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A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7
- Source :
- Journal of Microbiological Methods. 82:85-89
- Publication Year :
- 2010
- Publisher :
- Elsevier BV, 2010.
-
Abstract
- A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 104 CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.
- Subjects :
- Microbiology (medical)
Virulence Factors
Cattle Diseases
Virulence
Escherichia coli O157
medicine.disease_cause
Polymerase Chain Reaction
Microbiology
law.invention
chemistry.chemical_compound
fluids and secretions
Shiga-like toxin
STX2
law
Multiplex polymerase chain reaction
medicine
Animals
Humans
Molecular Biology
Escherichia coli
Escherichia coli Infections
Polymerase chain reaction
biology
Escherichia coli Proteins
biology.organism_classification
Enterobacteriaceae
Molecular biology
chemistry
bacteria
Cattle
Primer (molecular biology)
Subjects
Details
- ISSN :
- 01677012
- Volume :
- 82
- Database :
- OpenAIRE
- Journal :
- Journal of Microbiological Methods
- Accession number :
- edsair.doi.dedup.....9b08d9bc73667832ba0bed2bf93ff107