DNA arrays applied to microorganisms detection

A DNA array for the identification of Gram+ and Gram- bacteria based on 16S rDNA, with specificity at genus and species level has been developed for industrial quality control, public health and biodefense applications. The chip was made with 30 oligonucleotide (20mer) sequences to which PCR amplification products were hybridized as probes. The labeling was made during the PCR reaction, using an excess of 16S-reverse primer in an asymmetric amplification reaction. The DNA extractions from various matrices were optimized depending on the microorganism to be tected. In order to extract DNA from microorganisms colonizing surfaces, such as Listeria species, cells were harvested from samples by repeated washings. Additional enrichment steps based on filtration were performed when necessary. Bacteria were lysed in the presence of lysozyme, also using proteinase K in some cases and DNA was captured on silica or magnetized particles. The DNA array protocol took 24 hours to perform all steps, showing to be a fast multiplex analysis when food samples of unknown microbial composition were tested.