An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern
Abstract The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs,...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-48647-8 |
| _version_ | 1797388396878888960 |
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| author | Padmapriya P. Banada Raquel Green Deanna Streck Rohini Kurvathi Robert Reiss Sukalyani Banik Naranjargal Daivaa Ibsen Montalvan Robert Jones Salvatore A. E. Marras Soumitesh Chakravorty David Alland |
| author_facet | Padmapriya P. Banada Raquel Green Deanna Streck Rohini Kurvathi Robert Reiss Sukalyani Banik Naranjargal Daivaa Ibsen Montalvan Robert Jones Salvatore A. E. Marras Soumitesh Chakravorty David Alland |
| author_sort | Padmapriya P. Banada |
| collection | DOAJ |
| description | Abstract The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance. |
| first_indexed | 2024-03-08T22:40:13Z |
| format | Article |
| id | doaj.art-3c2196e8408b4b54b00852c01e9066c5 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-08T22:40:13Z |
| publishDate | 2023-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-3c2196e8408b4b54b00852c01e9066c52023-12-17T12:12:25ZengNature PortfolioScientific Reports2045-23222023-12-0113111110.1038/s41598-023-48647-8An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concernPadmapriya P. Banada0Raquel Green1Deanna Streck2Rohini Kurvathi3Robert Reiss4Sukalyani Banik5Naranjargal Daivaa6Ibsen Montalvan7Robert Jones8Salvatore A. E. Marras9Soumitesh Chakravorty10David Alland11Rutgers New Jersey Medical School, Public Health Research InstituteRutgers New Jersey Medical School, Public Health Research InstituteInstitute of Genomic Medicine, Rutgers New Jersey Medical SchoolInstitute of Genomic Medicine, Rutgers New Jersey Medical SchoolDivision of Infectious Diseases, Department of Medicine, Rutgers New Jersey Medical SchoolRutgers New Jersey Medical School, Public Health Research InstituteRutgers New Jersey Medical School, Public Health Research InstituteUniversity HospitalCraic Computing LLCRutgers New Jersey Medical School, Public Health Research InstituteRutgers New Jersey Medical School, Public Health Research InstituteRutgers New Jersey Medical School, Public Health Research InstituteAbstract The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.https://doi.org/10.1038/s41598-023-48647-8 |
| spellingShingle | Padmapriya P. Banada Raquel Green Deanna Streck Rohini Kurvathi Robert Reiss Sukalyani Banik Naranjargal Daivaa Ibsen Montalvan Robert Jones Salvatore A. E. Marras Soumitesh Chakravorty David Alland An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern Scientific Reports |
| title | An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern |
| title_full | An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern |
| title_fullStr | An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern |
| title_full_unstemmed | An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern |
| title_short | An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern |
| title_sort | expanded rt pcr melting temperature coding assay to rapidly identify all known sars cov 2 variants and sub variants of concern |
| url | https://doi.org/10.1038/s41598-023-48647-8 |
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