Blueprint for a Pop-up SARS-CoV-2 Testing Lab

Jennifer A. Doudna

Across the six dilutions tested, we observed a mean Ct value for MS2 of 24, with little variability among samples, indicating consistent nucleic acid RNA extraction ( Fig. 2A) . At our lowest concentration tested, 1 x 10 2 copies/ml, we were not able to detect amplification of any of the three SARS-CoV-2 primer-probe sets in any of our replicate samples, despite consistent MS2 detection, indicating that 1 x 10 2 copies/ml is below our detection limit ( Fig. 2A, left) . At 5 x 10 2 we observed amplification of each of the three target SARS-CoV-2 genes, however these values were not reproducible across triplicates ( Fig. 2A, ORF1ab is undetected for a single replicate at 5 x 10 2 copies/ml). Thus, the limit of detection was identified as 1 x 10 3 copies/ml, the lowest concentration tested where all target genes were detected for all replicates. Extraction and PCR 14 . CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We next sought to confirm the measurement 1 x 10 3 genomic copies/ml as the LOD by performing the assay in 20 technical replicates. For each of the 20 replicates, we detect all three SARS-CoV-2 genes, as well as MS2 internal control, as demonstrated by consistent, positive Ct values (Fig. 3, left) . To control for non-specific amplification, we extracted human RNA from 293T cells and subjected it to the same RNA extraction and RT-qPCR workflow. We observed no cross reactivity from any of the four primer-probe sets (Fig. 3, full workflow controls) . Nucleic acid extraction and PCR controls showed expected values, with no detectable amplification of SARS-CoV-2 genes in MS2 nucleic acid only or water only samples (Fig. 3 , PCR controls). The SARS-CoV-2 positive control from Thermo Fisher exhibited expected amplification of all three SARS-CoV-2 genes targeted, as well as the expected absence of MS2 nucleic acid. Together, our results show that we were able to detect 20/20 of our replicates, exceeding the required 19 out of 20 as defined by the FDA, and establishing 1 x 10 3 genomic copies/ml as our LoD.