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Stratakos A.C.,Queen's University of Belfast | Linton M.,Food Microbiology Branch Agriculture | Millington S.,OptiGene Ltd Horsham West Sussex UK | Grant I.R.,Queen's University of Belfast
Journal of Applied Microbiology | Year: 2017

Aim: To develop a multiplex loop-mediated isothermal amplification (LAMP) assay capable of quantifying Escherichia coli and differentiating verocytotoxigenic E. coli (VTEC). Methods and 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. Significance and Impact of the Study: 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. © 2017 The Society for Applied Microbiology.

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