Introduction The identification of bacterial milk pathogens, responsible for mastitis disease is of importance for the associated economic losses to producers. In recent years the development of DNA microarray technology has provided a promising option for the rapid identification of bacteria, analyzing thousands of DNA sequences in a relatively short time. A microarray platform was developed in this study and used to identified pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus spp. (Coagulase Negative Staphylococci), Streptococcus bovis, Streptococcus equi subsp. zooepidemicus, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus parauberis, Streptococcus uberis and Mycoplasma spp. Materials and Methods The universal array platform developed in this work is based on the discriminative properties of the DNA ligation detection reaction (LDR) as described in Castiglioni et al. (1) consisting of a set of synthetic oligonucleotides named "ZipCodes" (as shown in Figure 1) with similar thermodynamic characteristics but different unique artificial sequences. The pathogens were identified on the basis of "specie-specific" polymorphisms. This procedure requires the design of two adjacent oligonucleotide probes that hybridize consecutively along the template and the thermostable DNA ligase joins their ends. The resulting product is then hybridized onto the universal array. Genomic DNA was isolated from ATCC reference strains following the protocol described in Cremonesi et al. (2) and the 16S rRNA genes were amplified with universal primer as described in literature (1). The LDR reaction was carried out in a final volume of 20 ?L containing the specific buffer 10X, 1 ?mol of each discriminating probe, 50 fmol of PCR products purified and quantified by the Agilent Bioanalyser 2100 and 4U of Pfu DNA ligase. After an initial denaturation at 94°C for 5 min, the LDR was cycled for 30 rounds at 94°C for 30 seconds and at 65°C for 4 minutes, followed by 2 minutes at 94°C to complete the reaction. After denaturation, the LDR mixture was applied to the slide. Hybridization was carried out in a chamber in the dark at 65°C and the fluorescent signal was acquired by using a ScanArray Lite (Perkin Elmer). Results The preliminary results obtained from the reference ATCC strains of Staphylococcus aureus and Mycoplasma bovis (figure 2) demonstrated a high discriminative power and the specificity of the probes tested. Conclusions The possibility to use hightroughput 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. References 1. Castiglioni, B., E. Rizzi, A. Frosini, K., Sivonen, P. Rajaniemi et al. 2004. Development of a Universal Microarray based on the Ligation Detection Reaction and 16S rRNA gene polymorphism to target diversity of Cyanobacteria. Appl. Env. Microbiol. 70: 7161 2. Cremonesi, P., B. Castiglioni, G. Malferrari, I. Biunno, C. Vimercati, P. Moroni, S. Morandi, M. Luzzana. 2006. Technical note: Improved method for rapid DNA extraction of mastitis pathogens directly from milk. J. Dairy Sci. 89: 163.
Development of a microarray platform for milk pathogens detection: preliminary results
Cremonesi P;Boettcher P;Castiglioni B;
2008
Abstract
Introduction The identification of bacterial milk pathogens, responsible for mastitis disease is of importance for the associated economic losses to producers. In recent years the development of DNA microarray technology has provided a promising option for the rapid identification of bacteria, analyzing thousands of DNA sequences in a relatively short time. A microarray platform was developed in this study and used to identified pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus spp. (Coagulase Negative Staphylococci), Streptococcus bovis, Streptococcus equi subsp. zooepidemicus, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus parauberis, Streptococcus uberis and Mycoplasma spp. Materials and Methods The universal array platform developed in this work is based on the discriminative properties of the DNA ligation detection reaction (LDR) as described in Castiglioni et al. (1) consisting of a set of synthetic oligonucleotides named "ZipCodes" (as shown in Figure 1) with similar thermodynamic characteristics but different unique artificial sequences. The pathogens were identified on the basis of "specie-specific" polymorphisms. This procedure requires the design of two adjacent oligonucleotide probes that hybridize consecutively along the template and the thermostable DNA ligase joins their ends. The resulting product is then hybridized onto the universal array. Genomic DNA was isolated from ATCC reference strains following the protocol described in Cremonesi et al. (2) and the 16S rRNA genes were amplified with universal primer as described in literature (1). The LDR reaction was carried out in a final volume of 20 ?L containing the specific buffer 10X, 1 ?mol of each discriminating probe, 50 fmol of PCR products purified and quantified by the Agilent Bioanalyser 2100 and 4U of Pfu DNA ligase. After an initial denaturation at 94°C for 5 min, the LDR was cycled for 30 rounds at 94°C for 30 seconds and at 65°C for 4 minutes, followed by 2 minutes at 94°C to complete the reaction. After denaturation, the LDR mixture was applied to the slide. Hybridization was carried out in a chamber in the dark at 65°C and the fluorescent signal was acquired by using a ScanArray Lite (Perkin Elmer). Results The preliminary results obtained from the reference ATCC strains of Staphylococcus aureus and Mycoplasma bovis (figure 2) demonstrated a high discriminative power and the specificity of the probes tested. Conclusions The possibility to use hightroughput 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. References 1. Castiglioni, B., E. Rizzi, A. Frosini, K., Sivonen, P. Rajaniemi et al. 2004. Development of a Universal Microarray based on the Ligation Detection Reaction and 16S rRNA gene polymorphism to target diversity of Cyanobacteria. Appl. Env. Microbiol. 70: 7161 2. Cremonesi, P., B. Castiglioni, G. Malferrari, I. Biunno, C. Vimercati, P. Moroni, S. Morandi, M. Luzzana. 2006. Technical note: Improved method for rapid DNA extraction of mastitis pathogens directly from milk. J. Dairy Sci. 89: 163.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
