SARS-CoV-2 RNA Extraction and qRT-PCR Detection

Five de-identified samples that had been assessed as positive by other methods were obtained from the New Zealand Auckland District Health Board. An aliquot of 80 µl of Viral Transport Media that had previously stored a nasopharyngeal swab from a patient infected with SARS-CoV-2 was used for RNA isolation using the QIAamp Viral RNA Mini spin kit (Qiagen, Cat No./ID: 52904) according to manufacturer specifications, with the following modifications: sample volume was brought up from 80 to 140 µl using 1× PBS, and RNA was eluted using two elutions with 40 µl Buffer AVE for a final volume of ∼80 µl.
To determine C_q values, we performed quantitative RT-PCR. We used the 2× SensiFAST Probe No-ROX One-Step Mix (Bioline, Cat No./ID: BIO-76005), according to manufacturer specifications using 2.5 µl RNA template. Primers and probes were from the CDC 2019-nCoV CDC Assay (IDT, Cat No./ID: 10006606) targeting the N1 and N2 regions of the nucleocapsid phosphoprotein of SARS-CoV-2. NP targets the RNase P gene for detection of human nucleic acids and serves as an internal control for sample integrity. Reactions were run at a total volume of 20 µl on a real-time PCR device. The results are shown in Table 2.