Compositions and methods for amplifying a nucleic acid sequence in a sample

Abstract

The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time. These methods are compatible with target oligonucleotides amplified using a NEAR reaction.

What is claimed is: 
     
         1 . A method of detecting a specific product in an amplification reaction, the method comprising:
 (a) contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, and a nicking enzyme, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule;   (b) generating amplicons comprising at least a portion of the target nucleic acid molecule sequence; and   (c) contacting the amplicon with a detectable polynucleotide probe and detecting a signal specific for oligonucleotide probe hybridization to the amplicon, wherein the signal indicates detection of the target nucleic acid molecule in the sample or an amplicon thereof.   
     
     
         2 . The method of  claim 1 , wherein the test sample comprises a pathogen. 
     
     
         3 . The method of  claim 2 , wherein the pathogen is a virus, bacteria, yeast or fungus. 
     
     
         4 . The method of  claim 1 , wherein the test sample is a biological sample. 
     
     
         5 . The method of  claim 4 , wherein the biological sample is a biological fluid, cell, or tissue sample. 
     
     
         6 . The method of  claim 5 , wherein the biological fluid is urine, semen, vaginal secretion, or stool. 
     
     
         7 . The method of  claim 1 , wherein the detectable polynucleotide probe is incorporated into a lateral flow device. 
     
     
         8 . The method of  claim 1 , wherein the method is carried out at a point of care. 
     
     
         9 . The method of  claim 1 , wherein the detecting is carried out at the endpoint of the amplification reaction. 
     
     
         10 . A method of detecting a specific product in an amplification reaction, the method comprising:
 (a) contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, and a nicking enzyme, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule;   (b) generating detectable amplicons comprising at least a portion of said target nucleic acid molecule sequence and a detectable moiety; wherein detection of a signal indicates detection of the specific product.   
     
     
         11 . The method of  claim 10 , wherein the detectable moiety is a radioactive isotope, magnetic bead, metallic bead, colloidal particle, fluorescent dye, electron-dense reagent, enzyme, biotin, digoxigenin, or a hapten. 
     
     
         12 . The method of  claim 10 , wherein the detectable moiety is detected via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. 
     
     
         13 . The method of  claim 10 , wherein the test sample comprises a pathogen. 
     
     
         14 . The method of  claim 13 , wherein the pathogen is a virus, bacteria, yeast or fungus. 
     
     
         15 . The method of  claim 10 , wherein the test sample is a biological sample. 
     
     
         16 . The method of  claim 15 , wherein the biological sample is a biological fluid, cell, or tissue sample. 
     
     
         17 . The method of  claim 16 , wherein the biological fluid is urine, semen, vaginal secretion, or stool. 
     
     
         18 . The method of  claim 10 , wherein the detectable amplicon is detected in a lateral flow device. 
     
     
         19 . The method of  claim 10 , wherein the method is carried out at a point of care. 
     
     
         20 . The method of  claim 10 , wherein the detecting is carried out at the endpoint of the amplification reaction.