Compositions and methods for detecting a nucleic acid sequence in a sample comprising a primer oligonucleotide with a 3′-terminal region comprising a 2′-modified nucleotide
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 test sample comprising 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 test 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 in a test sample 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.