Improving the specificity of nucleic acid detection with endonuclease-actuated degradation

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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“…The development of highly sensitive, rapid, and low-cost analytical methods for biological substances, such as nucleic acids, proteins, bacteria, and viruses, has been a focused research area for decades due to its critical role in advancing biomedicine and healthcare technologies . The detection and quantification of biomarkers and pathogens at low concentrations are essential for screening and early diagnosis of diseases, which can offer improved treatment outcomes of fatal diseases and prevention of the spread of infectious diseases.…”