Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay
ABSTRACT In this report, we describe the development of a reverse transcription-quantitative PCR (RT-qPCR) assay, termed Alpha-Delta assay, which can detect all severe acute respiratory syndrome coronavirus 2 (SC-2) variants and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants....
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2022-04-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02176-21 |
| _version_ | 1811303448453316608 |
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| author | Oran Erster Ella Mendelson Areej Kabat Virginia Levy Batya Mannasse Hadar Assraf Roberto Azar Yaniv Ali Efrat Bucris Dana Bar-Ilan Orna Mor Michal Elul Michal Mandelboim Danit Sofer Shay Fleishon Neta S. Zuckerman Itay Bar-Or |
| author_facet | Oran Erster Ella Mendelson Areej Kabat Virginia Levy Batya Mannasse Hadar Assraf Roberto Azar Yaniv Ali Efrat Bucris Dana Bar-Ilan Orna Mor Michal Elul Michal Mandelboim Danit Sofer Shay Fleishon Neta S. Zuckerman Itay Bar-Or |
| author_sort | Oran Erster |
| collection | DOAJ |
| description | ABSTRACT In this report, we describe the development of a reverse transcription-quantitative PCR (RT-qPCR) assay, termed Alpha-Delta assay, which can detect all severe acute respiratory syndrome coronavirus 2 (SC-2) variants and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants. The Alpha- and Delta-specific reactions in the assay target mutations that are strongly linked to the target variant. The Alpha reaction targets the D3L substitution in the N gene, and the Delta reaction targets the spike gene 156 to 158 mutations. Additionally, we describe a second Delta-specific assay that we use as a confirmatory test for the Alpha-Delta assay that targets the 119 to 120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15 to 25 copies per reaction, similar to the sensitivity of commercial SC-2 detection tests. The Alpha-Delta assay and the Orf8119del assay were successfully used to classify clinical samples that were subsequently analyzed by whole-genome sequencing. Lastly, the capability of the Alpha-Delta assay and Orf8119del assay to identify correctly the presence of Delta RNA in wastewater samples was demonstrated. This study provides a rapid, sensitive, and cost-effective tool for detecting and classifying two worldwide dominant SC-2 variants. It also highlights the importance of a timely diagnostic response to the emergence of new SC-2 variants with significant consequences on global health. IMPORTANCE The new assays described herein enable rapid, straightforward, and cost-effective detection of severe acute respiratory syndrome coronavirus 2 (SC-2) with immediate classification of the examined sample as Alpha, Delta, non-Alpha, or non-Delta variant. This is highly important for two main reasons: (i) it provides the scientific and medical community with a novel diagnostic tool to rapidly detect and classify any SC-2 sample of interest as Alpha, Delta, or none and can be applied to both clinical and environmental samples, and (ii) it demonstrates how to respond to the emergence of new variants of concern by developing a variant-specific assay. Such assays should improve our preparedness and adjust the diagnostic capacity to serve clinical, epidemiological, and research needs. |
| first_indexed | 2024-04-13T07:48:17Z |
| format | Article |
| id | doaj.art-9d8cad06e8e246c0ac9bcd3f622be03c |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-13T07:48:17Z |
| publishDate | 2022-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-9d8cad06e8e246c0ac9bcd3f622be03c2022-12-22T02:55:38ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-04-0110210.1128/spectrum.02176-21Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR AssayOran Erster0Ella Mendelson1Areej Kabat2Virginia Levy3Batya Mannasse4Hadar Assraf5Roberto Azar6Yaniv Ali7Efrat Bucris8Dana Bar-Ilan9Orna Mor10Michal Elul11Michal Mandelboim12Danit Sofer13Shay Fleishon14Neta S. Zuckerman15Itay Bar-Or16Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelCentral Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Ramat Gan, IsraelABSTRACT In this report, we describe the development of a reverse transcription-quantitative PCR (RT-qPCR) assay, termed Alpha-Delta assay, which can detect all severe acute respiratory syndrome coronavirus 2 (SC-2) variants and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants. The Alpha- and Delta-specific reactions in the assay target mutations that are strongly linked to the target variant. The Alpha reaction targets the D3L substitution in the N gene, and the Delta reaction targets the spike gene 156 to 158 mutations. Additionally, we describe a second Delta-specific assay that we use as a confirmatory test for the Alpha-Delta assay that targets the 119 to 120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15 to 25 copies per reaction, similar to the sensitivity of commercial SC-2 detection tests. The Alpha-Delta assay and the Orf8119del assay were successfully used to classify clinical samples that were subsequently analyzed by whole-genome sequencing. Lastly, the capability of the Alpha-Delta assay and Orf8119del assay to identify correctly the presence of Delta RNA in wastewater samples was demonstrated. This study provides a rapid, sensitive, and cost-effective tool for detecting and classifying two worldwide dominant SC-2 variants. It also highlights the importance of a timely diagnostic response to the emergence of new SC-2 variants with significant consequences on global health. IMPORTANCE The new assays described herein enable rapid, straightforward, and cost-effective detection of severe acute respiratory syndrome coronavirus 2 (SC-2) with immediate classification of the examined sample as Alpha, Delta, non-Alpha, or non-Delta variant. This is highly important for two main reasons: (i) it provides the scientific and medical community with a novel diagnostic tool to rapidly detect and classify any SC-2 sample of interest as Alpha, Delta, or none and can be applied to both clinical and environmental samples, and (ii) it demonstrates how to respond to the emergence of new variants of concern by developing a variant-specific assay. Such assays should improve our preparedness and adjust the diagnostic capacity to serve clinical, epidemiological, and research needs.https://journals.asm.org/doi/10.1128/spectrum.02176-21SARS-COV-2RT-qPCRAlpha (B.1.1.7)Delta (B.1.617.2)classificationdiagnostic assay |
| spellingShingle | Oran Erster Ella Mendelson Areej Kabat Virginia Levy Batya Mannasse Hadar Assraf Roberto Azar Yaniv Ali Efrat Bucris Dana Bar-Ilan Orna Mor Michal Elul Michal Mandelboim Danit Sofer Shay Fleishon Neta S. Zuckerman Itay Bar-Or Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay Microbiology Spectrum SARS-COV-2 RT-qPCR Alpha (B.1.1.7) Delta (B.1.617.2) classification diagnostic assay |
| title | Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay |
| title_full | Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay |
| title_fullStr | Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay |
| title_full_unstemmed | Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay |
| title_short | Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay |
| title_sort | specific detection of sars cov 2 variants b 1 1 7 alpha and b 1 617 2 delta using a one step quantitative pcr assay |
| topic | SARS-COV-2 RT-qPCR Alpha (B.1.1.7) Delta (B.1.617.2) classification diagnostic assay |
| url | https://journals.asm.org/doi/10.1128/spectrum.02176-21 |
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