A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces

Stratakos, A. Ch; Stratakos, A.Ch.; Linton, M.; Millington, S.; Grant, I. R.

Abstract

© 2016 The Society for Applied Microbiology 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.

• Journal Article Type: Article
• Acceptance Date: Dec 13, 2016
• Online Publication Date: Dec 19, 2016
• Publication Date: Jan 1, 2017
• Deposit Date: Jun 18, 2019
• Publicly Available Date: Jun 18, 2019
• Journal: Journal of Applied Microbiology
• Print ISSN: 1364-5072
• Electronic ISSN: 1365-2672
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 122
• Issue: 3
• Pages: 817-828
• DOI: https://doi.org/10.1111/jam.13381
• Public URL: https://uwe-repository.worktribe.com/output/1494383
• Publisher URL: http://doi.org/10.1111/jam.13381
• Additional Information: Additional Information : This is the peer reviewed version of the following article: [Stratakos, A. , Linton, M. , Millington, S. and Grant, I. (2017) A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenicEscherichia coliin beef and bovine faeces. Journal of Applied Microbiology, 122 (3). pp. 817-828. ISSN 1364-5072], which has been published in final form at http://doi.org/10.1111/jam.13381. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
• Contract Date: Jun 18, 2019