Compositions and methods for detecting and identifying nucleic acid sequences in biological samples
The invention is directed to compositions and methods for isolating, detecting, amplifying, and quantitating pathogen-specific nucleic acids in a biological sample. The invention also provides diagnostic kits containing specific amplification primers, and labeled detection probes that specifically bind to the amplification products obtained therefrom. Also disclosed are compositions and methods for the isolation and characterization of nucleic acids that are specific to one or more pathogens, including for example Influenza virus and Mycobacterium tuberculosis , from a wide variety of samples including those of biological, environmental, clinical and/or veterinary origin.
1. A method for detection of a microorganism in a biological sample comprising:
contacting the biological sample to the composition to form a mixture, said composition comprising as components;
a thermostable polymerase present in an amount from about 0.05 U to about 1 U;
a mix of deoxynucleotide tri phosphates comprising about equivalent amounts of dATP, dCTP, dGTP and dTTP, collectively present in the composition at a concentration of about 0.1 mM to about 1 mM;
a chelating agent selected from the group consisting of ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, potassium citrate, magnesium citrate, ferric ammonium citrate, lithium citrate, and any combination thereof, present in the composition at a concentration of about 0.01 mM to about 1 mM;
a PCR osmolarity agent selected from the group consisting of N,N,N-trimethylglycine (betaine), dimethyl sulfoxide (DMSO), foramide, glycerol, nonionic detergents, polyethylene glycol, tetramethylammonium chloride, and any combination thereof, present in the composition at a concentration of about 1 mM to about 1 M;
an albumin selected from the group consisting of bovine serum albumin, human serum albumin, goat serum albumin, mammalian albumin, and any combination thereof, present in the composition at a concentration of about 5 ng/ml to about 100 ng/ml;
at least two salts, the first being a potassium salt selected from the group consisting of potassium chloride and potassium glutamate and the second being a magnesium salt selected from the group consisting of magnesium chloride and magnesium sulfate, collectively present in the composition at a concentration of about 50 mM to about 1 M;
one or more dyes; and
a buffer selected from the group consisting of tris(hydroxymethyl) aminomethane (Tris), citrate, 2-(N-morpholino)ethanesulfonic acid (MES), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 1,3-bis(tris(hydroxymethyl) methylamino)propane (Bis-Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N,N-bis(2-hydroxyethyl) glycine (Bicine), N-[tris(hydroxymethyl)methyl]glycine (Tricine), N-2-acetamido-2-iminodiacetic acid (ADA), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), bicarbonate, phosphate, and any combination thereof, present in the composition at a concentration of about 1 mM to about 1 M and with a pH of about 6.5 to about 9.0;
wherein the components are combined with nuclease-free water;
performing multiple thermal cycling steps on the mixture with a pair of PCR primers to form an amplification product that is derived from the nucleic acid that is specific for the microorganism, wherein one primer of the pair of the PCR primers comprises the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, and the other primer of the pair of PCR primers comprises the nucleic acid sequence of SEQ ID NO 3 or SEQ ID NO: 6; and
detecting the presence or absence of the amplification product to determine the presence or absence of the microorganism in the biological sample.
2. The method of claim 1 , further comprising detecting an amplified sequence of a control nucleic acid and determining the quality or quantity of amplification which occurred from the multiple thermal cycling steps.
3. The method of claim 1 , wherein the biological sample comprises biological material obtained from an individual.
4. A method of providing for detection of a microorganism in a biological sample comprising:
providing a PCR-ready composition containing as components:
a thermostable polymerase;
a mix of deoxynucleotide tri phosphates comprising about equivalent amounts of dATP, dCTP, dGTP and dTTP;
one or more chelating agents;
one or more PCR osmolarity agents;
one or more albumin proteins;
one or more salts;
one or more dyes; and
one or more buffers present in the composition at a pH of about 6.5 to about 9.0,
wherein the components are combined with nuclease-free water; and
providing a pair of PCR primers wherein one primer of the pair of PCR primers comprises the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, and the other primer of the pair of PCR primers comprises the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6.
5. The method of claim 4 , wherein the pKa of the buffer is within about 1.0 unit of the pH at an ambient temperature.
6. The method of claim 4 , further comprising contacting the biological sample with the composition and performing a thermal cycling reaction on the mixture.
7. The method of claim 4 , wherein the one or more chelating agents comprise ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, potassium citrate, magnesium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof.
8. The method of claim 4 , wherein the one or more PCR osmolarity agents comprise N,N,N-trimethylglycine (betaine), dimethyl sulfoxide (DMSO), foramide, glycerol, nonionic detergents, deoxyinosine, glycerine, 7-deaza deoxyguanosine triphosphate, sodium hydroxide, polyethylene glycol, tetramethylammonium chloride, or any combination thereof.
9. The method of claim 4 , wherein the one or more albumins comprises bovine serum albumin, human serum albumin, goat serum albumin, mammalian albumin or any combination thereof.
10. The method of claim 4 , wherein the one or more salts comprise potassium chloride, potassium glutamate, magnesium chloride, magnesium sulfate, and any combination thereof.
11. The method of claim 4 , wherein the one or more buffers comprise tris(hydroxymethyl) aminomethane (Tris), citrate, 2-(N-morpholino)ethanesulfonic acid (MES), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 1,3-bis(tris(hydroxymethyl) methylamino)propane (Bis-Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N,N-bis(2-hydroxyethyl) glycine (Bicine), N-[tris(hydroxymethyl)methyl]glycine (Tricine), N-2-acetamido-2-iminodiacetic acid (ADA), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), bicarbonate, phosphate, or any combination thereof.
12. The method of claim 1 , further comprising a reverse transcriptase.
13. The method of claim 4 , further comprising a reverse transcriptase.
14. The method of claim 1 , wherein the thermostable polymerase is a Platinum Taq polymerase, Taq polymerase, a high fidelity polymerase, a Pfu polymerase, a hot start polymerase, or a next gen polymerase.
15. The method of claim 1 , wherein the one or more dyes are selected from the group consisting of fluorescein, 5-carboxy-X-rhodamine and ROX.
16. The method of claim 4 , wherein the one or more dyes are selected from the group consisting of fluorescein, 5-carboxy-X-rhodamine and ROX.
17. The method of claim 1 , wherein the pH of the composition is from about 6.5 to about 7.5.
18. The method of claim 1 , wherein the pKa of the buffer is within 0.5 of the pH of the buffer at ambient temperature.
19. The method of claim 1 , wherein the pKa of the buffer is within 0.2 of the pH of the buffer at ambient temperature.
20. The method of claim 1 , wherein the pair of PCR primers is present in the composition at a concentration of about 0.5 μM to about 50 μM.
21. The method of claim 1 , wherein the microorganism is bacteria, virus, fungus or a parasite.
22. The method of claim 21 , wherein the bacteria is mycobacteria.
23. The method of claim 21 , wherein the virus is influenza virus.
24. The method of claim 23 , wherein the influenza virus is H1N1, H2N2, H3N3, or H5N1.
25. The method of claim 1 , further comprising a control nucleic acid present in the concentration at a concentration of about 1 fg to about 1 ng, wherein the control nucleic acid provides a qualitative or quantitative measure of PCR amplification.
26. The method of claim 25 , wherein the control nucleic acid comprises the sequence of SEQ ID NO 8, the sequence of SEQ ID NO 12 or the sequence of SEQ ID NO 21.
27. The method of claim 1 , further comprising a detection probe that binds to a PCR amplified nucleic acid sequence that is specific to the microorganism.
28. The method of claim 1 , which comprises about 50 mM of TRIS; about 70 mM of potassium chloride; about 3 mM of magnesium sulfate; about 45 mM of betaine; about 0.03 μg/mL of bovine serum albumin; about 0.1 mM of EDTA; about 0.05 μM of dye; about 8 μM of the pair of PCR primers.
29. The method of claim 27 , wherein the detection probe is a Mycobacterium -specific sequence and comprises the sequence of SEQ ID NO 4 or SEQ ID NO 7.
30. The method of claim 5 , wherein the pH of the composition is from about 6.5 to about 7.5.
31. The method of claim 5 , wherein the pKa of the buffer is within 0.5 of the pH at ambient temperature.
32. The method of claim 5 , wherein the pKa of the buffer is within 0.2 of the pH at ambient temperature.
33. The method of claim 4 , wherein the microorganism is bacteria, virus, fungus or a parasite.
34. The method of claim 33 , wherein the bacteria is mycobacteria.
35. The method of claim 33 , wherein the virus is influenza virus.
36. The method of claim 33 , wherein the influenza virus is H1N1, H2N2, H3N3, or H5N1.
37. The method of claim 4 , further comprising a control nucleic acid present in the concentration at a concentration of about 1 fg to about 1 ng, wherein the control nucleic acid provides a qualitative or quantitative measure of PCR amplification.
38. The method of claim 37 , wherein the control nucleic acid comprises the sequence of SEQ ID NO 8, the sequence of SEQ ID NO 12 or the sequence of SEQ ID NO 21.
39. The method of claim 4 , further comprising a detection probe that binds to a PCR amplified nucleic acid sequence that is specific to the microorganism.
40. The method of claim 39 , wherein the detection probe is a Mycobacterium -specific sequence and comprises the sequence of SEQ ID NO 4 or SEQ ID NO 7.
41. The method of claim 4 , which comprises about 50 mM of TRIS; about 70 mM of potassium chloride; about 3 mM of magnesium sulfate; about 45 mM of betaine; about 0.03 μg/mL of bovine serum albumin; about 0.1 mM of EDTA; about 0.05 μM of dye; about 8 μM of the pair of PCR primers.
42. A method of providing for detection of a microorganism in a biological sample containing nucleic acids forming a PCR-ready composition comprising:
providing a composition containing:
a mix of deoxynucleotide tri phosphates comprising about equivalent amounts of dATP, dCTP, dGTP and dTTP, collectively present in the composition at a concentration of about 0.1 mM to about 10 mM;
a chelating agent present in the composition at a concentration of about 0.01 mM to about 1 mM;
an osmolarity agent present in the composition at a concentration of about 1 mM to about 1 M;
an albumin protein present in the composition at a concentration of about 5 ng/ml to about 250 ng/ml;
at least two salts together present in the composition at a concentration of about 50 mM to about 1 M;
a pair of PCR primers wherein one primer of the pair of PCR primers comprises the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, and the other primer of the pair of PCR primers comprises the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6;
a dye at a concentration of from about 0.01 μM to about 1 μM; and
a buffer present in the composition at a concentration of about 100 mM to about 1 M and with a pH of about 6.5 to about 9.0,
combining the composition with nuclease-free water to form the PCR-ready composition; and
combining the PCR-ready composition with nucleic acids of the biological sample.
43. The method of claim 42 , wherein the pH of the composition is from about 6.5 to about 7.5.
44. The method of claim 42 , wherein the pKa of the buffer is within 1.0 unit of the pH at ambient temperature.
45. The method of claim 44 , wherein the pKa of the buffer is within 0.5 units of the pH at ambient temperature.
46. The method of claim 42 , wherein the PCR-ready composition contains as the chelating agent EDTA, EGTA, HEDTA, DTPA, NTA and/or APCA, as the osmolarity agent betaine, as the two salts a potassium salt and a magnesium salt, as the dye ROX, and as the buffer HEPES, Tris, MOPS and/or PIPES.
47. The method of claim 46 , wherein the PCR-ready composition contains the chelating agent at a concentration of from about 0.1 mM to 1 mM, the osmolarity agent at a concentration of from about 10 mM to 50 mM, the potassium salt at a concentration of from about 50 mM to 100 mM, the magnesium salt at a concentration of from about 1 mM to 10 mM, the dye at a concentration of from about 0.1 μM to 1 μM, and the buffer at a concentration from about 50 mM to 100 mM.
48. The method of claim 42 , further comprising a thermostable polymerase.
49. The method of claim 48 , wherein the thermostable polymerase is present in an amount from about 0.05 U to about 1 U.
50. The method of claim 45 , wherein the pKa of the buffer is within about 0.2 units of the pH at a selected temperature.
51. The method of claim 4 , wherein the thermostable polymerase is present in the composition in an amount of from about 0.05 U to about 1 U.
52. The method of claim 4 , wherein the mix of deoxynucleotide tri phosphates are collectively present in the composition at a concentration of from about 0.1 mM to about 1 mM.
53. The method of claim 4 , wherein the one or more chelating agents are present in the composition at a concentration of from about 0.01 mM to about 1 mM.
54. The method of claim 4 , wherein the one or more PCR osmolarity agents are present in the composition at a concentration of from about 1 mM to about 1 M.
55. The method of claim 4 , wherein the one or more albumins are present in the composition at a concentration of from about 5 ng/ml to about 100 ng/ml.
56. The method of claim 4 , wherein the one or more salts are collectively present in the composition at a concentration of from about 50 mM to about 1 M.
57. The method of claim 4 , wherein the one or more dyes are present in the composition at a concentration of from about 0.01 μM to about 1 μM.
58. The method of claim 4 , wherein the one or more buffers are present in the composition at a concentration of from about 1 mM to about 1 M.