title: Application of real-time PCR and RT-PCR assays for the detection and quantitation of VT 1 and VT 2 toxin genes in E. coli O157:H7.
abstract: Real-time PCR assays, based on hybridisation probes and LightCycler technology, were developed for VT 1 and VT 2 genes and applied to the detection and quantitation of DNA and mRNA of Escherichia coli O157:H7. The qualitative consensus PCR assay for the detection of VT 1 and/or VT 2 genes had a detection limit of 100 fg of E. coli O157:H7 genomic DNA and did not detect DNA from 13 non-VTEC isolates. When E. coli O157:H7 was inoculated into minced beef, enriched and recovered by immunomagnetic separation, the real-time consensus PCR assay had a detection limit of log(10)3.5 ml(-1) E. coli O157:H7 cells. Nineteen E. coli O157:H7 isolates, derived from food, bovine samples and human faeces, were analysed and compared for mRNA expression of three genes, VT 1, VT 2 and gapA (housekeeping gene), using quantitative real-time PCR assays. While there was no statistically significant difference for the expression of the VT 1 (p=0.134) or VT 2 (p=0.52) mRNA in the E. coli O157:H7 isolates from food, bovine and human sources, three clinical isolates did show lower expression of VT 2 compared to other isolates in the study. The study indicates that the consensus qualitative real-time PCR assay for VT 1 and VT 2 is rapid and sensitive and that the quantitative assays reported here have the potential to be used as an alternative method to more conventional methods for studying VT 1 and VT 2 virulence gene expression in E. coli O157:H7 with potential application in other pathogenic E. coli species.
authors.affiliation: The National Diagnostics Centre, DNA Diagnostics, BioResearch Ireland, National University of Ireland, Galway, Ireland.
authors.forename: J
authors.initials: J
authors.lastname: Fitzmaurice
authors.forename: M
authors.initials: M
authors.lastname: Glennon
authors.forename: G
authors.initials: G
authors.lastname: Duffy
authors.forename: J J
authors.initials: JJ
authors.lastname: Sheridan
authors.forename: C
authors.initials: C
authors.lastname: Carroll
authors.forename: M
authors.initials: M
authors.lastname: Maher
mesh_terms: D016172:DNA Fingerprinting / Q000379:methods; D015342:DNA Probes / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D005453:Fluorescence; D008460:Meat / Q000032:analysis / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification; D022622:Shiga Toxin 1 / Q000235:genetics*; D022641:Shiga Toxin 2 / Q000235:genetics*
chemical_list: D015342:DNA Probes; D018076:DNA, Complementary; D012333:RNA, Messenger; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2