Detection of recombinase polymerase amplification (RPA) amplicons via FEN1 cleavage
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.
1 . A method for detecting presence of a target nucleic acid in a sample, comprising:
amplifying the target nucleic acid via a recombinase polymerase amplification (RPA) reaction at 42° C. or less that generates amplicons, wherein the RPA reaction comprises one or more RPA proteins, a forward primer, a reverse primer, and a strand displacing polymerase; and
detecting the generated amplicons via a FEN1 cleavage detection reaction,
wherein the FEN1 cleavage detection reaction uses FEN1 cleavage detection reagents that are added after the RPA reaction, and
wherein the FEN1 cleavage detection reagents comprise an initiator oligonucleotide, a FEN1 probe oligonucleotide, a FEN1 endonuclease, and a FEN1 reporter oligonucleotide, which are used in two successive steps:
a first step comprising deactivating the RPA proteins at about 95° C., binding of the initiator oligonucleotide and the FEN1 probe oligonucleotide to the target nucleic acid, and cleaving off a detection flap of the FEN1 probe oligonucleotide, and
a second step comprising binding of the detection flap from the first step to the FEN1 reporter oligonucleotide, and
wherein the binding of the detection flap to the FEN1 reporter oligonucleotide generates a fluorescent signal indicative of the presence of the target nucleic acid in the sample.
2 . The method of claim 1 , wherein the target nucleic acid is an RNA-dependent RNA polymerase (RdRP) gene.
3 . The method of claim 1 , wherein the target nucleic acid is associated with a disease.
4 . The method of claim 3 , wherein the disease is COVID-19.
5 . The method of claim 1 , wherein the target nucleic acid is a gene or fragment thereof from a virus.
6 . The method of claim 5 , wherein the virus is SARS-COV-2.
7 . The method of claim 1 , further comprising a denaturing step that includes deactivating the RPA proteins.
8 . The method of claim 1 , wherein the RPA proteins comprise at least one recombinase enzyme selected from the group consisting of uvsX recombinase, RecA, RadA, and Rad51.
9 . The method of claim 1 , wherein the RPA proteins comprise at least one DNA binding protein selected from the group consisting of gp32, a bacterial single-strand binding protein (SSB), and replication protein A.
10 . The method of claim 1 , wherein the RPA proteins comprise a recombinase loading protein selected from the group consisting of uvsY, RecOR, and Rad52.
11 . The method of claim 1 , wherein the forward primer comprises SEQ ID NO: 2 and the reverse primer comprises SEQ ID NO: 1, or the forward primer comprises SEQ ID NO: 6 and the reverse primer comprises SEQ ID NO: 7.
12 . The method of claim 1 , wherein the RPA reaction comprises a reverse transcriptase.
13 . The method of claim 1 , wherein the FEN1 initiator oligonucleotide comprises SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, or a combination thereof.
14 . The method of claim 1 , wherein the FEN1 probe oligonucleotide comprises SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 12, or a combination thereof.
15 . The method of claim 1 , wherein the FEN1 probe oligonucleotide comprises SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 14, or a combination thereof.
16 . A method for detecting the presence of a target nucleic acid in a sample, comprising:
a. mixing the sample, RPA reaction reagents, and FEN1 cleavage detection reaction reagents together in a vessel, the RPA reaction reagents comprising RPA proteins,
b. amplifying the target nucleic acid via the RPA reaction at 42° C. or less thereby generating amplicons, and
c. detecting the generated amplicons via a FEN1 cleavage detection reaction, wherein the FEN1 cleavage detection reaction comprises using an initiator oligonucleotide, a FEN1 probe oligonucleotide, a FEN1 endonuclease, and a FEN1 reporter oligonucleotide in two successive steps,
d. deactivating the RPA proteins at about 95° C., binding of the initiator oligonucleotide and the FEN1 probe oligonucleotide to the target nucleic acid, and cleaving off a detection flap of the FEN1 probe oligonucleotide, and
e. binding of the detection flap to the FEN1 reporter oligonucleotide, and
wherein the binding of the detection flap to the FEN1 reporter oligonucleotide generates a fluorescent signal indicative of the presence of the target nucleic acid in the sample, and
wherein the amplifying and detecting are performed within the same vessel.
17 . The method of claim 16 , wherein the RPA reaction reagents comprise a forward primer, a reverse primer, and a strand displacing polymerase, and the FEN1 cleavage detection reaction comprises a FEN1 enzyme, a FEN1 probe oligonucleotide, and a FEN1 reporter oligonucleotide.
18 . The method of claim 17 , wherein the FEN1 probe oligonucleotide is blocked by one of phosphorylation, a C3 spacer, or an inverted dT at the 3′ end.
19 . The method of claim 17 , wherein the FEN1 probe oligonucleotide includes one or more base pair mismatches.
20 . The method of claim 16 , wherein an RPA primer is used as an initiator oligonucleotide for the FEN1 cleavage detection reaction.
21 . The method of claim 16 , wherein the method further comprises a reverse transcription reaction.
22 . The method of claim 21 , wherein reagents for the reverse transcription reaction are added during step (a).
23 . The method of claim 21 , wherein the reverse transcription reaction is carried out within the same vessel as the RPA reaction and FEN1 cleavage detection reaction.
24 . The method of claim 16 , wherein the target nucleic acid is a gene or fragment thereof from a virus.
25 . The method of claim 24 , wherein the virus is SARS-COV-2.
26 . The method of claim 16 , wherein the target nucleic acid is associated with a disease.
27 . The method of claim 26 , wherein the disease is COVID-19.
28 . The method of claim 16 , wherein the FEN1 cleavage detection reaction occurs at a temperature between 58° C. and 70° C.