Qiacube ht system

Sputum samples were collected from each patient in the hospital. The samples were subsequently divided into two aliquots and stored at − 80 °C until required for the assay. Nucleic acids were extracted from 200-µL samples using the cador Pathogen 96 QIAcube HT Kit (Qiagen) for automated viral DNA and RNA extraction with the QIAcube HT System. The samples were tested individually by qRT-PCR using specific primers and probes targeting different genomes according to our previously reported qRT-PCR methods [18 (link)]. The qRT-PCR assays were used to detect 16 different respiratory viruses: CoV (229E, NL63, OC43 and HKU1), PIV1–4, IV types A and B, RSV types A and B, HRV/EV, HMPV, human adenovirus (ADV), and human bocavirus (HBoV).

For viral RNA extraction, briefly, 200 μL of sample was extracted with the QIAamp 96 Virus QIAcube HT kit (QIAGEN) on the QIAcube HT System (QIAGEN) according to manufacturer’s instructions. Purified nucleic acid was then immediately converted to cDNA by reverse transcription with random hexamers using the SensiFAST cDNA Synthesis Kit (Bioline Reagents) as per manufacturer’s instructions. cDNA was used immediately in the real-time reverse transcription PCR (rRT-PCR) or stored at –20°C. A total of 3 μL of cDNA was added to a commercial real-time PCR master mix (PrecisionFast qPCR Master Mix; Primer Design) in a 20 μL reaction mix containing primers and probe (final concentration of 0.9 mM primer and 0.2 mM probe, respectively). Samples were tested for the presence of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes using previously described primers and probes (44 (link), 45 (link)). Thermal cycling and rRT-PCR analyses for all assays were performed on the ABI 7500 FAST real-time PCR system (Applied Biosystems) with the following thermal cycling profile: 95°C for 2 minutes, followed by 45 PCR cycles of 95°C for 5 seconds and 60°C for 25 seconds for N gene and 95°C for 2 minutes, followed by 45 PCR cycles of 95°C for 5 seconds and 55°C for 25 seconds for RdRp/Hel gene and S gene.

For viral RNA extraction, 200 μl of nasal swab sample was extracted with the QIAamp 96 Virus QIAcube HT kit (Qiagen, Germany) on the QIAcube HT System (Qiagen) according to the manufacturer’s instructions. Purified nucleic acid was then immediately converted to complementary DNA (cDNA) by reverse transcription with random hexamers using the SensiFAST cDNA Synthesis Kit (Bioline Reagents, UK) according to the manufacturer’s instructions. cDNA was used immediately in the real-time reverse transcription (rRT)–PCR or stored at −20°C. Three microliters of cDNA was added to a commercial real-time PCR master mix (PrecisionFast qPCR Master Mix; Primer Design, UK) in a 20-μl reaction mix containing primers and probes with a final concentration of 0.8 and 0.1 μM for each primer and the probe, respectively. Samples were tested for the presence of SARS-CoV-2 N genes using previously described primers and probes (28 (link), 29 (link)). Thermal cycling and rRT-PCR analyses for all assays were performed on the ABI 7500 FAST real-time PCR system (Applied Biosystems, USA) with the following thermal cycling profile: 95°C for 2 min, followed by 45 PCR cycles of 95°C for 5 s and 60°C for 30 s for N gene.