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Improving the specificity of nucleic acid detection with endonuclease-actuated degradation.
Zou, Roger S; Gavrilov, Momcilo; Liu, Yang; Rasoloson, Dominique; Conte, Madison; Hardick, Justin; Shen, Leo; Chen, Siqi; Pekosz, Andrew; Seydoux, Geraldine; Manabe, Yukari C; Ha, Taekjip.
  • Zou RS; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Gavrilov M; Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Liu Y; Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Rasoloson D; Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
  • Conte M; Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Hardick J; Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Shen L; Howard Hughes Medical Institute, Baltimore, MD, USA.
  • Chen S; Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Pekosz A; Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Seydoux G; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Manabe YC; Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Ha T; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Commun Biol ; 5(1): 290, 2022 03 31.
Article in English | MEDLINE | ID: covidwho-1774000
ABSTRACT
Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleic Acids / Nucleic Acid Amplification Techniques / Molecular Diagnostic Techniques / COVID-19 Type of study: Diagnostic study Limits: Humans Language: English Journal: Commun Biol Year: 2022 Document Type: Article Affiliation country: S42003-022-03242-x

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleic Acids / Nucleic Acid Amplification Techniques / Molecular Diagnostic Techniques / COVID-19 Type of study: Diagnostic study Limits: Humans Language: English Journal: Commun Biol Year: 2022 Document Type: Article Affiliation country: S42003-022-03242-x