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

作者：Stratakos A Ch; Linton M; Millington S; Grant I R^*

来源：Journal of Applied Microbiology, 2017, 122(3): 817-828.

DOI：10.1111/jam.13381

• beef
• detection
• Escherichia coli (all pathogenic types)
• loop-mediated isothermal amplification
• rapid methods

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 28 (95% CI 24-33), 32 (95% CI 25-39) and 28-32 (95% CI 21-35) 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 125-147% 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.

• 出版日期2017-3