Methods for multiplexing recombinase polymerase amplification

1. A method comprising:

providing a liquid sample comprising a target nucleic acid;

combining the liquid sample with a recombinase polymerase amplification (RPA) reagent composition to provide a reaction mixture, wherein the RPA reagent composition comprises:

a recombinase;

a polymerase;

a first nucleic acid primer;

a second nucleic acid primer;

a third extension-blocked primer comprising an elongation blocking group at a 3′ end, an internal tetrahydrofuran (THF) residue, and an internal residue non-complementary to the target nucleic acid; and

a nuclease;

performing RPA on the reaction mixture to produce an amplicon indicative of the presence of the target nucleic acid; and

detecting the presence of the amplicon using a lateral flow strip.

2. The method of claim 1 , wherein the lateral flow strip is configured to form a visible signal at a specific position on the lateral flow strip indicating the presence of the target nucleic acid.

3. The method of claim 1 , wherein the lateral flow strip comprises a sample pad, a detection line, and a control line, wherein said detection line is positioned upstream of a control line.

4. The method of claim 3 , further comprising transferring all or part of the reaction mixture to the sample pad;

flowing the reaction mixture to the detection line; and

capturing an amplicon indicating the presence of the target nucleic acid at the detection line.

5. The method of claim 1 , wherein said first nucleic acid primer is labeled with a first detectable label selected from the group consisting of enzymes, enzyme substrates, coenzymes, enzyme inhibitors, fluorescent markers, chromophores, luminescent markers, radioisotopes, and a member of a binding pair; and/or said second nucleic acid primer is labeled with a second detectable label selected from the group consisting of enzymes, enzyme substrates, coenzymes, enzyme inhibitors, fluorescent markers, chromophores, luminescent markers, radioisotopes, and a member of a binding pair.

6. The method of claim 5 , wherein an amplicon comprises the first detectable label and/or the second detectable label(s).

7. The method of claim 6 , wherein said amplicon comprising the first detectable label and/or the second detectable label(s) produces a visible signal on the lateral flow strip.

8. The method of claim 5 , wherein said first nucleic acid primer and/or said second nucleic acid primer is/are labeled with a member of a binding pair that is a biotin, an avidin, a streptavidin, an antigen, an antibody, a ligand, a receptor, an analog or a mutant of a biotin, an analog or a mutant of an avidin, an analog or a mutant of a streptavidin, an analog or a mutant of an antigen, an analog or a mutant of an antibody, an analog or a mutant of a ligand, or an analog or a mutant of a receptor.

9. The method of claim 7 , wherein the visible signal is a fluorescent signal.

10. The method of claim 1 , wherein performing RPA comprises maintaining said reaction mixture at a temperature of at least 30° C., at least 35° C., or at least 40° C.

11. The method of claim 1 , wherein performing RPA comprises maintaining said reaction mixture at a temperature less than 45° C., less than 40° C., less than 35° C., or less than 30° C.

12. The method of claim 1 , wherein performing RPA comprises maintaining said reaction mixture at a temperature for a time period of approximately 5 minutes, approximately 15 minutes, approximately 30 minutes, or approximately 15-60 minutes.

13. The method of claim 5 wherein said first nucleic acid primer and said second nucleic acid primer are labeled with different detectable labels.

14. The method of claim 1 wherein said lateral flow strip comprises an antibody.

15. The method of claim 1 wherein said liquid sample comprises blood.

16. The method of claim 1 wherein said liquid sample comprises less than 100 copies of said target nucleic acid.