Development of a random genomic DNA microarray for the detection and identification of Listeria monocytogenes in milk

Abstract

We developed a DNA microarray that contains random genomic DNA fragments of Listeria monocytogenes, validated its diagnostic abilities using cells grown in laboratory media and milk, and established enrichment conditions for detection of a low population of L. monocytogenes in milk. Genomic DNA of L. monocytogenes strain ATCC 19111 was fractionated by agarose gel electrophoresis after being cleaved using several different pairs of restriction enzymes. Sixty DNA fragments of different sizes were randomly selected and spotted onto an amine-coated glass slide. To validate diagnostic ability, probes on the DNA microarray were hybridized with genomic DNA extracted from L. monocytogenes, other Listeria spp., and foodborne pathogenic bacteria belonging to other genera grown in laboratory media. The DNA microarray showed 98-100% positive hybridization signals for the 16 strains of L. monocytogenes tested, 7-85% positive signals for 9 strains of other Listeria spp., and 0-32% positive signals for 13 strains of other types of foodborne pathogens. In milk, the detection limit of the DNA microarray was approximately 8 log CFU/mL. When milk contained L. monocytogenes (3-4 log CFU/mL) with other types of bacteria (Bacillus spp., B. cereus, Salmonella Montevideo, Peudomonas aeruginosa, and Yersinia enterocolitica; ca. 3 log CFU/mL each), L. monocytogenes enriched in UVM modified Listeria enrichment broth at 37°C for 24h was successfully detected by the DNA microarray. Results indicate that the DNA microarray can detect L. monocytogenes and distinguish it from other Listeria spp. and other foodborne pathogens in laboratory media and milk. This platform will be useful when developing a DNA microarray to rapidly and simultaneously detect and identify various foodborne pathogens in foods.