Fig. 1: Overview of the IGI SARS-CoV-2 Testing Consortium assay.

Diagram of the IGI’s testing workflow and assay for SARS-CoV-2 using the automated method. Our test begins with a custom specimen collection kit for nasopharyngeal (NP) or oropharyngeal (OP) swabs (step 1). This kit uses alternative collection tubes and a chaotropic agent, DNA/RNA Shield, to inactivate the virus for transport to our facility (step 2). Barcodes are supplied to connect patient intake forms with specimens. Samples are transported to the IGI by courier where they are visually inspected for rejection criteria before being decontaminated in a biosafety cabinet and placed into trays for arraying. In step 3, a Hamilton STARlet liquid handler scans the sample barcodes, accessioning them into our LIMS, and arrays them into 96-well deep-well plates, recording the corresponding positions in the LIMS. Step 4 is conducted by a Hamilton Vantage liquid handler in our automated method and manually in our semi-automated method. The MS2 spike-in control is added and RNA extraction is performed using Thermo Fisher’s MagMAX Viral/Pathogen Nucleic Acid Isolation Kit. Following extraction, the Hamilton Vantage performs RT-qPCR reaction setup using Thermo Fisher’s TaqPath RT-PCR COVID-19 Kit by consolidating four 96-well plates into one 384-well plate preloaded with RT-qPCR master mix (this step performed manually in our semi-automated approach and using 96-well plates only). After the Hamilton Vantage adds positive and negative controls, the plate is moved to an ABI QuantStudio 6 Flex Real-Time PCR System for RNA detection. In our semi-automated approach, RT-qPCR is performed on an Applied Biosystems 7500 Fast Real-Time PCR system. Step 6 involves interpretation of the RT-qPCR data. Thermo Fisher’s TaqPath RT-PCR COVID-19 Kit targets three SARS-CoV-2 genes shown in blue on the diagram of the SARS-CoV-2 genome (NCBI NC_045512.2). The open reading frame targeted by Thermo Fisher’s kit, ORF1ab, encodes non-structural proteins for replication, whereas the spike (S) and nucleocapsid (N) genes encode two structural proteins. A diagram of qPCR amplification curves demonstrates the criteria by which sample data are interpreted; Ct, cycle threshold. Further criteria for data interpretation are detailed in “Methods” on Figshare, and details of the workflow are diagrammed in “Semi-Automated and Automated Workflow Equipment” on Figshare. Portions of figure created using BioRender.com.