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

The aim of this study was to develop a multiplex loop-mediated isothermal amplification (LAMP) method capable of detecting Escherichia coli generally and verocytotoxigenic E. coli (VTEC) specifically in beef and bovine faeces. The LAMP assay developed was highly specific (100%) and able to distinguish between E. coli 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 E. coli 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 E.coli 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.

Crack propagation in non-homogenous materials: Evaluation of mixed-mode SIFs, T-stress and kinking angle using a variant EFG method

A new variant of the Element-Free Galerkin (EFG) method, that combines the diffraction method, to characterize the crack tip solution, and the Heaviside enrichment function for representing discontinuity due to a crack, has been used to model crack propagation through non-homogenous materials. In the case of interface crack propagation, the kink angle is predicted by applying the maximum tangential principal stress (MTPS) criterion in conjunction with consideration of the energy release rate (ERR). The MTPS criterion is applied to the crack tip stress field described by both the stress intensity factor (SIF) and the T-stress, which are extracted using the interaction integral method. The proposed EFG method has been developed and applied for 2D case studies involving a crack in an orthotropic material, crack along an interface and a crack terminating at a bi-material interface, under mechanical or thermal loading; this is done to demonstrate the advantages and efficiency of the proposed methodology. The computed SIFs, T-stress and the predicted interface crack kink angles are compared with existing results in the literature and are found to be in good agreement. An example of crack growth through a particle-reinforced composite materials, which may involve crack meandering around the particle, is reported.