DEVELOPMENT OF LDR-BASED MICROARRAYS FOR THE DETECTION OF MICROBIAL PATHOGENS

The advent of genetic-based technologies makes feasible developing a sensitive and specific screening test for the detection of microbial pathogens. Microarray-based technologies represent an advance in nucleic acid testing. The major advantages of this technology, including miniaturization, high performance, the ability to process samples in parallel, and ease of automation, have expended its application area in this decade. The advantage of microarray-based detection is that it can combine powerful nucleic acid amplification strategies with a massive screening capability, resulting in a high level of sensitivity, specificity, and throughput capacity. Microarrays allow the characterization of microorganisms by providing information for specific identification of isolates; moreover, they allow understanding the pathogenesis based on the presence of virulence genes and indicating how new pathogenic strains evolved epidemiologically and phylogenetically. Our research deals with the development and the validation of a Ligation Detection Reaction-Universal Array (LDR-UA) platform for the direct identification and pathogenic characterization of microbial pathogens. The high specificity and the multiplexing potential achieved in the enzymatic reaction make the LDR-UA approach particularly suitable to target a relatively small number of species and marker genes. The LDR technique uses a pair of probes for each target: a) a Discriminating Oligonucleotide (DS) whose 3?- end insists on a position capable of discriminating the target sequences from non-target ones; b) a Common Probe (CP), designed to be immediately 3?-downstream of the DS, which is used to complete the ligation process. The Universal Array format is based on a set of artificial sequences, called ZipCodes, designed to be different from any biological sequence, and it can be used to address specifically ligated products to pre-determined positions of the array. This innovative technique has been recently applied to pathogen detection in food safety applications and environmental screenings successfully exhibiting high specificity and sensitivity. In particular, a DNA chip based on the use of a PCR-LDR-UA assay was implemented to detect, directly from milk samples, microbial pathogens known to cause bovine, ovine, and caprine mastitis or to be responsible for foodborne intoxication or infection, or both. Specific probes on the 16S rRNA gene were employed for the identification of 15 different bacterial groups at different phylogenetic levels. Furthermore, a LDR-UA microarray was developed to detect pathogenic and spoilage bacteria in seafood. The potential for high specificity and sensitivity makes ligation-based detection techniques a promising tool for characterizing complex microbial communities.