Wollschlaeger P1, Todt D2, Gerlitz N1, Pfaender S3, Bollinger T4, Sing A5, Dangel A5, Ackermann N5, Korn K6, Ensser A6, Steinmann E3, Buhl M1, Steinmann J7. 1. Institute of Clinical Hygiene, Medical Microbiology and Infectiology, Paracelsus Medical University, Nuremberg, Germany. 2. Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany; European Virus Bioinformatics Center (EVBC), 07743 Jena, Germany. 3. Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany. 4. Microbiology, Laboratory Medicine, Klinikum Bayreuth GmbH, Bayreuth, Germany. 5. Bavarian Health and Food Safety Authority (LGL), Oberschleissheim, Germany. 6. Institute of Clinical and Molecular Virology, University Hospital of Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany. 7. Institute of Clinical Hygiene, Medical Microbiology and Infectiology, Paracelsus Medical University, Nuremberg, Germany. Electronic address: joerg.steinmann@klinikum-nuernberg.de.
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 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.
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.
Authors: Kurt J Vandegrift; Michele Yon; Meera Surendran-Nair; Abhinay Gontu; Saranya Amirthalingam; Ruth H Nissly; Nicole Levine; Tod Stuber; Anthony J DeNicola; Jason R Boulanger; Nathan Kotschwar; Sarah Grimké Aucoin; Richard Simon; Katrina Toal; Randall J Olsen; James J Davis; Dashzeveg Bold; Natasha N Gaudreault; Juergen A Richt; James M Musser; Peter J Hudson; Vivek Kapur; Suresh V Kuchipudi Journal: bioRxiv Date: 2022-02-07
Authors: Huanyu Wang; Sophonie Jean; Sarah A Wilson; Jocelyn M Lucyshyn; Sean McGrath; Richard K Wilson; Vincent Magrini; Amy L Leber Journal: Diagn Microbiol Infect Dis Date: 2021-12-29 Impact factor: 2.983