key: cord-0874343-njeetiaz
authors: Wollschlaeger, P.; Gerlitz, N.; Todt, D.; Pfaender, S.; Bollinger, T.; Sing, A.; Dangel, A.; Ackermann, N.; Korn, K.; Ensser, A.; Steinmann, E.; Buhl, M.; Steinmann, J.
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 widely used commercial multiplex PCR assay
date: 2021-03-26
journal: nan
DOI: 10.1101/2021.03.23.21254171
sha: 5947ed6fcd7c2f0805c5bb49b4f92fd0dbf99e98
doc_id: 874343
cord_uid: njeetiaz

Objectives. Increased importance in detection and surveillance of SARS-CoV-2 has been demonstrated due to the emergence of variants of concern (VOCs). In this study we evaluated if a commercially available real-time SARS-CoV-2 PCR assay can identify B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared to the S or RdRP gene. Methods. Patients samples with confirmed B.1.1.7 variant by whole-genome sequencing and variant-specific PCR (n=48) and non-B.1.1.7 samples (n=53) were tested by the Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay for presence of S, RdRP and N gene of SARS CoV-2. The N gene coding sequence of SARS-CoV-2 with and without D3L mutation (specific for B.1.1.7) were cloned into pCR-TOPO vectors and Allplex SARS-CoV-2/FluA/FluB/RSV PCR assay was performed. Results. All studied B.1.1.7 patient samples showed significantly higher Ct values (delta 6-10, N-gene dropout on Ct values >29) in the N gene compared to the respective values of S and RdRP gene. Receiver operating characteristic (ROC) curve analysis resulted in 100% sensitivity and specificity for delta Ct N/RdRP and delta Ct N/S. As a result of the reversed genetic experiments we found also the shift in Ct values for the 3L variant N-gene. Conclusions. N gene dropout or Ct value shift is specific for B.1.1.7 positive samples using 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.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of the life-threating 60 COVID-19 disease, has rapidly spread rapidly worldwide since December 2019. This positive-61 sense single-stranded RNA virus, a member of the beta-coronavirus subfamily. Since 62 transmission to humans the virus undergoes adaptive evolution, resulting in genetic variation that 63 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.23.21254171 doi: medRxiv preprint can be a challenge particularly in molecular diagnostics, which are based on the detection of small 64 regions of the viral genome. To this date, several major variants of concern (VOCs) of SARS-CoV-65 2 have been reported. Currently, (i) B. Multiplex PCR is considered as gold standard for SARS-CoV-2 detection, and dual or triple target 77 approaches are recommended. However, identification of viral variants is not intended with regular 78 PCR kits. The presence of SARS-CoV-2 variants in a patient sample can potentially change the 79 performance of the SARS-CoV-2 assay. Occasional dropouts of various genes included as targets 80 in SARS-CoV-2 multiplex PCR approaches have been reported, affecting the S gene [6, 7] , N gene 81 [8, 9] or E gene [10], which may allow presumptive identification of specific lineages. While rapid 82 spread of VOC lineages is an imminent danger in Europe and worldwide, rapid detection of VOCs 83 is limited by the turnaround time and costs or availability of methods such as next-generation 84 sequencing (NGS) or variant specific PCR, respectively. Here, we report an approach which 85 allows for presumptive identification of the B.1. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.23.21254171 doi: medRxiv preprint

Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 193 (SARS-CoV-2) lineage with multiple spike mutations in South Africa

Genomic 196 characterisation of an emergent SARS-CoV-2 lineage in Manaus: preliminary findings

Preliminary genomic characterisation of an 199 emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations Preliminary 200 genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set 201 of spike mutations

community-tested cases of SARS-CoV

Estimated 206 transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England

S-variant SARS-CoV-2 209 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath 210 RT-QPCR

A novel point 218 mutation in the N gene of SARS-CoV-2 may affect the detection of the virus by RT-qPCR

A recurrent 221 mutation at position 26,340 of SARS-CoV-2 is associated with failure of the E-gene qRT-PCR 222 utilised in a commercial dual-target diagnostic assay

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

Approach Can Predict Candidate Targets for Immune Responses 230 to SARS-CoV-2

Isolation of virus from a SARS 232 patient and genome-wide analysis of genetic mutations related to pathogenesis and 233 epidemiology from 47 SARS-CoV isolates

No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted

All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (gray line and 99% prediction band). No significant difference in slope is determined, y-intercepts 276 (elevations) differ significantly (**** p < 0.0001). 277 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.23.21254171 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.23.21254171 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.23.21254171 doi: medRxiv preprint