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

Aims: 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 to 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 g-1 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 quantitation 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.