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Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2
Mariana Abdulnoor1,2,3, AliReza Eshaghi1, Stephen J Perusini1
1Public Health Ontariogrid.415400.4, Toronto, Ontario, Canada.
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.
Insights
A new real-time PCR assay efficiently screens for the N501Y mutation in SARS-CoV-2 variants of concern (VOCs). This rapid screening tool aids in surveillance, quickly identifying potential VOCs for further genomic sequencing and public health monitoring.
Area of Science:
- Virology
- Molecular Biology
- Public Health
Background:
- The emergence of SARS-CoV-2 variants of concern (VOCs) like B.1.1.7, B.1.351, and P.1, characterized by the N501Y mutation in the spike gene, poses a significant public health challenge.
- Effective surveillance is crucial for controlling the spread of COVID-19 and managing the threat posed by VOCs.
- Existing methods for identifying SARS-CoV-2 lineages with the N501Y mutation, such as Sanger or whole-genome sequencing, are time-consuming, labor-intensive, and costly.
Purpose of the Study:
- To develop and validate a rapid screening tool for the N501Y mutation in SARS-CoV-2.
- To create a single nucleotide polymorphism (SNP) real-time reverse transcription PCR (RT-PCR) assay for efficient surveillance of VOCs.
- To differentiate SARS-CoV-2 lineages possessing the N501Y mutation from those without it.
Main Methods:
- Development of a SNP real-time RT-PCR assay utilizing allelic discrimination for the N501Y mutation in the SARS-CoV-2 spike gene.
- Validation of the assay using 160 clinical specimens previously characterized by Sanger sequencing for the N501Y mutation status (mutant or wild type).
- Comparative analysis of the developed RT-PCR assay against Sanger sequencing for accuracy in SNP detection.
Main Results:
- The N501Y SNP real-time RT-PCR assay demonstrated 100% positive percent agreement for all 57 specimens identified as N501Y mutant (A23063T) by Sanger sequencing.
- The assay achieved 100% negative percent agreement for all 103 specimens identified as N501 wild type (A23063) by Sanger sequencing.
- The assay successfully identified one specimen with a mixed signal for wild type and mutant N501Y.
Conclusions:
- The developed SNP real-time RT-PCR assay is a highly accurate and efficient tool for screening SARS-CoV-2 specimens for the presence of the N501Y mutation.
- This assay facilitates high-volume screening of potential VOCs, enabling focused selection of specimens for confirmatory sequencing.
- The assay significantly aids in SARS-CoV-2 surveillance efforts by reducing turnaround times and optimizing resource allocation for public health response.
