key: cord-0858688-iioni81h
authors: 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
title: 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
date: 2021-05-24
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2021.05.025
sha: 6bd47a3a5aa77f58a4b78f5607154af6c213ce5b
doc_id: 858688
cord_uid: iioni81h

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.

Multiplex PCR is considered as gold standard for SARS-CoV-2 detection, and dual or triple 79 target approaches are recommended [6, 7] . However, identification of viral variants is not 80 intended with regular PCR kits. The presence of SARS-CoV-2 variants in a patient sample can 81 potentially change the performance of the SARS-CoV-2 assay. Some studies have reported 82 dropouts of various genes targeted by SARS-CoV-2 multiplex PCR approaches affecting the S 83 gene [8, 9] , N gene [10, 11] or E gene [12] , which may allow presumptive identification of specific 84 lineages. While spread of VOCs is an imminent danger worldwide, rapid detection of VOCs is 85 limited by the turnaround time and costs, or availability of methods such as next-generation 86 sequencing (NGS) or variant-specific PCR, respectively. Here, we report an approach which To elucidate the underlying molecular mechanism resulting in the observed N gene dropout or 160

Ct value shift, we cloned the N gene coding sequence with and without D3L mutation into 161 pCR®-TOPO vectors which were then used for PCR analysis. 

Emergence and rapid spread of a new severe acute respiratory syndrome-related 235

SARS-CoV-2) lineage with multiple spike mutations in South Africa

Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7

Estimated 248 transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England

Detection of 251 2019 novel coronavirus ( 2019-nCoV ) by

Diagnostics for SARS-CoV-2 infections

S-variant SARS-CoV-2 is 257 associated with significantly higher viral loads in samples tested by ThermoFisher 258

S gene dropout 260 patterns in SARS-CoV-2 tests suggest spread of the H69del/V70del mutation in the US

A novel point 266 mutation in the N gene of SARS-CoV-2 may affect the detection of the virus by RT-qPCR

A recurrent 269 mutation at position 26,340 of SARS-CoV-2 is associated with failure of the E-gene qRT

PCR utilised in a commercial dual-target diagnostic assay

Characterization of a novel coronavirus associated with severe acute respiratory 276 syndrome

Approach Can Predict Candidate Targets for Immune 279 Responses to SARS-CoV-2

Isolation of virus from a SARS 282 patient and genome-wide analysis of genetic mutations related to pathogenesis and 283 epidemiology from 47 SARS-CoV isolates

Could mutations of SARS-CoV-2 suppress diagnostic detection? Nat