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Pathogen detection in milk samples by ligation detection reaction-mediated universal array method

P. Cremonesi^*,1, G. Pisoni, M. Severgnini, C. Consolandi, P. Moroni^,, M. Raschetti^*,# and B. Castiglioni^*

^* Institute of Agricultural Biology and Biotechnology–Italian National Research Council, Via Bassini 15, 20133 Milan, Italy
Department of Veterinary Pathology, Hygiene and Public Health, University of Milan, Via Celoria 10, 20133 Milan, Italy
Institute of Biomedical Technologies–Italian National Research Council, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy
Centro Interdipartimentale di Studi sulla Ghiandola Mammaria (CISMA), Via Celoria 10, 20133 Milan, Italy
^# Department of Veterinary Science and Technology for Food Safety, University of Milan, Via Trentacoste 2, 20134 Milan, Italy

¹ Corresponding author: paola.cremonesi{at}unimi.it

This paper describes a new DNA chip, based on the use of a ligation detection reaction coupled to a universal array, developed to detect and analyze, 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. Probes were designed for the identification of 15 different bacterial groups: Staphylococcus aureus, Streptococcus agalactiae, nonaureus staphylococci, Streptococcus bovis, Streptococcus equi, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus parauberis, Streptococcus uberis, Streptococcus pyogenes, Mycoplasma spp., Salmonella spp., Bacillus spp., Campylobacter spp., and Escherichia coli and related species. These groups were identified based on the 16S rRNA gene. For microarray validation, 22 strains from the American Type Culture Collection or other culture collections and 50 milk samples were tested. The results demonstrated high specificity, with sensitivity as low as 6 fmol. Moreover, the ligation detection reaction-universal array assay allowed for the identification of Mycoplasma spp. in a few hours, avoiding the long incubation times of traditional microbiological identification methods. The universal array described here is a versatile tool able to identify milk pathogens efficiently and rapidly.

Key Words: foodborne pathogen • ligase detection reaction • deoxyribonucleic acid array • mastitis