Legionella strains were cultured onto buffered charcoal yeast extract (BCYE) agar plates (Hope Bio-technology Co., Ltd, Qingdao, China) and incubated in a 5% CO2 incubator at 37°C for 2–4 days.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted from pure cultures using bacterial genomic DNA purification kit (Tiangen Biotech Co., Ltd., Beijing, China).
Label
Cy3
Label protocol
To label the PCR products, 10 µL of the PCR products generated from the first run and the reverse primer and 0.3 µL of 25 nM Cy3-dUTP were added to the PCR mixture, and PCR was carried out using the same PCR conditions described for the first step.
Hybridization protocol
All labeled PCR products were precipitated using 100% cold ethanol, centrifuged at 13,000 g for 10 min, washed with 75% ethanol, and dried at room temperature. The dried, labeled DNA was diluted in 16 µL of hybridization buffer (50% formamide, 6× SSC, 5× Denhardt, and 0.5% SDS) and then hybridized with the prepared microarray at 45°C for 12 h. After hybridization, the slide was washed with solution A (1× SSC and 0.1% SDS) for 3 min, solution B (0.05× SSC) for 3 min, and solution C (95% ethanol) for 1.5 min.
Scan protocol
The microarray was dried under a gentle air stream and scanned with a laser beam of 532 nm using the GenePix biochip scanner 4100A (Axon Instruments, CA, USA) set to the following parameters: photomultiplier tube gain, 600, and pixel size, 5 µm.
Data processing
The signal-to-noise ratio (SNR) was calculated for each spot using the built-in software, GenePix Pro 6.0, with the threshold set at 3.0. A signal was considered positive when 70% of the probes to a respective target gene generated hybridization signals above the SNR threshold.
