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Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.
Sherrill-Mix, Scott; Hwang, Young; Roche, Aoife M; Glascock, Abigail; Weiss, Susan R; Li, Yize; Haddad, Leila; Deraska, Peter; Monahan, Caitlin; Kromer, Andrew; Graham-Wooten, Jevon; Taylor, Louis J; Abella, Benjamin S; Ganguly, Arupa; Collman, Ronald G; Van Duyne, Gregory D; Bushman, Frederic D.
  • Sherrill-Mix S; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Hwang Y; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Roche AM; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Glascock A; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Weiss SR; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Li Y; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Haddad L; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Deraska P; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Monahan C; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Kromer A; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Graham-Wooten J; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Taylor LJ; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Abella BS; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Ganguly A; Department of Emergency Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Collman RG; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Van Duyne GD; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
  • Bushman FD; Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. vanduyne@pennmedicine.upenn.edu.
Genome Biol ; 22(1): 169, 2021 06 03.
Article in English | MEDLINE | ID: covidwho-1388811
Preprint
This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
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ABSTRACT

BACKGROUND:

Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.

RESULTS:

Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.

CONCLUSIONS:

LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: RNA, Viral / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Genome Biol Journal subject: Molecular Biology / Genetics Year: 2021 Document Type: Article Affiliation country: S13059-021-02387-Y

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Full text: Available Collection: International databases Database: MEDLINE Main subject: RNA, Viral / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: English Journal: Genome Biol Journal subject: Molecular Biology / Genetics Year: 2021 Document Type: Article Affiliation country: S13059-021-02387-Y