Abstract
AbstractNucleic 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.
Funder
U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health
National Science Foundation
Howard Hughes Medical Institute
JHU COVID-19 Research and Response Program Fund Sherrilyn and Ken Fisher Center for Environmental Infectious Diseases Discovery Program
Publisher
Springer Science and Business Media LLC
Subject
General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)
Cited by
3 articles.
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