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. 2021 Nov 10;16(11):e0258263.
doi: 10.1371/journal.pone.0258263. eCollection 2021.

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

Elizabeth C Stahl  1   2 , Allan R Gopez  2 , Connor A Tsuchida  1   2 , Vinson B Fan  1 , Erica A Moehle  1   2 , Lea B Witkowsky  1   2 , Jennifer R Hamilton  1   2 , Enrique Lin-Shiao  1   2 , Matthew McElroy  2 , Shana L McDevitt  1   2 , Alison Ciling  1   2 , C Kimberly Tsui  1 , Kathleen Pestal  1 , Holly K Gildea  1 , Amanda Keller  2 , Iman A Sylvain  2 , Clara Williams  2 , Ariana Hirsh  1   2 , Alexander J Ehrenberg  1 , Rose Kantor  1   3 , Matthew Metzger  1   3 , Kara L Nelson  1   2   3 , Fyodor D Urnov  1   2 , Bradley R Ringeisen  1   2 , Petros Giannikopoulos  2 , Jennifer A Doudna  1   2   4 ; IGI Testing Consortium
Affiliations

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

Elizabeth C Stahl et al. PLoS One. .

Abstract

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a 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 (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

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Conflict of interest statement

The authors have read the journal’s policy and the authors of this manuscript have the following 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. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 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.
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, c) Full and half reaction volumes on the QuantStudio-6 with 4x master mix. Samples which failed to amplify are denoted as “not detected” (ND). Plasmid DNA was added directly into the master mix at 200,000 copies/mL (2-4x Saliva Direct limit of detection) in triplicate. Full reactions (15μL master mix with 5μL sample input), half reactions (7.5μL master mix with 5μL sample input).
Fig 2
Fig 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. Full reactions (15μL master mix with 5μL sample input), half reactions (7.5μL master mix with 5μL sample input). 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 was selected as the final formulation for LuNER.
Fig 3
Fig 3. Limit of detection and reproducibility.
a) The limit of detection was defined by extracting RNA from heat-inactivated virus at concentrations ranging from 10.24 to 0.01 TCID50/mL and performing RT-qPCR with the LuNER reagents. b) Representative plate controls for the LuNER assay are shown from the LoD titration experiment, including negative buffer-only control (NC), human RNA control (HC), RT-qPCR negative (blank), and RT-qPCR positive controls (synthetic SARS-CoV-2 RNA genome). c) An independent experiment showing reproducibility of the LoD at 0.64, 1.28, and 2.56 TCID50/mL.
Fig 4
Fig 4. Clinical concordance.
a) One hundred and twenty samples were pooled into thirty wells, each expected to contain one positive sample, and tested for SARS-CoV-2 with the LuNER assay, which flagged all thirty wells for testing individually, b) One hundred and twenty independent samples were pooled into thirty wells, each expected to contain zero positive samples, and tested for SARS-CoV-2 with the LuNER assay, which flagged one well for testing individually, c) Individual testing results for 124 samples flagged from pooled wells (pink lines denote inconclusive results, asterisk denotes expected negative pool 6), d) Four samples that amplified only N-gene were retested and resulted accordingly. Overall clinical concordance with the previous sample result was 99.6%, with one additional positive sample identified with LuNER.
Fig 5
Fig 5. Wastewater testing with LuNER.
a) PCR efficiencies of LuNER measured on the QuantStudio-3 using control plasmids ranging from 2.5–50,000 copies per reaction. Slopes between -3.1 and -3.6 giving reaction efficiencies between 90 and 110% are typically acceptable, b) Nine wastewater sites around the San Francisco Bay Area were surveilled for SARS-CoV-2 with LuNER on the QuantStudio-6, c) Comparing Ct Values for N1 between the test currently deployed by the pop-up wastewater lab and LuNER for each of the nine sites.
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