Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations
ABSTRACT Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred...
| Main Authors: | , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2023-02-01
|
| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03591-22 |
| _version_ | 1811164403643449344 |
|---|---|
| author | Katja Spiess Vithiagaran Gunalan Ellinor Marving Sofie Holdflod Nielsen Michelle G. P. Jørgensen Anna S. Fomsgaard Line Nielsen Alonzo Alfaro-Núñez Søren M. Karst Shila Mortensen Morten Rasmussen Ria Lassaunière Maiken Worsøe Rosenstierne Charlotta Polacek Jannik Fonager Arieh S. Cohen Claus Nielsen Anders Fomsgaard |
| author_facet | Katja Spiess Vithiagaran Gunalan Ellinor Marving Sofie Holdflod Nielsen Michelle G. P. Jørgensen Anna S. Fomsgaard Line Nielsen Alonzo Alfaro-Núñez Søren M. Karst Shila Mortensen Morten Rasmussen Ria Lassaunière Maiken Worsøe Rosenstierne Charlotta Polacek Jannik Fonager Arieh S. Cohen Claus Nielsen Anders Fomsgaard |
| author_sort | Katja Spiess |
| collection | DOAJ |
| description | ABSTRACT Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark. |
| first_indexed | 2024-04-10T15:20:58Z |
| format | Article |
| id | doaj.art-dd8fc4256706407c99f17cdfe3a4d51f |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-10T15:20:58Z |
| publishDate | 2023-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-dd8fc4256706407c99f17cdfe3a4d51f2023-02-14T14:15:50ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-02-0111110.1128/spectrum.03591-22Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 MutationsKatja Spiess0Vithiagaran Gunalan1Ellinor Marving2Sofie Holdflod Nielsen3Michelle G. P. Jørgensen4Anna S. Fomsgaard5Line Nielsen6Alonzo Alfaro-Núñez7Søren M. Karst8Shila Mortensen9Morten Rasmussen10Ria Lassaunière11Maiken Worsøe Rosenstierne12Charlotta Polacek13Jannik Fonager14Arieh S. Cohen15Claus Nielsen16Anders Fomsgaard17Department of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkTest Center Denmark, Statens Serum Institut, Copenhagen, DenmarkTest Center Denmark, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkTest Center Denmark, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkQlife ApS Symbion, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkTest Center Denmark, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkDepartment of Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, DenmarkABSTRACT Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.https://journals.asm.org/doi/10.1128/spectrum.03591-22SARS-CoV-2signature mutationsvariant of concernvariant PCRlarge-scale screeningwhole-genome sequencing |
| spellingShingle | Katja Spiess Vithiagaran Gunalan Ellinor Marving Sofie Holdflod Nielsen Michelle G. P. Jørgensen Anna S. Fomsgaard Line Nielsen Alonzo Alfaro-Núñez Søren M. Karst Shila Mortensen Morten Rasmussen Ria Lassaunière Maiken Worsøe Rosenstierne Charlotta Polacek Jannik Fonager Arieh S. Cohen Claus Nielsen Anders Fomsgaard Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations Microbiology Spectrum SARS-CoV-2 signature mutations variant of concern variant PCR large-scale screening whole-genome sequencing |
| title | Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations |
| title_full | Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations |
| title_fullStr | Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations |
| title_full_unstemmed | Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations |
| title_short | Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations |
| title_sort | rapid and flexible rt qpcr surveillance platforms to detect sars cov 2 mutations |
| topic | SARS-CoV-2 signature mutations variant of concern variant PCR large-scale screening whole-genome sequencing |
| url | https://journals.asm.org/doi/10.1128/spectrum.03591-22 |
| work_keys_str_mv | AT katjaspiess rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT vithiagarangunalan rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT ellinormarving rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT sofieholdflodnielsen rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT michellegpjørgensen rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT annasfomsgaard rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT linenielsen rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT alonzoalfaronunez rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT sørenmkarst rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT shilamortensen rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT mortenrasmussen rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT rialassauniere rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT maikenworsøerosenstierne rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT charlottapolacek rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT jannikfonager rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT ariehscohen rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT clausnielsen rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations AT andersfomsgaard rapidandflexiblertqpcrsurveillanceplatformstodetectsarscov2mutations |