Open AccessRapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica
Author(s) -
A. Vishnubhatla,
Daniel Y. C. Fung,
Richard D. Oberst,
Michael P. Hays,
T. G. Nagaraja,
S. J. A. Flood
Publication year - 2000
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.66.9.4131-4135.2000
Subject(s) - yersinia enterocolitica , virulence , biology , microbiology and biotechnology , taqman , yersinia , salmonella , yersinia ruckeri , serotype , polymerase chain reaction , virology , bacteria , gene , fish <actinopterygii> , genetics , fishery , rainbow trout
We have developed a rapid procedure for the detection of virulentYersinia 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 chromosomalyst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes ofY. enterocolitica , other species ofYersinia (Y. aldovae ,Y. pseudotuberculosis ,Y. mollaretti ,Y. intermedia ,Y. bercovieri ,Y. ruckeri ,Y. frederiksenii , andY. kristensenii ), and other enteric bacteria (Escherichia ,Salmonella ,Citrobacter , andFlavobacterium ). The assay was 100% specific in identifying the pathogenic strains ofY. enterocolitica . The sensitivity of the assay was found to be ≥102 CFU/ml in pure cultures and ≥103 CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked withY. 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 virulentY. 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.