Tags

Type your tag names separated by a space and hit enter

A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept.
mBio. 2021 05 18; 12(3)MBIO

Abstract

The ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2, the limits of detection were established as 25 pg of NP or 20 infectious units (IU) and 875 pg of SP or 625 IU. Testing reverse transcription-PCR (RT-PCR)-positive (n = 48, with cycle threshold [CT ] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with CT values of <25 and for a single RT-PCR-negative sample. Virus isolation from the RT-PCR-positive nasopharyngeal swabs showed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation and 77.1% (37/48) sensitivity and 99.0% (95/96) specificity in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity, and the assay is relatively simple to set up as an "in-house" test. Here, SARS-CoV-2 served as the model pathogen, but the assay principle is applicable to other viral infections, and the test format could easily be adapted to high-throughput testing.IMPORTANCE PCR is currently the gold standard for the diagnosis of many acute infections. While PCR and its variants are highly sensitive and specific, the time from sampling to results is measured in hours at best. Antigen tests directly detect parts of the infectious agent, which may enable faster diagnosis but often at lower sensitivity and specificity. Here, we describe a technique for rapid antigen detection and demonstrate the test format's potential using SARS-CoV-2 as the model pathogen. The 10-min test, performed in a buffer that readily inactivates SARS-CoV-2, from nasopharyngeal samples identified 97.4% (37/38) of the samples from which we could isolate the virus. This suggests that the test performs well in identifying patients potentially shedding the virus. Although SARS-CoV-2 served as the model pathogen to demonstrate proof of concept, the test principle itself would be applicable to a wide variety of infectious and perhaps also noninfectious diseases.

Authors+Show Affiliations

University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland.University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland. University of Helsinki, Faculty of Veterinary Medicine, Department of Veterinary Biosciences, Helsinki, Finland.University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland.University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland.University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland.HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki, Helsinki, Finland. Helsinki University Hospital, Helsinki, Finland.HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki, Helsinki, Finland. Helsinki University Hospital, Helsinki, Finland.HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki, Helsinki, Finland. Helsinki University Hospital, Helsinki, Finland.University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland.University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland. HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki, Helsinki, Finland. Helsinki University Hospital, Helsinki, Finland.University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland. University of Helsinki, Faculty of Veterinary Medicine, Department of Veterinary Biosciences, Helsinki, Finland. HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki, Helsinki, Finland. Helsinki University Hospital, Helsinki, Finland.University of Helsinki, Faculty of Medicine, Medicum, Department of Virology, Helsinki, Finland jussi.hepojoki@helsinki.fi. University of Zürich, Vetsuisse Faculty, Institute of Veterinary Pathology, Zürich, Switzerland.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

34006662

Citation

Rusanen, Juuso, et al. "A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept." MBio, vol. 12, no. 3, 2021.
Rusanen J, Kareinen L, Szirovicza L, et al. A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept. mBio. 2021;12(3).
Rusanen, J., Kareinen, L., Szirovicza, L., Uğurlu, H., Levanov, L., Jääskeläinen, A., Ahava, M., Kurkela, S., Saksela, K., Hedman, K., Vapalahti, O., & Hepojoki, J. (2021). A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept. MBio, 12(3). https://doi.org/10.1128/mBio.00902-21
Rusanen J, et al. A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept. mBio. 2021 05 18;12(3) PubMed PMID: 34006662.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept. AU - Rusanen,Juuso, AU - Kareinen,Lauri, AU - Szirovicza,Leonora, AU - Uğurlu,Hasan, AU - Levanov,Lev, AU - Jääskeläinen,Anu, AU - Ahava,Maarit, AU - Kurkela,Satu, AU - Saksela,Kalle, AU - Hedman,Klaus, AU - Vapalahti,Olli, AU - Hepojoki,Jussi, Y1 - 2021/05/18/ PY - 2021/5/19/entrez PY - 2021/5/20/pubmed PY - 2021/6/2/medline KW - COVID-19 KW - SARS-CoV-2 KW - TR-FRET KW - antigen test KW - mix and read KW - rapid diagnostic test JF - mBio JO - mBio VL - 12 IS - 3 N2 - The ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2, the limits of detection were established as 25 pg of NP or 20 infectious units (IU) and 875 pg of SP or 625 IU. Testing reverse transcription-PCR (RT-PCR)-positive (n = 48, with cycle threshold [CT ] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with CT values of <25 and for a single RT-PCR-negative sample. Virus isolation from the RT-PCR-positive nasopharyngeal swabs showed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation and 77.1% (37/48) sensitivity and 99.0% (95/96) specificity in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity, and the assay is relatively simple to set up as an "in-house" test. Here, SARS-CoV-2 served as the model pathogen, but the assay principle is applicable to other viral infections, and the test format could easily be adapted to high-throughput testing.IMPORTANCE PCR is currently the gold standard for the diagnosis of many acute infections. While PCR and its variants are highly sensitive and specific, the time from sampling to results is measured in hours at best. Antigen tests directly detect parts of the infectious agent, which may enable faster diagnosis but often at lower sensitivity and specificity. Here, we describe a technique for rapid antigen detection and demonstrate the test format's potential using SARS-CoV-2 as the model pathogen. The 10-min test, performed in a buffer that readily inactivates SARS-CoV-2, from nasopharyngeal samples identified 97.4% (37/38) of the samples from which we could isolate the virus. This suggests that the test performs well in identifying patients potentially shedding the virus. Although SARS-CoV-2 served as the model pathogen to demonstrate proof of concept, the test principle itself would be applicable to a wide variety of infectious and perhaps also noninfectious diseases. SN - 2150-7511 UR - https://www.unboundmedicine.com/medline/citation/34006662/A_Generic_Scalable_and_Rapid_Time_Resolved_Förster_Resonance_Energy_Transfer_Based_Assay_for_Antigen_Detection_SARS_CoV_2_as_a_Proof_of_Concept_ L2 - https://doi.org/10.1128/mBio.00902-21 DB - PRIME DP - Unbound Medicine ER -