IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2
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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 Lu na 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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SciScore for 10.1101/2020.12.10.20247338: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: We confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Limit of Detection: Heat-inactivated virus was generated in the BSL3 laboratory at the University of California, Berkeley. Briefly, SARS-CoV-2 was cultured in Vero E6 cells and titers were determined using cytopathic effect (CPE) assays. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resou… SciScore for 10.1101/2020.12.10.20247338: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: We confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Limit of Detection: Heat-inactivated virus was generated in the BSL3 laboratory at the University of California, Berkeley. Briefly, SARS-CoV-2 was cultured in Vero E6 cells and titers were determined using cytopathic effect (CPE) assays. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources Ct values were exported into Excel version 16.42 (Microsoft Office, Redmond WA) and plotted with Prism 8.4.3 (Graphpad, San Diego, CA) as the average value ± standard deviation. Excelsuggested: NonePrismsuggested: (PRISM, RRID:SCR_005375)Ct values were exported into Excel version 16.42 (Microsoft Office, Redmond WA) and plotted with Prism 8.4.3 (Graphpad, San Diego, CA) as the average value ± standard deviation. Reproducibility: Eight clinically reported negative samples (OP/mid-turbinate swabs in 4mL of 1x RNA/DNA shield) were pooled to generate a negative sample matrix for spike-in experiments. Graphpadsuggested: (GraphPad, RRID:SCR_000306)Clinical Concordance: 91 OP/mid-turbinate samples with clinically reported results (61 positive, 30 negative), based on original RNA extraction with the MagMax Viral/Pathogen Nucleic Acid Isolation Kit and the TaqPath RT-PCR COVID-19 kit (ThermoFisher), were arrayed from the original tubes (containing residual volume of approximately 3.5mL of 1x DNA/RNA shield) into a 96-well deep-well plate on the Microlab STARlet. ThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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