
This publication has 15 references indexed in Scilit:
- Multiplex riboprobes for the detection of virulent Yersinia enterocolitica and simple methods for their preparationJournal of Applied Bacteriology, 1995
- Structure-Specific Endonucleolytic Cleavage of Nucleic Acids by Eubacterial DNA PolymerasesScience, 1993
- Allelic discrimination by nick-translation PCR with fluorgenic probesNucleic Acids Research, 1993
- Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.Proceedings of the National Academy of Sciences, 1991
- Yersinia enterocolitica in food hygieneInternational Journal of Food Microbiology, 1991
- Detection of pathogenic Yersinia eriterocolitica by the polymerase chain reactionThe Lancet, 1990
- Yersinia enterocoliticaO:3 Infections in Infants and Children, Associated with the Household Preparation of ChitterlingsNew England Journal of Medicine, 1990
- Yersinia EnterocoliticaNew England Journal of Medicine, 1989
- Analysis of the yopA gene encoding the Yop1 virulence determinants of Yersinia spp.Molecular Microbiology, 1989
- Plasmid-mediated tissue invasiveness in Yersinia enterocoliticaNature, 1980