Tianamp genomic dna extraction kit

After we received informed consent from patients, we acquired 4ml of peripheral blood for DNA extraction. Genomic DNA was extracted from this peripheral blood mixed with ethylenediaminetetraacetic acid according to standard methods using TIANamp Genomic DNA Extraction Kits (Tiangen Biotech Corporation, Beijing, China), according to the manufacturer’s instructions.
The genotyping of HLA class I (-A, -B, and–C) and class II (-DR, -DQ, and–DP) was performed using polymerase chain reaction-sequence-based typing (PCR-SBT) methods, as described in a previous study [25 (link)]. In total, 20 alleles for HLA-A, 36 alleles for HLA-B, 22 alleles for HLA-C, 29 alleles for HLA-DRB1, 16 alleles for HLA-DQB1, and 13 alleles for HLA-DPB1 were detected. Amino acid sequences for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 with a resolution of 4 digits were obtained from the international ImMunoGeneTics references [26 (link)]. Analysis of variant amino acids was performed across all HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 alleles present in the genotyping results.

Genomic DNA from heart tissue was isolated using the TIANamp Genomic DNA Extraction Kit according to the manufacturer’s protocol (Tiangen Biotech, Beijing). Whole blood was centrifuged quickly at 1350 ± 150 RCF for 10 min at room temperature. Plasma was carefully transferred to a new tube and then stored at − 20 °C until DNA extraction. Cell-free DNA was isolated from plasma using the Nucleic Acid Purification/Magnetic Beads Kit (GENESHINE, Shanghai, China) following the manufacturer’s recommendations. DNA concentrations were quantitated using the Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific).

DNA was extracted with the TIANamp genomic DNA extraction kit (Tiangen, #DP304) following manufacturer’s instructions. Quantitative real-time PCR reactions were performed in a total volume of 20 µL using 10 µL SYBR Green Master Mix (Vazyme, #Q111-02), 2 µL DNA and 200 nM of COXII (mitochondrial DNA) or RPL13A (genomic DNA) primers (Supplementary Table 1). Reactions were run on a Roche 480II with an initial step of 30 s at 95 °C, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Relative differences between mitochondrial and genomic DNA were calculated by the 2^-ΔΔCt method.

The constructed infectious clones p15A-cm-H/H/R188I, p15A-cm-O/O/I188R, and p15A-cm-H/O/I188R were linearized with PmeI (NEB, USA, catalog no. R0560S) overnight at 37°C and purified by ethanol precipitation before transfection to LMH cells. LMH cells were seeded in T25 flasks a day before transfection. The transfection mixture was carried out with 5 μg of the linearized infectious clones and Lipofectamine 3000 according to the manufacturer’s instruction (Thermo Fisher Scientific, USA). The cells were harvested when obvious cytopathic effects occurred. The harvested viruses were centrifuged at 10,000 rpm for 1 min, and the supernatant was aliquoted and stored at −80°C. The viral DNA was extracted using TIANamp genomic DNA extraction kit (Tiangen, Beijing, China, catalog no. DP304) for sequencing identification. The successfully rescued recombinant viruses were named H/H/R188I, O/O/I188R, and H/O/I188R, respectively.

To determine the viral loads in heart, liver, spleen, lung, kidney, cecal tonsil, bursa of Fabricius, duodenum, proventriculus and pancreas samples and the viral shedding in the cloacal swab samples, total DNA was extracted from 100 mg of tissue samples or 200 μL of the cloacal swab samples with the TIANamp genomic DNA extraction kit (Tiangen, Beijing, China, Cat. no. DP304). The viral copy numbers were determined with previously established standard curve quantitative real-time PCR (qRT-PCR) methods [22 (link)]. Briefly, the ORF14 genes of FAdV-4 and FAdV-8b were cloned into the pMD18-T vector to construct standard plasmids pMD18T-ORF14/4 and pMD18T-ORF14/8b and used as indicators for the presence of the viruses. The measurement of viral copy number in organs and cloacal swab samples by qRT-PCR was carried out as previously described [22 (link)]. The number of viral genomes was expressed as log₁₀ (copy number/mg).