Methods and devices related to toehold-based strand displacement with loop-mediated isothermal amplification
Disclosed are compositions and methods for isothermal nucleic acid amplification and detection.
1. A method of detecting a nucleic acid, the method comprising
a) amplifying a target nucleic acid using an isothermal amplification reaction, wherein the isothermal amplification reaction produces at least one loop product, wherein at least part of the single-stranded portion of the loop product represents the target nucleic acid;
b) exposing the loop product of step a) to a strand displacement reporter, wherein the strand displacement reporter comprises single-stranded and double-stranded nucleic acid, and further wherein a portion of the single-stranded nucleic acid of the strand displacement reporter is complementary to at least a portion of the single-stranded nucleic acid of the loop product representing the target nucleic acid; and further wherein the strand displacement reporter is not a primer of the target nucleic acid and comprises one or more features that deters polymerase extension of 3′ end of the single-stranded nucleic acid portion of the strand displacement reporter;
c) allowing the loop product and the strand displacement reporter to interact, wherein interaction between the strand displacement reporter and the target nucleic acid portion of the loop product produces a detectable signal, wherein the step of amplifying in step a) and interaction between the strand displacement reporter and the loop product happens simultaneously, and further wherein the signal indicates the presence of the target nucleic acid.
2. The method of claim 1 , wherein the isothermal amplification reaction is loop-mediated isothermal amplification (LAMP).
3. The method of claim 2 , wherein LAMP is conducted with 5 primers.
4. The method of claim 1 , wherein the strand displacement reporter is one step toehold displacement (OSD) reporter.
5. The method of claim 1 , wherein detection of the target nucleic acid takes place in real time.
6. The method of claim 1 , wherein the strand displacement reporter comprises a secondary structure, such as a hairpin or loop structure.
7. The method of claim 1 , wherein multiple target nucleic acids can be detected simultaneously.
8. The method of claim 1 , wherein the strand displacement reporter comprises one or more modified nucleic acids.
9. The method of claim 7 , wherein primers are used with the isothermal amplification reaction, and further wherein these primers bind a primer binding region of the target nucleic acid.
10. The method of claim 1 , wherein interaction between the strand displacement reporter and the target nucleic acid portion of the loop product produces a detectable signal, which signal can be captured using a camera.
11. The method of claim 10 , wherein the signal captured by the camera can be further analyzed using computational analysis.
12. The method of claim 1 , wherein the one or more features that deters polymerase extension of the molecule comprises a primary detection label, or a chemically modifiable moiety.
13. The method of claim 12 , wherein the primary detection label comprises fluorescent, colored or luminescent dyes.
14. The method of claim 12 , wherein the primary detection label comprises a quencher or a fluorophore.
15. The method of claim 13 , wherein the fluorescent dye comprises fluorescent lanthanide complexes, fluorescein, fluorescein isothiocyanate, carboxyfluorescein (FAM), dichlorotriazinylamine fluorescein, rhodamine, tetramethylrhodamine, umbelliferone, eosin, erythrosin, coumarin, methyl-coumarin, pyrene, Malacite green, stilbene, Lucifer Yellow, dansyl chloride, phycoerythin, green fluorescent protein (GFP).
16. The method of claim 12 , wherein the chemically modifiable moiety comprises an amino group, carboxy group, maleimide group, oxo group, or thiol group.
17. The method of claim 8 , wherein the modified nucleic acid is an inverted dT.