Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays
BackgroundGenomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/o...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-05-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2022.894613/full |
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| author | Gwynngelle A. Borillo Ron M. Kagan Elizabeth M. Marlowe |
| author_facet | Gwynngelle A. Borillo Ron M. Kagan Elizabeth M. Marlowe |
| author_sort | Gwynngelle A. Borillo |
| collection | DOAJ |
| description | BackgroundGenomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants.MethodsSARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology (PlexPrime® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant.ResultsA total of 398 SAR-CoV-2 RT-PCR positive and 39 negative specimens previously tested by WGS were re-tested by RT-PCR genotyping. PCR detection of spike gene mutations N501Y, E484K, and S982A correlated 100% with WGS for the 29 lineages represented, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results.ConclusionsThese data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management. |
| first_indexed | 2024-12-12T02:53:21Z |
| format | Article |
| id | doaj.art-624fe96a93c14701b1897a885889d56d |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-12-12T02:53:21Z |
| publishDate | 2022-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-624fe96a93c14701b1897a885889d56d2022-12-22T00:40:49ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882022-05-011210.3389/fcimb.2022.894613894613Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR AssaysGwynngelle A. BorilloRon M. KaganElizabeth M. MarloweBackgroundGenomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants.MethodsSARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology (PlexPrime® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant.ResultsA total of 398 SAR-CoV-2 RT-PCR positive and 39 negative specimens previously tested by WGS were re-tested by RT-PCR genotyping. PCR detection of spike gene mutations N501Y, E484K, and S982A correlated 100% with WGS for the 29 lineages represented, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results.ConclusionsThese data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management.https://www.frontiersin.org/articles/10.3389/fcimb.2022.894613/fullSARS-CoV-2COVID-19variantsgenotypingRT-PCR |
| spellingShingle | Gwynngelle A. Borillo Ron M. Kagan Elizabeth M. Marlowe Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays Frontiers in Cellular and Infection Microbiology SARS-CoV-2 COVID-19 variants genotyping RT-PCR |
| title | Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays |
| title_full | Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays |
| title_fullStr | Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays |
| title_full_unstemmed | Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays |
| title_short | Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays |
| title_sort | rapid and accurate identification of sars cov 2 variants using real time pcr assays |
| topic | SARS-CoV-2 COVID-19 variants genotyping RT-PCR |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2022.894613/full |
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