Detection of recombinase polymerase amplification (RPA) amplicons via FEN1 cleavage
Inventors
Gerber, Kyle • COCKRELL, Lisa • Nelson, William • Armantrout, Kyle
Assignees
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Publication Number
US-12668836-B2
Publication Date
2026-06-30
Expiration Date
Abstract
Systems and methods for detecting the presence of a target nucleic acid in a sample via a recombinase polymerase amplification (RPA) reaction followed by a FEN1 cleavage detection reaction are disclosed. One aspect of the present disclosure relates to systems involving a sample collection device for collecting a sample and performing an RPA reaction on the sample, followed by the detection of the amplified product via a two-step FEN1 cleavage detection reaction which generates a fluorescent signal indicative of the presence of amplified product.
Core Innovation
The disclosed method detects a target nucleic acid by combining recombinase polymerase amplification with flap endonuclease 1 cleavage detection. The resulting cleavage reaction produces a fluorescent signal indicative of the presence of the target nucleic acid. Certain embodiments perform amplification and detection within the same vessel, and utilize sample-collection devices configured to initiate amplification.
The method achieves target identification without reliance on conventional thermal cycling equipment. Documented examples demonstrate target detection for SARS-CoV-2 RdRP and N-gene sequences, including verification within contrived nasal samples and assays utilizing an internal control. Reported assay performance indicates that the N3 assay provides higher sensitivity than the RdRp assay and exhibits sensitivity comparable to polymerase chain reaction assays.
Claims Coverage
The claims define two independent claims covering two inventive features: target amplification followed by cleavage detection, and target amplification and cleavage detection performed within the same vessel.
RPA amplification followed by FEN1 cleavage detection
Independent claim 1 covers amplifying a target nucleic acid using recombinase polymerase amplification and detecting the resulting amplicons through FEN1 cleavage detection. The detection reagents include an initiator oligonucleotide, a FEN1 probe oligonucleotide, a FEN1 endonuclease, and a FEN1 reporter oligonucleotide, generating a fluorescent signal indicative of the target's presence.
Same-vessel amplification and FEN1 cleavage detection
Independent claim 16 covers combining the sample and reaction reagents in a vessel, amplifying the target nucleic acid using recombinase polymerase amplification, and detecting the resulting amplicons through FEN1 cleavage detection. Amplification and detection are performed within the same vessel to produce a fluorescent signal indicative of the target's presence.
The independent claims cover recombinase polymerase amplification followed by fluorescent FEN1 cleavage detection, with claim 16 specifically requiring amplification and detection in the same vessel.
Stated Advantages
Rapid nucleic-acid detection without reliance on conventional thermal cycling equipment.
The N3 assay provides higher sensitivity than the RdRp assay and exhibits sensitivity comparable to polymerase chain reaction assays.
Documented Applications
Detection of SARS-CoV-2 RdRP and N-gene targets, including assays with an internal control.
Detection of target nucleic acids in contrived nasal samples through target-dependent fluorescent signaling.
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