Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay
Throughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Heal...
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MDPI AG
2023-01-01
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| author | Andreas C. Chrysostomou Antonia Aristokleous Johana Hezka Rodosthenous Christina Christodoulou Georgia Stathi Leondios G. Kostrikis |
| author_facet | Andreas C. Chrysostomou Antonia Aristokleous Johana Hezka Rodosthenous Christina Christodoulou Georgia Stathi Leondios G. Kostrikis |
| author_sort | Andreas C. Chrysostomou |
| collection | DOAJ |
| description | Throughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Health Organization as variants of concern (VOC) because they have resulted in an increased number of cases, posing a strong risk to public health. Thus far, five VOCs have been designated, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), including their sublineages. Next-generation sequencing (NGS) can produce a significant amount of information facilitating the study of variants; however, NGS is time-consuming and costly and not efficient during outbreaks, when rapid identification of VOCs is urgently needed. In such periods, there is a need for fast and accurate methods, such as real-time reverse transcription PCR in combination with probes, which can be used for monitoring and screening of the population for these variants. Thus, we developed a molecular beacon-based real-time RT-PCR assay according to the principles of spectral genotyping. This assay employs five molecular beacons that target ORF1a:ΔS3675/G3676/F3677, S:ΔH69/V70, S:ΔE156/F157, S:ΔΝ211, S:ins214EPE, and S:ΔL242/A243/L244, deletions and an insertion found in SARS-CoV-2 VOCs. This assay targets deletions/insertions because they inherently provide higher discrimination capacity. Here, the design process of the molecular beacon-based real-time RT-PCR assay for detection and discrimination of SARS-CoV-2 is presented, and experimental testing using SARS-CoV-2 VOC samples from reference strains (cultured virus) and clinical patient samples (nasopharyngeal samples), which have been previously classified using NGS, were evaluated. Based on the results, it was shown that all molecular beacons can be used under the same real-time RT-PCR conditions, consequently improving the time and cost efficiency of the assay. Furthermore, this assay was able to confirm the genotype of each of the tested samples from various VOCs, thereby constituting an accurate and reliable method for VOC detection and discrimination. Overall, this assay is a valuable tool that can be used for screening and monitoring the population for VOCs or other emerging variants, contributing to limiting their spread and protecting public health. |
| first_indexed | 2024-03-11T08:33:21Z |
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| spelling | doaj.art-34345986ed8e43daa73c29ea9135f45f2023-11-16T21:39:25ZengMDPI AGLife2075-17292023-01-0113230410.3390/life13020304Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping AssayAndreas C. Chrysostomou0Antonia Aristokleous1Johana Hezka Rodosthenous2Christina Christodoulou3Georgia Stathi4Leondios G. Kostrikis5Department of Biological Sciences, University of Cyprus, Aglantzia, 2109 Nicosia, CyprusDepartment of Biological Sciences, University of Cyprus, Aglantzia, 2109 Nicosia, CyprusDepartment of Biological Sciences, University of Cyprus, Aglantzia, 2109 Nicosia, CyprusDepartment of Biological Sciences, University of Cyprus, Aglantzia, 2109 Nicosia, CyprusDepartment of Biological Sciences, University of Cyprus, Aglantzia, 2109 Nicosia, CyprusDepartment of Biological Sciences, University of Cyprus, Aglantzia, 2109 Nicosia, CyprusThroughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Health Organization as variants of concern (VOC) because they have resulted in an increased number of cases, posing a strong risk to public health. Thus far, five VOCs have been designated, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), including their sublineages. Next-generation sequencing (NGS) can produce a significant amount of information facilitating the study of variants; however, NGS is time-consuming and costly and not efficient during outbreaks, when rapid identification of VOCs is urgently needed. In such periods, there is a need for fast and accurate methods, such as real-time reverse transcription PCR in combination with probes, which can be used for monitoring and screening of the population for these variants. Thus, we developed a molecular beacon-based real-time RT-PCR assay according to the principles of spectral genotyping. This assay employs five molecular beacons that target ORF1a:ΔS3675/G3676/F3677, S:ΔH69/V70, S:ΔE156/F157, S:ΔΝ211, S:ins214EPE, and S:ΔL242/A243/L244, deletions and an insertion found in SARS-CoV-2 VOCs. This assay targets deletions/insertions because they inherently provide higher discrimination capacity. Here, the design process of the molecular beacon-based real-time RT-PCR assay for detection and discrimination of SARS-CoV-2 is presented, and experimental testing using SARS-CoV-2 VOC samples from reference strains (cultured virus) and clinical patient samples (nasopharyngeal samples), which have been previously classified using NGS, were evaluated. Based on the results, it was shown that all molecular beacons can be used under the same real-time RT-PCR conditions, consequently improving the time and cost efficiency of the assay. Furthermore, this assay was able to confirm the genotype of each of the tested samples from various VOCs, thereby constituting an accurate and reliable method for VOC detection and discrimination. Overall, this assay is a valuable tool that can be used for screening and monitoring the population for VOCs or other emerging variants, contributing to limiting their spread and protecting public health.https://www.mdpi.com/2075-1729/13/2/304COVID-19SARS-CoV-2variants of concernmolecular beacons |
| spellingShingle | Andreas C. Chrysostomou Antonia Aristokleous Johana Hezka Rodosthenous Christina Christodoulou Georgia Stathi Leondios G. Kostrikis Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay Life COVID-19 SARS-CoV-2 variants of concern molecular beacons |
| title | Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay |
| title_full | Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay |
| title_fullStr | Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay |
| title_full_unstemmed | Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay |
| title_short | Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay |
| title_sort | detection of circulating sars cov 2 variants of concern vocs using a multiallelic spectral genotyping assay |
| topic | COVID-19 SARS-CoV-2 variants of concern molecular beacons |
| url | https://www.mdpi.com/2075-1729/13/2/304 |
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