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Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica.

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.

SUBMITTER: Vishnubhatla A

PROVIDER: S-EPMC92271 | biostudies-literature | 2000 Sep

REPOSITORIES: biostudies-literature

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Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica.

Vishnubhatla A A Fung D Y DY Oberst R D RD Hays M P MP Nagaraja T G TG Flood S J SJ

Applied and environmental microbiology 20000901 9

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 no ...[more]

PMID: 10966441

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Project description:BACKGROUND: Yersinia enterocolitica is a gastrointestinal foodborne pathogen found worldwide and which especially affects infants and young children. While different bioserotypes have been associated with varying pathogenicity, research on Y. enterocolitica is mainly conducted on the highly virulent mouse-lethal strains of biotype 1B and serotype O:8. We demonstrate here that two Y. enterocolitica bioserotype 1B/O:8 strains, 8081 and WA-314, display different virulence and fitness properties in a mouse model. In vivo co-infection experiments revealed that strain WA-314 overcomes strain 8081 in the colonization of spleen and liver. To trace the reasons of this incongruity, we present here the first high-quality sequence of the whole genome of strain WA-314 and compare it to the published genome of strain 8081. RESULTS: Regions previously accepted as unique to strain 8081, like the YAPI and YGI-3 genomic islands, are absent from strain WA-314, confirming their strain-specificity. On the other hand, some fitness- and bacterial competition-associated features, such as a putative colicin cluster and a xenobiotic-acyltransferase-encoding gene, are unique to strain WA-314. Additional acquisitions of strain WA-314 are seven prophage-like regions. One of these prophages, the 28-kb P4-like prophage YWA-4, encodes a PilV-like protein that may be used for adhesion to and invasion of the intestinal cells. Furthermore, a putative autotransporter and two type 1 fimbrial proteins of strain WA-314 show a sequence similarity <50% with the orthologous proteins in strain 8081. The dissimilar sequences of these proteins indicate possible different functions or interaction modes, reflecting the specific adhesion properties of Y. enterocolitica strains 8081 and WA-314 and thus the different efficiency of host colonization. Further important differences were found in two pYV plasmid-encoded virulence factors, YopM and YscP. The impact of these differences on virulence is discussed. CONCLUSIONS: Our study emphasizes that the virulence of pathogens can be increased, by acquiring new genes and/or improving the function of essential virulence proteins, resulting in permanently hyper-virulent strains. This work also highlights the importance of addressing genetic and phenotypic variations among closely related bacterial strains, even those belonging to the same bioserotype.

Dataset Information

Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica.

Publications

Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica.

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