Abi 7500 fast dx

N1 and N2 nucleocapsid primers from the CDC assay were obtained from Integrated DNA Technologies (IDT). PCR cycling conditions including reverse transcription were run per the CDC EUA-approved protocol⁵ with the Promega GoTaq Probe 1-Step RT-qPCR system (Catalog # A6120). In brief, a 20 μL reaction comprising 10 μL GoTaq, 0.4 μL GoScript, 1.5 μL primer master mix, 3.1 μL water, and 5 μL input RNA (or crude lysate) was reverse transcribed by incubating at 45 °C for 15 min followed by 95 °C for 2 min per CDC protocol. The reaction was then thermocycled for 45 cycles of 95 °C for 20 s (denaturation) and 55 °C for 30 s (annealing) on either the ABI 7500 Fast DX (Applied Biosystems) or the QuantStudio 5 Real-Time PCR System (Thermo Fisher).

Sanger sequencing (SINOMD, Beijing, China) was used to determine the frequency of mutations in IDH1 and IDH2. Sanger sequencing were performed using an ABI‐3130 DNA Analyzer (Applied Biosystems).
Methylation-specific quantitative PCR (qMSP) was applied to detect the status of MGMT promoter methylation. Genomic DNA was extracted from paraffin section and converted by bisulfite. The QIAamp DNA FFPE tissue Kit (Tiangen Biotech, Beijing, China) and DNA Bisulfite Conversion Kit (Tiangen Biotech, Beijing, China) were used according to the manufacturer’s instructions. The converted DNA was mixed with MGMT methylation mixture (SINOMD, Beijing, China). Real-time quantitative polymerase chain reaction was performed by ABI 7500 Fast Dx (Applied Biosystems Co. Ltd., US) at 95°C for 3 min, then 45 cycles of 95°C for 15 s and 60°C for 45 s.

Plasma was inactivated in TRIzol LS (ThermoFisher Scientific, Waltham, MA), and total RNA was isolated using the QIAamp Viral RNA Mini Kit (QIAGEN, Germantown, MD) in accordance with manufacturer’s instructions. Briefly, 70 μL of TRIzol LS-inactivated sample was added to 280 μL of QIAGEN Buffer AVL containing carrier RNA. Sample was eluted in 70 μL of Buffer AVE, aliquoted, and frozen until assayed. Plasma viral RNA was quantified using BEI Resources Critical Reagents Program EZ1 RT-qPCR (TaqMan) assay kit on an ABI 7500 FastDx (Applied Biosystems, ThermoFisher Scientific) in accordance with manufacturer’s instructions using a synthetic RNA standard curve and reported as viral RNA copies (log₁₀) per mL of sample.

Genomic DNA was extracted from surgically excised fresh solid tumor tissues. The TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) were used according to the manufacturer's instructions.
For each sample, mutations of KRAS exons 2 (codon 12 and 13) were detected by Human KRAS Gene Seven Mutation Detection Kit (YZY Medical Science & Technology Co., Ltd., Wuhan, China); mutations of NRAS exons 2 (codon 12 and 13), exons 3 (codon 59 and 61), exons 4 (codon 117 and 146) were detected by Human NRAS Gene Mutation Detection Kit (YZY Medical Science & Technology Co., Ltd., Wuhan, China); mutations of BRAF exons 15 (codon 600) was detected by Human BRAF Gene V600E Mutation Detection Kit (YZY Medical Science & Technology Co., Ltd., Wuhan, China). All operations were strictly performed in accordance with the kit manual. Specifically, diluted 30 ng of total DNA sample to 2 μl, then mixed with 0.2 μl polymerase. The mixture was then added to a tube preloaded with a dual fluorescent probe primer. Real-time quantitative polymerase chain reaction was performed by ABI 7500 Fast Dx (Applied Biosystems Co. Ltd., US) as 37°C for 10 min, 95°C for 5 min, then 40 cycles of 95°C for 15 s and 60°C for 60 s.

To detect SARS-CoV-2 viral infection, a one-step Real-Time PCR assay was performed using STANDARD M nCoV Real-Time detection kit (SD Biosensor, Korea), targeting the nCoV2 specific ORF1ab (RdRp) and pan-sarbeco specific E genes on LightCycler^® 480 System (Roche) and ABI 7500 Fast DX (Applied Biosystems) as per the manufacturer’s instructions.