Milk samples were cultured immediately on arrival at the laboratory by spreading 50 μl on the surface of SBA plates and Edward’s agar plates followed by incubation at 37°C for 48 hrs. Bacterial colonies resembling Streptococcus spp. were subcultured onto BHI agar and incubated at 37°C for 24 hrs. If multiple morphologically different colonies grew on a primary culture plate, each of these was subcultured to BHI agar.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted from the different S. uberis strains using a Wizard® Genomic DNA Purification Kit in accordance with the manufacturer’s instructions.
Label
Biotin/Steptavidin
Label protocol
The pooled tester and driver (Target DNA) as well as the 29 S. uberis strains were digested with Rsa 1 and subsequently purification on a column using QIAquick PCR Purification Kit. Approximately 200 ng of restriction digested gDNA fragments were then labeled with Biotin-11-dUTP using the Biotin DecaLabelTM DNA Labeling Kit in accordance with manufacturer’s instructions. However, the reaction was incubated at 37oC for 20 h, and stopped with 1 µL of 0.5M EDTA, pH 8.0 and no further purification was performed.
Hybridization protocol
The SDA slides were pre-hybridized for 45 min at 42°C in a pre-warmed solution containing 5X standard saline citrate (SSC), 0.1% sodium dodecyl sulphate (SDS), 1% bovine serum albumin (BSA) and 25% formamide. The slides were rinsed twice with sterile MilliQ water and immediately dried with an air gun. The biotin-labelled targets (dried to 16 μL) were added to 17.5 μL of fresh 2X Hybridization buffer (250 μL of formamide, 250 μL of 10X SSC, 10 μL of 10% SDS), 0.5 μL of 5 μg/μL Human Cot1 DNA (Sigma-Aldrich, St Louis, MO), 0.5 μL of 10 mg/mL Poly A (Sigma-Aldrich) and 0.5 μL of 10 mg/mL salmon sperm DNA (Sigma-Aldrich). The mixture was denatured at 100°C for 2 min and immediately applied onto the array under a 22 × 25-mm lifter slip (Grale Scientific, Victoria, Australia). The slides were then placed in waterproof, humidified hybridization chambers (Corning Incorporated Life Sciences) and incubated overnight in a 42°C water bath. Following hybridization, the slides were washed twice for 5 min in 500 mL Wash buffer 1 (1X SSC with 0.1% SDS), once for 5 min in 500 mL Wash buffer 3 (0.1X SSC with 0.1% SDS), and once for 5 min in 500 mL Wash buffer 4 (0.1X SSC). Subsequently the slides were transferred to 500 mL of 6X SSPE-T buffer (0.9 M NaCl, 0.06 M NaH2PO4.H2O, 0.006 M EDTA, 0.005% Triton X-100, pH 7.4) without allowing them to dry. The biotinylated DNA targets bound on the array were then labelled with fluorescent FluoroLink™ streptavidin-labelled Cy3 dye (Amersham Pharmacia, UK) using a biotin–streptavidin system. Briefly, 200 μL of a Detection solution (0.5 µL of 0.8 µg/µL streptavidin-labelled Cy3, 0.8 µl of 25 µg/µL BSA, made to 200 µL with 6X SSPE-T) was applied directly onto the array surface and a 22 × 25-mm lifter slip was placed over it to evenly distribute the solution on the array. The slides were placed in hybridization chambers, wrapped in aluminum foil and incubated at 37oC for 1 h in the dark. Finally, the slides were washed thrice in 6X SSPE-T for 5 min and rinsed with sterile MilliQ water before being dried with an air gun. All hybridizations were performed with six technical replicates (corresponding to six sub-arrays) and two biological replicates, resulting in 12 data points for each array feature.
Scan protocol
Slides were scanned with a ScanArray Gx (PerkinElmer Life and Analytical Sciences, Downers Grove, IL) microarray scanner in conjunction with the supplied software. The slides were scanned at 10 μm resolutions at 532 nm (Cy3, green laser) and 50% photomultiplicator (PMT) gain to keep background noise low. The quantitation of scanned array was performed using PerkinElmer ScanArray Express software v 2.0. The designed grid which was provided with the program was fully adjusted to match and fit all the printed spots on the array image, and the signal intensities were quantified using the adaptive circle and LOWESS functions.
Data processing
The scanning software flagged Probes which did not hybridize automatically and labelled as ‘bad’ while some other abnormal spots were flagged manually. ‘Good’ probes were accepted as having a mean ‘signal to noise ratio’ (SNR) value of a minimum 5, all the quantified data was then exported to Microsoft Excel Sheet. Data analysis included subtracting the background from median signal intensity for each feature, log2 transformation and combining technical replicates by taking average.
Virulence associated genes in Streptococcus uberis
Data table header descriptions
ID_REF
VALUE
Signal intensities and flag values of the three biological replicates were first compared before calculating the average of signal intensities for only those features that were flagged ‘Good’ in all three replicates. The values of features were converted to zero when even just one of the replicates had a ‘Bad’ flag.
