Title
Combining CRISPR–Cas12a with terminal deoxynucleotidyl transferase dependent reporter elongation for pathogen detection using lateral flow test strips
Author
Berghuis, N.F.
Mars-Groenendijk, R.H.
Busker, R.W.
Paauw, A.
van Leeuwen, H.C.
Publication year
2022
Abstract
CRISPR–Cas (CC)-based detection technologies have some exceptional features, which hold the promise of developing into the nextgeneration diagnostic platforms. One of these features is the ability to trigger non-specific single-stranded DNA/RNA cleavage activity after specific target recognition and Cas enzyme activation. This cleavage activity can be visualized either by single-stranded DNA/RNA fluorescence resonance energy transfer quenching reporters or via lateral flow strips, which separate and detect the cleaved reporters. In a previous study, we reported coupling CC-cleavage activity with the enzyme terminal deoxynucleotidyl transferase (TdT) that elongates cleaved ssDNA reporter fragments with dTTP nucleotides. These elongated poly(thymine) tails then act as scaffolds for the formation of copper nanoparticles which generate a bright fluorescent signal upon UV excitation. In the current study, we visualize the poly(thymine) tails on lateral flow strips, using different combinations of biotinylated or fluorescein-labeled nucleotides, various reporters, and capture oligos. One particular approach, using a fluorescein reporter, reached a target sensitivity of
Subject
CRISPR–CAS
DNA detection
Lateral flow assay
Point of care
Pathogen
FRET
To reference this document use:
http://resolver.tudelft.nl/uuid:e3a995e9-646b-43dd-99e2-9513fced14c1
DOI
https://doi.org/10.1093/biomethods/bpac015
TNO identifier
977290
Publisher
Oxford University Press
ISSN
2396-8923
Source
Biology Methods and Protocols, 7 (7), bpac015
Document type
article