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Detection of SARS-CoV-2 RNA by multiplex RT-qPCR

Eriko Kudo, Benjamin Israelow, Chantal B.F. Vogels, Peiwen Lu, View ORCID ProfileAnne L. Wyllie, Maria Tokuyama, Arvind Venkataraman, Doug E Brackney, Isabel M. Ott, Mary E. Petrone, Rebecca Earnest, Sarah Lapidus, M. Catherine Muenker, Adam J. Moore, Arnau Casanovas-Massana, Yale IMPACT Research Team, Saad B. Omer, Charles S. Dela Cruz, Shelli F. Farhadian, Albert I. Ko, Nathan D. Grubaugh, View ORCID ProfileAkiko Iwasaki
doi: https://doi.org/10.1101/2020.06.16.155887
Eriko Kudo
1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520, USA
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Benjamin Israelow
1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520, USA
2Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, 06510, USA
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Chantal B.F. Vogels
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Peiwen Lu
1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520, USA
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Anne L. Wyllie
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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  • ORCID record for Anne L. Wyllie
Maria Tokuyama
1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520, USA
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Arvind Venkataraman
1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520, USA
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Doug E Brackney
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Isabel M. Ott
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Mary E. Petrone
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Rebecca Earnest
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Sarah Lapidus
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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M. Catherine Muenker
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Adam J. Moore
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Arnau Casanovas-Massana
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Saad B. Omer
2Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, 06510, USA
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
4Yale Institute of Global Health, New Haven, CT 06510, USA
5Yale School of Nursing, New Haven, CT 06510, USA
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Charles S. Dela Cruz
6Department of Internal Medicine, Section of Pulmonary, Critical Care, and Sleep Medicine, Yale School of Medicine, New Haven, CT, 06510, USA
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Shelli F. Farhadian
2Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, 06510, USA
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Albert I. Ko
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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Nathan D. Grubaugh
3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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  • For correspondence: nathan.grubaugh@yale.edu akiko.iwasaki@yale.edu
Akiko Iwasaki
1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, 06520, USA
7Howard Hughes Medical Institute, Chevy Chase, MD, 20815, USA
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  • ORCID record for Akiko Iwasaki
  • For correspondence: nathan.grubaugh@yale.edu akiko.iwasaki@yale.edu
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Abstract

The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. To simplify testing and reduce the volume of required reagents, we developed a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the CDC, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the singleplex assay adapted for research purposes. Low copies (>500 copies / reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current singleplex diagnostics by saving reagents, costs, time and labor.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵A Research was supported in part by the National Institute of Health grants, 2T32AI007517-16 (to BI), AI054359 and AI127429 (to AI), the Japan Society for the Promotion of Science (to EK), Women’s Health Research at Yale Pilot Project Program, Emergent Ventures at the Mercatus Center, George Mason University, Mathers Foundation and the Ludwig Family Foundation. A.I. is an Investigator of the Howard Hughes Medical Institute.

  • ↵B None of the authors have any conflicts of interest to declare.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 17, 2020.
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Detection of SARS-CoV-2 RNA by multiplex RT-qPCR
Eriko Kudo, Benjamin Israelow, Chantal B.F. Vogels, Peiwen Lu, Anne L. Wyllie, Maria Tokuyama, Arvind Venkataraman, Doug E Brackney, Isabel M. Ott, Mary E. Petrone, Rebecca Earnest, Sarah Lapidus, M. Catherine Muenker, Adam J. Moore, Arnau Casanovas-Massana, Yale IMPACT Research Team, Saad B. Omer, Charles S. Dela Cruz, Shelli F. Farhadian, Albert I. Ko, Nathan D. Grubaugh, Akiko Iwasaki
bioRxiv 2020.06.16.155887; doi: https://doi.org/10.1101/2020.06.16.155887
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Detection of SARS-CoV-2 RNA by multiplex RT-qPCR
Eriko Kudo, Benjamin Israelow, Chantal B.F. Vogels, Peiwen Lu, Anne L. Wyllie, Maria Tokuyama, Arvind Venkataraman, Doug E Brackney, Isabel M. Ott, Mary E. Petrone, Rebecca Earnest, Sarah Lapidus, M. Catherine Muenker, Adam J. Moore, Arnau Casanovas-Massana, Yale IMPACT Research Team, Saad B. Omer, Charles S. Dela Cruz, Shelli F. Farhadian, Albert I. Ko, Nathan D. Grubaugh, Akiko Iwasaki
bioRxiv 2020.06.16.155887; doi: https://doi.org/10.1101/2020.06.16.155887

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