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

P, Wollschlaeger; D, Todt; N, Gerlitz; S, Pfaender; T, Bollinger; A, Sing; A, Dangel; N, Ackermann; K, Korn; A, Ensser; E, Steinmann; M, Buhl; J, Steinmann

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 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 to 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 gene. N gene coding sequence of SARS-CoV-2 with and without D3L mutation (specific for B.1.1.7) was cloned into pCR®-TOPO vectors to validate polymorphism dependent N gene dropout with 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 (Δ 6-10, N gene dropout on Ct values >29) of N gene compared to 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 AUC = 1) in a receiver operating characteristic (ROC) curve analysis. Identical Ct shifts (Δ 7-10) were detected in reverse genetic experiments, using isolated plasmids containing N gene coding sequences corresponding to D3 or 3L variants. CONCLUSIONS: A 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.