title: A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces.
abstract: OBJECTIVE
  To develop a multiplex loop-mediated isothermal amplification (LAMP) assay capable of quantifying Escherichia coli and differentiating verocytotoxigenic E. coli (VTEC).
  
  
  RESULTS
  Primer sets were selected to amplify the phoA gene (all E. coli strains) and stx1 and/or stx2 genes (VTEC strains only). LAMP calibration curves demonstrated good quantification capability compared with conventional culture. The limits of detection 50% (LOD50) of the multiplex LAMP assay were 2·8 (95% CI 2·4-3·3), 3·2 (95% CI 2·5-3·9) and 2·8-3·2 (95% CI 2·1-3·5) log CFU per g for the phoA, stx1 and stx2 genes, respectively. When validated by testing retail beef and bovine faeces samples, good correlation between E. coli counts indicated by the LAMP assay and culture was observed; however, false-negative LAMP assay results were obtained for 12·5-14·7% of samples.
  
  
  CONCLUSIONS
  A rapid, multiplex LAMP assay for direct quantification of E. coli and specific detection of VTEC in beef and faeces was successfully developed. Further optimisation of the assay would be needed to improve detection sensitivity.
  
  
  CONCLUSIONS
  The multiplex LAMP assay represents a rapid alternative to culture for monitoring E. coli levels on beef for hygiene monitoring purposes, and, potentially, a method for detection of VTEC in beef and faeces.
authors.affiliation: Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Belfast, UK.
authors.forename: A Ch
authors.initials: AC
authors.lastname: Stratakos
authors.affiliation: Food Microbiology Branch, Agriculture, Food and Environmental Science Division, Agri-Food and Biosciences Institute for Northern Ireland, Belfast, UK.
authors.forename: M
authors.initials: M
authors.lastname: Linton
authors.affiliation: OptiGene Limited, Horsham, West Sussex, UK.
authors.forename: S
authors.initials: S
authors.lastname: Millington
authors.affiliation: Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Belfast, UK.
authors.forename: I R
authors.identifier: http://orcid.org/0000-0001-5860-3769
authors.initials: IR
authors.lastname: Grant
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D002417:Cattle; D017931:DNA Primers; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D005243:Feces / Q000382:microbiology*; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D000069466:Red Meat / Q000382:microbiology*; D037521:Virulence Factors / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D037521:Virulence Factors
keywords: Escherichia coli (all pathogenic types); beef; detection; loop-mediated isothermal amplification; rapid methods