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.
Keywords: B.1.1.7; Diagnostic quantitative RT-PCR; Mutation; SARS-CoV-2; VOC.
【저자키워드】 SARS-CoV-2, Mutation, B.1.1.7, VOC., Diagnostic quantitative RT-PCR, 【초록키워드】 whole-genome sequencing, Mutation, VoC, qRT-PCR, Genetic, polymorphism, variants of concern, detection, diagnostics, variants, B.1.1.7 lineage, PCR, Surveillance, B.1.1.7, vector, VOCs, Patient, Lineage, Ct value, RdRP, N gene, characteristic, Quantitative, RdRp gene, Analysis, Plasmid, Ct values, receiver operating characteristic, PCR assay, N genes, SARS-CoV-2 S, quantitative real-time PCR, plasmids, coding sequence, positive sample, SARS-CoV-2 B.1.1.7, Genes, approach, shown, identify, cloned, evaluated, analysed, can be used, significantly higher, experiments, Confirmed, 【제목키워드】 Multiplex PCR, SARS-CoV-2 N gene,