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Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern.
Microbiol Spectr. 2022 02 23; 10(1):e0068121.MS

Abstract

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole-genome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times.

Authors+Show Affiliations

Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada. Division of Infectious Diseases, Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Dalla Lana School of Public Health, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Microbiology, Sinai Health/University Health Network, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada.Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada. Department of Laboratory Medicine and Pathobiology, University of Torontogrid.17063.33, Toronto, Ontario, Canada. Division of Infectious Diseases, Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Validation Study

Language

eng

PubMed ID

35170989

Citation

Abdulnoor, Mariana, et al. "Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated With B.1.1.7 Variant of Concern." Microbiology Spectrum, vol. 10, no. 1, 2022, pp. e0068121.
Abdulnoor M, Eshaghi A, Perusini SJ, et al. Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern. Microbiol Spectr. 2022;10(1):e0068121.
Abdulnoor, M., Eshaghi, A., Perusini, S. J., Broukhanski, G., Corbeil, A., Cronin, K., Fittipaldi, N., Forbes, J. D., Guthrie, J. L., Kus, J. V., Li, Y., Majury, A., Mallo, G. V., Mazzulli, T., Melano, R. G., Olsha, R., Sullivan, A., Tran, V., Patel, S. N., ... Gubbay, J. B. (2022). Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern. Microbiology Spectrum, 10(1), e0068121. https://doi.org/10.1128/spectrum.00681-21
Abdulnoor M, et al. Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated With B.1.1.7 Variant of Concern. Microbiol Spectr. 2022 02 23;10(1):e0068121. PubMed PMID: 35170989.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern. AU - Abdulnoor,Mariana, AU - Eshaghi,AliReza, AU - Perusini,Stephen J, AU - Broukhanski,George, AU - Corbeil,Antoine, AU - Cronin,Kirby, AU - Fittipaldi,Nahuel, AU - Forbes,Jessica D, AU - Guthrie,Jennifer L, AU - Kus,Julianne V, AU - Li,Ye, AU - Majury,Anna, AU - Mallo,Gustavo V, AU - Mazzulli,Tony, AU - Melano,Roberto G, AU - Olsha,Romy, AU - Sullivan,Ashleigh, AU - Tran,Vanessa, AU - Patel,Samir N, AU - Allen,Vanessa G, AU - Gubbay,Jonathan B, Y1 - 2022/02/16/ PY - 2022/2/17/pubmed PY - 2022/3/1/medline PY - 2022/2/16/entrez KW - COVID-19 KW - SARS-CoV-2 KW - VOC KW - real-time RT-PCR SP - e0068121 EP - e0068121 JF - Microbiology spectrum JO - Microbiol Spectr VL - 10 IS - 1 N2 - The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole-genome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times. SN - 2165-0497 UR - https://www.unboundmedicine.com/medline/citation/35170989/Real_Time_RT_PCR_Allelic_Discrimination_Assay_for_Detection_of_N501Y_Mutation_in_the_Spike_Protein_of_SARS_CoV_2_Associated_with_B_1_1_7_Variant_of_Concern_ DB - PRIME DP - Unbound Medicine ER -