SARS-CoV-2 N gene dropout and N gene Ct value shift as indicator for the presence of B.1.1.7 lineage in a commercial multiplex PCR assay

Wollschläger P, Todt D, Gerlitz N, Pfaender S, Bollinger T, Sing A, Dangel A, Ackermann N, Korn K, Enßer A, Steinmann E, Buhl M, Steinmann J (2021)

Publication Type: Journal article

Publication year: 2021

Journal

Clinical Microbiology and Infection Wiley-Blackwell

DOI: 10.1016/j.cmi.2021.05.025

Abstract

Objectives: Detection and surveillance of SARS-CoV-2 is of eminent importance, particularly due to the rapid emergence of variants of concern (VOCs). In this study we evaluated if a commercially available quantitative real-time PCR (qRT-PCR) assay can identify SARS-CoV-2 B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared with the S or RdRp gene. Methods: VOC B.1.1.7 and non-B.1.1.7 SARS-CoV-2-positive patient samples were identified via whole-genome sequencing and variant-specific PCR. Confirmed B.1.1.7 (n = 48) and non-B.1.1.7 samples (n = 58) were analysed using the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay for presence of SARS-CoV-2 S, RdRp and N genes. The N gene coding sequence of SARS-CoV-2 with and without the D3L mutation (specific for B.1.1.7) was cloned into pCR™II-TOPO™ vectors to validate polymorphism-dependent N gene dropout with the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. Results: All studied B.1.1.7-positive patient samples showed significantly higher Ct values in qRT-PCR (Δ6–10, N gene dropout on Ct values > 29) of N gene than the corresponding values of S (p ≤ 0.0001) and RdRp (p ≤ 0.0001) genes. The assay reliably discriminated B.1.1.7 and non-B.1.1.7 positive samples (area under the curve = 1) in a receiver operating characteristic curve analysis. Identical Ct value shifts (Δ7–10) were detected in reverse genetic experiments, using isolated plasmids containing N gene coding sequences corresponding to D3 or 3L variants. Discussion: An N gene dropout or Ct value shift is shown for B.1.1.7-positive samples in the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.

Authors with CRIS profile

Klaus Korn Institute of Clinical and Molecular Virology Armin Enßer Medizinische Fakultät

Involved external institutions

Paracelsus Medizinische Privatuniversität, Nürnberg DE Germany (DE) Ruhr-Universität Bochum (RUB) DE Germany (DE) Klinikum Bayreuth DE Germany (DE) Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL) DE Germany (DE)

How to cite

APA:

Wollschläger, P., Todt, D., Gerlitz, N., Pfaender, S., Bollinger, T., Sing, A.,... Steinmann, J. (2021). SARS-CoV-2 N gene dropout and N gene Ct value shift as indicator for the presence of B.1.1.7 lineage in a commercial multiplex PCR assay. Clinical Microbiology and Infection. https://doi.org/10.1016/j.cmi.2021.05.025

MLA:

Wollschläger, Paul, et al. "SARS-CoV-2 N gene dropout and N gene Ct value shift as indicator for the presence of B.1.1.7 lineage in a commercial multiplex PCR assay." Clinical Microbiology and Infection (2021).

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