Qiaamp 96 virus qiacube ht kit

Mouse organs were homogenized in RNALater, and viral RNA was isolated using the QIAamp 96 virus QIAcube HT kit (Qiagen). Two microliters of RNA was used as a template for the amplification of selected genes by real-time quantitative PCR (qRT-PCR) using HiSxript II one-step qRT-PCR SYBR green kit (Vazyme). Average values from duplicates of each gene were used to calculate the viral genomic copies. The primers based on the SARS-CoV-2 S gene were RBD-qF1, 5′-CAATGGTTTAACAGGCACAGG-3′, and RBD-qR1, 5′-CTCAAGTGTCTGTGGATCACG-3′. For qRT-PCR, the 10-μL mix contained 5 μL 2× one-step SYBR green mix, 1.9 μL nuclease-free water, 0.5 μL one-step SYBR green enzyme mix, 0.2 μL 50× ROX reference dye 1, 0.2 μL of each primer (10 m M), and 2 μL template RNA. Amplification was performed at 50°C for 3 min, 95°C for 30 s followed by 40 cycles consisting of 95°C for 10 s and 60°C for 30 s, and a default melting curve step in a Step-One Plus real-time PCR machine (ABI).

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.

The nasopharyngeal swabs were collected from suspected COVID-19 infected patients, between 1 July 2021 and 31 January 2022, from several hospitals in Apulia (Italy) and subsequently sent to Genetic and Molecular Epidemiology Laboratory of Experimental Zooprophylactic Institute of Apulia and Basilicata (IZSPB). No information about symptomatology and vaccination status was available.
Viral RNA was extracted from nasopharyngeal swabs in virus transport medium (COPAN’s Collection & Transport Kits for COVID-19). According to the manufacturer’s instructions, RNA extraction was performed from 200 µL of the virus transport medium using the QIAamp 96 Virus QIAcube HT Kit (Qiagen). Before RNA extraction, we added 20 µL of PEDV (Porcine Epidemic Diarrhea Virus), a Positive Control (PC) previously diluted in 200 µL of TE [28 (link)], which consists of the intact and inactive virus, to each sample. This PC was used as external control during the RNA extraction procedure. The extracted RNA was stored at −80 °C until the assay was performed.