COMPOSITIONS AND METHODS FOR AMPLIFYING A NUCLEIC ACID SEQUENCE IN A SAMPLE
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.
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.