SARS-CoV-2 Viral Load Quantification by RT-qPCR

To measure viral load in SARS-CoV-2-infected patients, we performed quantitative reverse transcription-PCR (RT-qPCR) in the midturbinate swabs. Total RNA was extracted from the swabs using a phenol-chloroform-based method. The swabs were placed in red 1.5-mL RINO screw-cap tubes (NextAdvance) prefilled with RNase-free zirconium oxide beads, and QIAzol lysis reagent (Qiagen) was added. Samples were then homogenized in a Bullet Blender 24 Gold (NextAdvance) system for 3 min at maximum speed. Following homogenization, genomic DNA was eliminated with genomic DNA (gDNA) eliminator columns (Qiagen), and RNA was purified using the RNeasy mini plus kit (Qiagen) following the manufacturer’s protocol. The RNA quality was measured using a 2100 bioanalyzer (Agilent Technologies). The United States Centers for Disease Control and Prevention primers and probes designed for the detection of SARS-CoV-2 (2019-nCoV) were purchased from Integrated DNA Technologies (IDT) (48 ). Both the SARS-CoV-2 nucleocapsid gene region 1 (N1) and nucleocapsid gene region 2 (N2) were targeted to detect SARS-CoV-2. RNase P was also examined as a measure of RNA quality and quantity. RT-qPCR was performed using the SuperScript III one-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen) as per manufacturer’s instructions on a CFX96 touch real-time PCR detection system (Bio-Rad). Plasmid controls for 2 SARS-CoV-2 nucleocapsids and RNase P were also ordered from IDT at a concentration of 66,666 copies/reaction. No-template controls and an extraction negative were used as negative controls. Reactions were prepared using 12.5 μL of SuperScript III master mix (ThermoFisher), 1 μL each of 400-nm forward and reverse primer, 1 μL of 400 nM FAM-labeled probe, 1 μL of Platinum Taq polymerase, 3 μL of template RNA, and 7.25 μL of PCR-certified water (Teknova). RNA was reverse transcribed at 50°C for 15 min, and PCR conditions were run on a 95°C denaturation step for 2 min, followed by 40 cycles of 95°C for 15 s and 55°C for 30 s. The cycle threshold (C_T) values were captured and calculated by the CFX Maestro (Bio-Rad) software and used as a measure of viral load.