Multi-site Evaluation of SARS-CoV-2 Spike Mutation Detection Using a Multiplex Real-time RT-PCR Assay
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Abstract
Background
SARS-CoV-2 causes COVID-19, which can be fatal and is responsible for a global pandemic. Variants with increased transmissibility or the potential to evade immunity have emerged and represent a threat to global pandemic control. Variants of concern (VOC) can be identified by sequencing of viral RNA, or by more rapid methods for detection of subsets of signature mutations.
Methods
We developed a multiplex, real-time RT-PCR assay (cobas ® SARS-CoV-2 Variant Set 1) for the qualitative detection and differentiation of three key SARS-CoV-2 mutations in the viral spike protein: del 69-70, E484K and N501Y. Analytical sensitivity and accuracy were evaluated at three testing sites using clinical specimens from patients infected with SARS-CoV-2 variants belonging to several different lineages, including B.1.1.7, B.1.351, and P.1.
Results
The limit of detection for E484K was between 180 and 620 IU/mL for the three different isolates tested. For N501Y, the LOD was between 270 and 720 IU/mL (five isolates), while for del 69-70, it was 80 - 92 IU/mL (two isolates). Valid test results were obtained with all clinical specimens that were positive using routine diagnostic tests. Compared to sequencing (Sanger and next-generation), test results were 100% concordant at all three loci; no false positive or false negative results were observed.
Conclusions
Data collected at three independent laboratories indicates excellent performance and concordance of cobas ® SARS-CoV-2 Variant Set 1 with sequencing. New sets of primers and probes that target additional loci can be rapidly deployed in response to the identification of other emerging variants.
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SciScore for 10.1101/2021.05.05.21254713: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Patient specimens were collected and used according to institutional review board regulations in effect at each site. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: The eight bacteria were bordetella pertussis, chlamydia pneumoniae, haemophilus influenzae, legionella pneumophila, mycobacterium tuberculosis, mycoplasma pneumoniae, streptococcus pyogenes, streptococcus pneumoniae. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Virus stocks for assay performance evaluation, including analytical sensitivity, were prepared by culturing virus from de-identified … SciScore for 10.1101/2021.05.05.21254713: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Patient specimens were collected and used according to institutional review board regulations in effect at each site. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: The eight bacteria were bordetella pertussis, chlamydia pneumoniae, haemophilus influenzae, legionella pneumophila, mycobacterium tuberculosis, mycoplasma pneumoniae, streptococcus pyogenes, streptococcus pneumoniae. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Virus stocks for assay performance evaluation, including analytical sensitivity, were prepared by culturing virus from de-identified patient specimens in Vero cells. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Experimental Models: Organisms/Strains Sentences Resources Clinical specimens: Residual de-identified clinical specimens from three routine testing sites were used: Labor Berlin (Berlin, Germany), Bioscentia Labor (Ingelheim am Rhein, Germany), and the Institute of Clinical Microbiology and Hygiene, University Hospital of Regensburg (Regensburg, Germany). Labor Berlinsuggested: NoneSoftware and Algorithms Sentences Resources The primers and probes were designed based on SARS-CoV-2 sequences in the GISAID and NCBI databases (total over 345,000 sequences) and were designed to provide maximum inclusivity for detecting the targets in each channel. NCBIsuggested: (NCBI, RRID:SCR_006472)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.
Results from scite Reference Check: We found no unreliable references.
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