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Pathogen detection in milk samples by ligation detection reaction-mediated universal array method
P Cremonesi1, G Pisoni, M Severgnini
1Institute of Agricultural Biology and Biotechnology-Italian National Research Council, Via Bassini 15, 20133 Milan, Italy. paola.cremonesi@unimi.it
Abstract:
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.
Insights
A novel DNA chip utilizing ligation detection reaction and a universal array rapidly identifies 15 milkborne bacterial pathogens directly from milk. This advancement offers high specificity and sensitivity, significantly reducing detection times compared to traditional methods.
Area of Science:
- Microbiology
- Molecular Biology
- Food Safety
Background:
- Bovine, ovine, and caprine mastitis pose significant challenges in animal husbandry.
- Foodborne illnesses are a major public health concern, often linked to contaminated dairy products.
- Traditional microbial identification methods are time-consuming and labor-intensive.
Purpose of the Study:
- To develop and validate a rapid DNA-based diagnostic tool for identifying key milkborne pathogens.
- To enable direct analysis of milk samples, bypassing lengthy culture procedures.
- To improve the efficiency and speed of mastitis and foodborne pathogen detection.
Main Methods:
- Development of a DNA chip integrating ligation detection reaction (LDR) with a universal array.
- Design of probes targeting the 16S rRNA gene for 15 specific bacterial groups.
- Validation using reference strains (ATCC) and real-world milk samples.
Main Results:
- The LDR-universal array assay demonstrated high specificity.
- Achieved a high sensitivity level as low as 6 fmol.
- Successfully identified Mycoplasma spp. within hours, outperforming traditional culture methods.
Conclusions:
- The developed universal array is a versatile and efficient tool for rapid milk pathogen identification.
- This technology offers a significant improvement over conventional methods for mastitis and foodborne pathogen detection.
- The assay provides a rapid, specific, and sensitive solution for ensuring milk safety and animal health.
