Sars cov 2 n1 n2 assay kit

The RNA of SARS-CoV-2 was quantified by RT–qPCR amplification of the nucleocapsid (N) and envelope (E) genes from the viral genome. The E gene was targeted by the E_Sarbeco primers and E_Sarbeco_P1 probe (Corman et al., 2020 (link)), and the N gene was targeted by the SARS-CoV-2 N1+N2 Assay Kit (Qiagen, Germany), which includes N1 and N2 primers and probes from the CDC design (Lu et al., 2020 (link)). The total volume of the RT–qPCR was 20 μL, and all measurements were performed in duplicate for each sample. The reaction mixture contained 5 μL of RNA, 10 μL of 2 × Reaction Mix from the SuperScript™ III One-Step RT–PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, USA), 16 nmol of additional MgSO₄, 0.5 μL of SuperScript™ III RT/Platinum™ Taq Mix, and either of the two primer and probe sets. E_Sarbeco primers and probes were used at final concentrations of 0.4 nM and 0.2 nM, respectively. The N1+N2 Assay Kit was used at a 20× dilution. The thermal cycling conditions for both E gene and N gene reactions were as described by Corman et al. (2020) (link). Serial dilutions of the synthetic RNA SARS-CoV-2 Positive Run Control (Exact Diagnostics, USA) were used to produce a standard curve.

During blood collection for flow cytometry experiments, quantitative real-time PCR (RT qPCR) RT qPCR was performed as described previously [18] (link). RNA was isolated from nasopharyngeal or oropharyngeal swabs, and RNA was extracted (QIAprep& Viral RNA UM Kit QIAGEN, USA) and amplified by one-step RT qPCR (SARS-CoV-2N1 + N2 Assay Kits - QIAGEN, USA). This procedure is essential to ensure no SARS-CoV-2 infection or reinfection in the HC and recovered patients, respectively.