Universal Array for Pathogens Detection in Milk and Cheese

The identification of bacterial milk pathogens responsible for mastitis is of importance not only for the associated economic losses to producers, but also for the hazard these could represent for consumers. Raw milk and dairy products made from non-pasteurized milk have been responsible for Staphylococcus aureus (heat-stable enterotoxins producer), Campylobacter, Salmonella, Bacillus, E. coli outbreaks expecially E. coli O157:H7 strain, very dangerous for human health. In recent years, DNA microarray technology has played an increasingly important role in the study of infectious diseases. A new DNA-chip, based on the use of a Ligation Detection Reaction (LDR) coupled to a universal array, has been developed to detect and analyze directly from milk and cheese samples 15 different bacterial groups such as Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus spp. (Coagulase Negative Staphylococci), Strep. bovis, Strep. equi subsp. zooepidemicus, Strep. canis, Strep. dysgalactiae, Strep. parauberis, Strep. uberis, Strep. pyogenes, Mycoplasma spp., Salmonella spp., Bacillus spp., Campylobacter spp. and Escherichia coli O157. The pathogens were identified from "species-specific" polymorphisms on the 16S rRNA gene. After the genomic DNA extraction from milk and cheese samples and the 16S rRNA gene amplification with universal primer, the LDR reaction was carried out by using two adjacent oligonucleotide probes specific for each target sequence. The two probes hybridize consecutively along the template and the thermostable DNA ligase joins their ends. The resulting product was then hybridized onto the universal array. For microarray validation, 34 strains from ATCC culture collections, 20 raw mastitis milk samples and 10 fresh semi-hard and hard cheese samples were tested. The results demonstrated high specificity and discriminative power, with sensitivity down to 6 fmol. The possibility to use high-throughput methods that are able to identify milk pathogens efficiently, rapidly and inexpensively could improve the treatment and prevention of mastitis, reducing a potential hazard for consumers and increasing the efficiency of milk production. The universal array described here is a versatile tool, as new probe pairs can be added to the system without further optimization, thus reducing costs and set up time.