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

Abstract

The aim of this study was to develop a method for detecting antibiotic resistance (AR) genes in cheese based on a combination of multiplex PCR and a DNA microarray hybridisation system. Twenty oligonucleotide probes were designed targeting 10 common AR genes, namely aac(6′)-Ie-aph(2″)-Ia, aadE, aphA-3, ermB, tet(L), tet(M), tet(O), tet(S), vanA and vanB. Specificity of the probes was tested by hybridising against DNA from Enterococcus strains harbouring known AR genes. DNA was labelled through two multiplex PCR reactions with fluorescent nucleotides and specific primers flanking the probe sequences. Sensitivity of the microarray was assessed by contamination of a cheese with an Enterococcus faecium strain carrying vanA gene. Two tetracycline resistance genes, tet(M) and tet(S), proved to be present in a series of retail cheeses, while genes aadE, aphA3, ermB, tet(L) and tet(O) were occasionally detected. This method is envisioned as a valuable tool for identification of AR genes in foods. © 2010 Elsevier Ltd.