A Loop-mediated Isothermal Amplification Method for Rapid Direct Detection and Differentiation of Non-pathogenic and Verocytotoxigenic <i>Escherichia coli </i>in Beef and Bovine Faeces

The aim of this study was to develop a multiplex loop-mediated isothermal amplification (LAMP) method capable of detecting <i>Escherichia coli </i>generally and verocytotoxigenic <i>E. coli</i> (VTEC) specifically in beef and bovine faeces. The LAMP assay developed was highly specific (100%) and able to distinguish between <i>E. coli </i>and VTEC based on the amplification of the phoA, and stx1 and/or stx2 genes, respectively. In the absence of an enrichment step, the limit of detection 50% (LOD50) of the LAMP assay was determined to be 2.83, 3.17 and 2.83-3.17 log CFU/g for <i>E. coli</i> with phoA, stx1 and stx2 genes, respectively, when artificially inoculated minced beef and bovine faeces were tested. The LAMP calibration curves generated with pure cultures, and spiked beef and faeces, suggested that the assay had good quantification capability. Validation of the assay, performed using retail beef and bovine faeces samples, demonstrated good correlation between counts obtained by the LAMP assay and by a conventional culture method, but suggested the possibility of false negative LAMP results for 12.5-14.7% of samples tested. The multiplex LAMP assay developed potentially represents a rapid alternative to culture for monitoring <i>E.coli</i> levels in beef or faeces and it would provide additional information on the presence of VTEC. However, some further optimisation is needed to improve detection sensitivity.