Methods and devices related to toehold-based strand displacement with loop-mediated isothermal amplification

Abstract

Disclosed are methods for isothermal nucleic acid amplification and detection.

What is claimed is: 
     
         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, and further wherein the isothermal reaction is conducted with six primers;   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;   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 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 1 , wherein the strand displacement reporter is one step toehold displacement (OSD) reporter. 
     
     
         4 . The method of  claim 1 , wherein detection of the target nucleic acid takes place in real time. 
     
     
         5 . The method of  claim 1 , wherein the signal is a fluorescent signal. 
     
     
         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, in step b), the strand displacement reporter is not a primer of the target nucleic acid. 
     
     
         8 . The method of  claim 1 , wherein in step b), the strand displacement reporter comprises one or more features that deters polymerase extension of 3′ end of the single-stranded nucleic acid portion of the strand displacement reporter. 
     
     
         9 . The method of  claim 8 , wherein the one or more features that deters polymerase extension of the molecule comprises a detection label, or a chemically modifiable moiety. 
     
     
         10 . The method of  claim 9 , wherein the detection label comprises fluorescent, colored or luminescent dyes. 
     
     
         11 . The method of  claim 10 , 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). 
     
     
         12 . The method of  claim 9 , wherein the detection label comprises a quencher or a fluorophore. 
     
     
         13 . The method of  claim 9 , wherein the chemically modifiable moiety comprises an amino group, carboxy group, maleimide group, oxo group, or thiol group. 
     
     
         14 . The method of  claim 1 , wherein the step of amplifying in step a) and interaction between the strand displacement reporter and the loop product in step c) happens simultaneously. 
     
     
         15 . The method of  claim 1 , wherein multiple target nucleic acids can be detected simultaneously. 
     
     
         16 . The method of  claim 1 , wherein the strand displacement reporter comprises one or more modified nucleic acids. 
     
     
         17 . The method of  claim 16 , wherein the modified nucleic acid is an inverted dT. 
     
     
         18 . 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. 
     
     
         19 . The method of  claim 1 , wherein the signal captured by the camera can be further analyzed using computational analysis. 
     
     
         20 . 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 and further wherein the isothermal reaction is conducted with at least 5 primers, and has accelerated amplification compared with an isothermal reaction where 4 or less primers are used;   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;   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 signal indicates the presence of the target nucleic acid.