Development and validation of a multiplex PCR-based DNA microarray hybridisation method for detecting bacterial antibiotic resistance genes in cheese

18 19 The aim of this study was to develop a method for detecting antibiotic resistance 20 (AR) genes in cheese based on a combination of multiplex PCR and a DNA microarray 21 hybridisation system. Twenty oligonucleotide probes were designed targeting 10 22 common AR genes, namely aac(6’)-Ie-aph(2’’)-Ia, aadE, aphA-3, ermB, tet(L), tet(M), 23 tet(O), tet(S), vanA and vanB. Specificity of the probes was tested by hybridising 24 against DNA from Enterococcus strains harbouring known AR genes. DNA was 25 labelled through two multiplex PCR reactions with fluorescence nucleotides and 26 specific primers flanking the probe sequences. Sensitivity of the microarray was 27 assessed by contamination of a cheese with an Enterococcus faecium strain carrying 28 vanA gene. Two tetracycline resistance genes, tet(M) and tet(S), proved to be present in 29 a series of retail cheeses, while genes aadE, aphA3, ermB, tet(L) and tet(O) were 30 occasionally detected. This method is envisioned as a valuable tool for identification of 31 AR genes in foods. 32 ______________________________________________________________________ 33