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

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.

Array Platform for Food Safety and Quality

“…In Volokhov et al (2003), signals that differed from the average background at a statistically significant level (P < 0.01) were considered positive; similarly, in Cremonesi et al (2009), for each oligonucleotide, we required the population of all the replicates to be above the intensity of "blank" spots (i.e., with no oligonucleotide spotted), plus two times their standard deviations by applying a one-sided Student t-test (P < 0.01). Typically, each zipcode population was made by quadruplicate spots, whereas the null distribution of the "blanks" was made up by six.…”

“…A new DNA chip based on the use of a PCR-LDR-UA assay was developed and validated by Cremonesi et al (2009) The bacterial pathogens on the assay panel were chosen to include (a) the organisms most frequently isolated from mastitis samples, such as Staph. aureus, Strep.…”

Advances in DNA Microarray Technology for the Detection of Foodborne Pathogens

“…In Volokhov et al (2003), signals that differed from the average background at a statistically significant level (p<0.01) were considered positive; similarly, in Cremonesi et al (2009), for each oligonucleotide, the population of all the replicates was tested by a one-sided Student's t test (p<0.01) against the intensity of "blank" spots (i.e., with no oligonucleotide spotted) plus 2 times their standard deviations.…”

“…The intra-group and the inter-group scores are used to retrieve a list of probes that best discriminate between the target and the non-target sequences, allowing the selection of probes covering the highest number of species of the positive set (intra-group score) and having the lowest interaction (inter-group score) with those of the negative set. This software is especially suited for probe design in enzymatic-related applications, such as the LDR-UA platform described in Cremonesi et al (2009).…”

Technological advances in bovine mastitis diagnosis

“…The detection limit of this method was 10 3 -10 5 CFU/mL. A previous study 14 also described a DNA chip, based on the use of a ligation detection reaction coupled to a universal array, developed to detect and analyze pathogens directly from milk samples. These bacterial groups were identified based on the 16S rRNA gene.…”