Luxscan 10k b microarray scanner

For sputum sample collection and processing, the first sputum sample was collected in the early morning. After a clear water gargle, we first asked the patient to produce a deep, hard cough to raise sputum; the sample was deposited in sterile sample containers, sealed, and inspected. The samples were incubated 1 to 2 times in 4% NaOH, with agitation. After 15 to 20 min, we added mixed phosphate buffer, pH 6.8. After centrifugation, the supernatant was precipitated, and the pellet was washed with 0.5–1 ml of mixed phosphate buffer. The precipitates were then applied to the GeneChip according to the manufacturer’s instructions. The results were obtained via semi-automatic scanning using a LuxScan 10 K.B microarray scanner (CapitalBio Technology Inc., Beijing, China) (see Fig. 3 for common results).Fig. 3

Common results of the CapitalBio™ DNA microarray detection spectra are shown for samples with mutation(s) at a: WT: wild-type. b: NTB: nontuberculous mycobacteria. c: rpoB gene codon 531 (TCG → TTG). d: rpoB gene codon 526 (CAC → TAC). e: katG gene codon 315 (AGC → ACC). f: inhA gene promoter − 15 (C → T)

Genomic DNA was extracted from whole blood samples (empty stomach on the early morning of the day after admission) with a Lab-Aid nucleic acid (DNA) magnetic bead separation kit (Zeesan, Xiamen) according to the manufacturer's instructions. The PEAR1 rs12041331 in human whole blood genomic DNA was detected by the combination of multiplex allele-specific PCR and universal array developed by CapitalBio Technology Corporation, Ltd. Using human whole blood genomic DNA as the template, amplicons from PEAR1 gene were multiplex PCR-amplified with allele-specific PCR primers. After amplification, the reaction mixture was hybridized with specific tag probes immobilized on a microarray chip in the CapitalBio BioMixerTM II Microarray Hybridization Station (CapitalBio Corporation, Beijing, China). Hybridization was stopped by washing the slide in a wash buffer. The chips were scanned and imaged using LuxScan 10K-B Microarray Scanner (CapitalBio Corporation, Beijing, China). The detection results of polymorphic loci were obtained.

Serum reaction against purified recombinant proteins was determined using well-type amine arrays as previously described [30 (link), 31 (link)]. For the assays, teflon tapes with holes were pasted on adhesion microscope slides (CITOTEST Scientific, Jiangsu, China) as protein microarrays; 1 μl of rPocDBP-RII (50 μg/μl) was spotted to each well of the arrays and incubated for 2 h at 37 °C. Array slides were blocked with 5% BSA in PBST at 37 °C for 1 h. Serum samples diluted to 1:250 from each of the four blood collection periods or a set of 1:2 serial dilution from 1:250 to 1:32,000 drawn from the third and fourth blood collection periods was added to the chip for 1 h at 37 °C. Pre-immune serum and serum from mice immunized with adjuvant alone served as negative controls. Finally, bound antibodies were visualized using Alexa Fluor 555-labeled Donkey Anti-Mouse IgG (H + L) (1:100 dilution; catalog no. A0460, Beyotime, Shanghai, China), scanned by LuxScanTM 10 K-B Microarray Scanner (catalog no. 100020, CapitalBio); fixed circle approach was used to quantify the mean fluorescence intensity (MFI).

Genetic analysis was conducted for all patients using genomic DNA from peripheral
blood samples. The genetic screening included nine common mutations in four common
non-syndromic hearing loss related genes, including GJB2(NG_008358.1), SLC26A4 (NG_008489.1), GJB3(NG_008309.1; NM_024009.2) and MT-RNR1 (NC_012920.1). The mutations
were detected by allele-specific polymerase chain reaction (PCR) and universal array
(BioMixer^TM, CapitalBio Corporation, Beijing, China) for simultaneously
screening the nine mutations leading to hearing impairment (GJB2:
c.235delC, c.35delG, c.176del16, c.299-300delAT; SLC26A4: c.919-2A
> G, c.2168A > G; GJB3: c.538C > T;
MT-RNR1: m.1555A > G, m.1494C > T). The multiplex
allele-specific PCR was performed as described previously (Qu et al., 2012 (link)) and the results of microarrays
were scanned and analyzed by a LuxScan^TM 10K/B Microarray Scanner
(CapitalBio). The results were also validated by Sanger sequencing for wild and
mutant types.