Colorimetric detection of nucleic acid amplification
Colorimetry is used to detect amplification reaction products. A sample is contacted with a reaction mix under conditions such that an amplification reaction occurs and produces an amplification reaction product if the sample contains a target nucleic acid template molecule. The reaction mix includes an enzyme for catalyzing the amplification reaction, and at least one halochromic agent. If the target nucleic acid template molecule is present, the amplification reaction changes the starting pH of the reaction mix to cause a detectable colorimetric change of the halochromic agent, thereby indicating the presence of the target nucleic acid. If the target nucleic acid template molecule is not present, the amplification reaction does not generate an adequate number of protons to sufficiently change the starting pH of the reaction mix to cause a detectable colorimetric change of the halochromic agent, thereby indicating that the amplification reaction product has not been produced.
1. A composition of matter for colorimetric detection of an amplification reaction product in a sample, the composition of matter comprising a reaction mix comprising:
a buffer comprising one or more buffering agents, the buffer having a net buffering capacity equivalent to Tris buffer at a concentration between 1.5 mM-19 mM in a solution having a pH of 8.0;
an enzyme for catalyzing an amplification reaction; and
a colorimetric agent having a transition pH range between a starting pH of the reaction mix and an expected ending pH of the reaction mix, the expected ending pH of the reaction mix affected by the amplification reaction.
2. The composition of matter of claim 1 , wherein a colorimetric change of the colorimetric agent caused by the amplification reaction is capable of being quantified at a cell path length of 50 μm.
3. The composition of matter of claim 1 , wherein the reaction mix comprises at least two colorimetric agents.
4. The composition of matter of claim 1 , wherein the amplification reaction is an isothermal reaction.
5. The composition of matter of claim 1 , wherein the colorimetric agent is at a concentration between 50 μM-260 μM.
6. The composition of matter of claim 1 , further comprising an acid or base.
7. The composition of matter of claim 1 , further comprising at least one of: dNTPs, primers, and a monovalent cation.
8. The composition of matter of claim 1 , wherein the enzyme is a reverse transcriptase, a DNA polymerase, a RNA polymerase, an RNase, a helicase, a recombinase, a ligase, a restriction endonuclease, a TAQ polymerase, or a single-strand binding protein.
9. The composition of matter of claim 1 , wherein the colorimetric agent comprises one or more of phenol red, bromocresol purple, bromothymol blue, neutral red, naphtholphthalein, cresol red, cresolphthalein, and phenolphthalein.
10. The composition of matter of claim 1 , wherein the transition pH of the colorimetric agent is between pH 5-10.
11. A composition of matter for colorimetric detection of an amplification reaction product in a sample, the composition of matter comprising a reaction mix comprising:
a buffer comprising one or more buffering agents, the buffer having a net buffering capacity equivalent to Tris buffer at a concentration between 1.5 mM-19 mM in a solution having a pH of 8.0;
an enzyme for catalyzing an amplification reaction; and
a halochromic agent.
12. The composition of matter of claim 11 , wherein the halochromic agent is at a concentration between 50 μM-260 μM.
13. The composition of matter of claim 11 , further comprising at least one of: dNTPs, primers, and a monovalent cation.
14. The composition of matter of claim 11 , wherein the enzyme is a reverse transcriptase, a DNA polymerase, a RNA polymerase, an RNase, a helicase, a recombinase, a ligase, a restriction endonuclease, a TAQ polymerase, or a single-strand binding protein.
15. The composition of matter of claim 11 , wherein the halochromic agent has a transition pH range between a starting pH of the reaction mix and an expected ending pH of the reaction mix, the expected ending pH of the reaction mix affected by the amplification reaction.
16. The composition of matter of claim 15 , wherein the transition pH of the halochromic agent is between pH 5-10.
17. The composition of matter of claim 4 , wherein the isothermal reaction is a strand displacement amplification, a multiple displacement amplification, a recombinase polymerase amplification, a helicase dependent amplification, a rolling circle amplification, or a loop mediated isothermal amplification.
18. The composition of matter of claim 1 , further comprising an acid.
19. The composition of matter of claim 1 , further comprising a base.
20. The composition of matter of claim 4 , wherein the isothermal reaction is a loop mediated isothermal amplification.
21. The composition of matter of claim 20 , further comprising at least one of: dNTPs, primers, and a monovalent cation.
22. The composition of matter of claim 20 , wherein the transition pH of the colorimetric agent is between pH 5-10.
23. The composition of matter of claim 20 , wherein the colorimetric agent is at a concentration between 50 μM-260 μM.
24. The composition of matter of claim 20 , wherein the enzyme is a DNA polymerase.
25. The composition of matter of claim 20 , wherein the enzyme is a RNA polymerase.
26. The composition of matter of claim 11 , wherein the amplification reaction is an isothermal reaction.
27. The composition of matter of claim 26 , wherein the isothermal reaction is a loop mediated isothermal amplification.
28. The composition of matter of claim 26 , further comprising at least one of: dNTPs, primers, and a monovalent cation.
29. The composition of matter of claim 26 , wherein the transition pH of the halochromic agent is between pH 5-10.
30. The composition of matter of claim 26 , wherein the halochromic agent is at a concentration between 50 μM-260 μM.
31. The composition of matter of claim 26 , wherein the enzyme is a DNA polymerase.
32. The composition of matter of claim 26 , wherein the enzyme is a RNA polymerase.
33. The composition of matter of claim 26 , wherein the halochromic agent is a fluorescent agent.