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[Preprint]. 2020 Dec 11:2020.12.10.20247338.
doi: 10.1101/2020.12.10.20247338.

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

Elizabeth C Stahl  1   2 , Connor A Tsuchida  1   2 , Jennifer R Hamilton  1   2 , Enrique Lin-Shiao  1   2 , Shana L McDevitt  1   2 , Erica A Moehle  1   2 , Lea B Witkowsky  1   2 , C Kimberly Tsui  1 , Kathleen Pestal  1 , Holly K Gildea  1 , Matthew McElroy  2 , Amanda Keller  2 , Iman Sylvain  2 , Clara Williams  2 , Ariana Hirsh  1   2 , Alison Ciling  1   2 , Alexander J Ehrenberg  1 ; IGI SARS-CoV-2 consortium; Fyodor D Urnov  1   2 , Bradley R Ringeisen  1   2 , Petros Giannikopoulos  2 , Jennifer A Doudna  1   2   3
Affiliations

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

Elizabeth C Stahl et al. medRxiv. .

Abstract

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.

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Conflict of interest statement

Competing Interests: The Regents of the University of California have patents issued and pending for CRISPR technologies on which J.A.D. is an inventor. J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, Algen Biotechnologies, Felix Biosciences and Inari. J.A.D. is a Director at Johnson & Johnson and has research projects sponsored by Biogen, Pfizer, AppleTree Partners and Roche. F.D.U. is a co-founder of Tune Therapeutics. P.G. is a cofounder and Director at NewCo Health. P.G. is the CLIA Laboratory Director for Coral Genomics and 3DMed. The other authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Implementation and adaptation of the Saliva Direct RT-qPCR assay to detect SARS-CoV-2 N1 and human RNase P with the QuantStudio-6. Plasmid DNA was added directly into the master mix at 200,000 copies/mL (2–4x Saliva Direct limit of detection) in triplicate. A) Full and half reaction volumes on the CFx96 with 2x master mix, B) Full and half reaction volumes on the QuantStudio-6 with 2x master mix, D) Full and half reaction volumes on the QuantStudio-6 with 4x master mix. Samples which failed to amplify are denoted as “not detected” (ND).
Figure 2.
Figure 2.
Evaluating E-Sarbeco and RdRp-SARSr primers and probes with RNase P on the QuantStudio-6. A) Full and half-sized reactions of E-Sarbeco on the QuantStudio-6 with 4x master mix B) Full and half-sized reactions of RdRp on the QuantStudio-6 with 4x master mix. Plasmid DNA was added directly into the master mix at 200,000 copies/mL (2–4x Saliva Direct limit of detection) in triplicate. C) Evaluation of corrected RdRp reverse primers at two different concentrations on pooled RNA eluted from positive clinical swab samples in triplicate. p<0.001 (*) between mismatch and corrected at 400nM and between both concentrations of primers. Not significant (ns). D) E-Sarbeco primers and probes multiplexed with N1 and RNase P in a single half-sized reaction on the QuantStudio-6 with 4x master mix.
Figure 3.
Figure 3.
Limit of detection and reproducibility. A) The limit of detection was defined by extracting RNA from heat inactivated virus at concentrations ranging from 50 TCID50/mL to 0.1 TCID50/mL and performing RT-qPCR with the LuNER reagents. B) Twenty replicates at 2x LoD (1 TCID50/mL) were prepared by extracting RNA from heat inactivated virus and performing RT-qPCR with the LuNER reagents to test for reproducibility. Samples were positive if either N gene or E gene was detected at a Ct value < 40. Samples that did not amplify or had Ct values >=40 are shown on the x-axis. C) Controls for the LuNER assay are valid, including negative buffer-only control (“NC”), human RNA control (“HC”), qPCR negative, and qPCR positive controls.
Figure 4.
Figure 4.
Clinical concordance. A) RNA was extracted from known clinical samples and reanalyzed with the TaqPath COVID-19 kit or IGI-LuNER assay. B) Ct value comparisons for the 61 expected positive samples show 9 negative samples in the IGI-LuNER assay (pink lines), which were also negative in the TaqPath COVID-19 kit. An additional 10 samples assayed with the TaqPath COVID-19 kit returned an alternative result. C) Ct value comparison for N-gene between the IGI-LuNER and TaqPath assays shows strong concordance. Pink lines reflect samples in the TaqPath kit that returned inconclusive or invalid results despite amplifying N-gene in the expected positive samples. Logic gating for possible sample results is listed in the Methods.
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