Enzyme preparations and reaction mixtures for nucleic acid amplification

Abstract

Disclosed are formulations and reaction mixtures comprising at least one polymerase, alpha-cyclodextrin, and polysorbate 20. Related methods for preparing the disclosed formulations and reaction mixtures, as well as related kits and methods for nucleic acid amplification, are also disclosed. The formulations and reaction mixtures are useful, for example, for mitigating the inhibitory effects of sample extraction detergents on nucleic acid amplification reactions. The formulation can be a lyophilized polymerase formulation. The formulation can further comprise a cryoprotectant.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 217,560, filed July 1, 2021, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on June 8, 2022, is named "GPR_7910 PC_20220608_Seq_Listing_ST 25" and is 4,974 bytes in size. [Background technology]

[0003] background Nucleic acid amplification methods are widely used in many bioscience applications, including, for example, in vitro detection assays. Methods for amplifying target nucleic acids from biological samples typically employ sample extraction steps that leave one or more residual detergents in the extracted sample that are often inhibitory to polymerase enzymes. The inhibitory effects of such residual detergents present challenges for the formulation of polymerase enzymes and amplification reaction mixtures. Summary of the Invention

[0004] Abstract In one aspect, the present invention provides aqueous polymerase formulations. In some embodiments, the formulations generally comprise at least one polymerase, α-cyclodextrin at a concentration of about 10 mg / mL to about 40 mg / mL, polysorbate 20 at a concentration of about 0.002% to about 0.05% (v / v), and at least one buffering agent. In certain variations, the formulation further comprises a cryoprotectant. A particularly suitable cryoprotectant is trehalose, which may be present, for example, at a concentration of about 0.2 M to about 0.4 M.

[0005] In another aspect, the invention provides a method for preparing a lyophilized polymerase formulation. The method generally comprises: (a) providing an aqueous formulation as described above; and (b) lyophilizing the aqueous formulation to form the lyophilized polymerase formulation. In another aspect, the invention provides a lyophilized polymerase formulation prepared by the aforementioned method.

[0006] In another aspect, the present invention provides a lyophilized polymerase formulation that allows for reconstitution into the aqueous formulation described above.

[0007] In another aspect, the invention provides methods for preparing an aqueous polymerase formulation, which generally include (a) providing a lyophilized polymerase formulation as described above, and (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation.

[0008] In another aspect, the invention provides a kit having a first sealed container containing the lyophilized polymerase formulation described above. In some embodiments, the kit further comprises a second sealed container containing a diluent.

[0009] In another aspect, the present invention provides amplification reaction mixtures. The reaction mixtures generally comprise at least one polymerase, α-cyclodextrin at a concentration of about 5 mg / mL to about 20 mg / mL, polysorbate 20 at a concentration of about 0.001% to about 0.025% (v / v), and at least one buffer. In some variations, the reaction mixture further comprises nucleotide triphosphates suitable for nucleic acid amplification. In other, non-mutually exclusive embodiments, the reaction mixture comprises at least one amplification oligomer configured to amplify a target region of a target nucleic acid and / or at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region. In still other, non-mutually exclusive embodiments, the reaction mixture further comprises the target nucleic acid to be amplified and / or at least one detergent, such as an anionic detergent. Particularly suitable anionic detergents include sodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS).

[0010] In another aspect, the present invention provides methods for preparing an amplification reaction mixture. In some embodiments, the methods generally comprise: (a) providing an aqueous polymerase formulation described above; and (b) combining the aqueous formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid. In some such variations, the methods further comprise combining the mixture produced in step (b) with a sample containing or suspected of containing the target nucleic acid. In other embodiments, the methods generally comprise: (a) providing a lyophilized polymerase formulation described above; (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation; and (c) combining the reconstituted formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid. In some such variations, the methods further comprise combining the mixture produced in step (c) with a sample containing or suspected of containing the target nucleic acid. In some of the above embodiments involving further mixing with a sample, the sample is an extracted sample containing at least one detergent, e.g., an anionic detergent. Particularly suitable anionic detergents include sodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS). In other non-mutually exclusive embodiments described above that involve further mixture with a sample, the sample containing or suspected of containing the target nucleic acid comprises at least about 20% or at least about 40% of the reaction mixture.

[0011] In another aspect, the present invention provides a method for performing an amplification reaction, which generally includes: (a) providing a sample containing or suspected of containing a target nucleic acid; (b) contacting the sample with an aqueous mixture containing at least one polymerase, α-cyclodextrin, polysorbate 20, at least one buffer, nucleotide triphosphates suitable for nucleic acid amplification, at least one cofactor, and at least one amplification oligomer configured to amplify a target region of the target nucleic acid, wherein contacting the sample with the aqueous mixture provides a reaction mixture in which the α-cyclodextrin is present at a concentration of about 5 mg / mL to about 20 mg / mL and the polysorbate 20 is present at a concentration of about 0.001% to about 0.025% (v / v); and (c) performing an in vitro nucleic acid amplification reaction using the reaction mixture, wherein the target nucleic acid, if present in the sample, is used as a template to generate one or more amplicons corresponding to the target region. In some embodiments, the method further includes detecting one or more amplicons (e.g., detecting one or more amplicons in real time); in some such variations, the detecting step includes contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within one or more amplicons. In some embodiments, the sample is an extracted sample containing at least one detergent, such as an anionic detergent (e.g., sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS)). In others, the sample constitutes at least about 20% or at least about 40% of the reaction mixture produced as step (b). DETAILED DESCRIPTION OF THE INVENTION

[0012] Representative embodiments of these aspects are further described below.

[0013] Embodiment Embodiment 1. An aqueous polymerase formulation comprising: at least one polymerase; α-cyclodextrin at a concentration of about 10 mg / mL to about 40 mg / mL; Polysorbate 20 at a concentration of about 0.002% to about 0.05% (v / v), at least one buffer; and 1. An aqueous polymerase formulation comprising:

[0014] Embodiment 2. The formulation of embodiment 1, wherein the α-cyclodextrin concentration is from about 16 mg / mL to about 30 mg / mL, from about 15 mg / mL to about 20 mg / mL, or from about 10 mg / mL to about 15 mg / mL.

[0015] Embodiment 3. The formulation of embodiment 1, wherein the α-cyclodextrin concentration is about 20 mg / mL, about 17.5 mg / mL, or about 12.5 mg / mL.

[0016] Embodiment 4. The formulation of any one of Embodiments 1 to 3, wherein the concentration of polysorbate 20 is about 0.003% to about 0.03% (v / v).

[0017] Embodiment 5. The formulation of any one of embodiments 1-3, wherein the polysorbate 20 concentration is about 0.0042% (v / v), about 0.0035% (v / v), about 0.0026% (v / v), or about 0.02% (v / v).

[0018] Embodiment 6. The formulation of any one of embodiments 1 to 5, wherein the at least one polymerase is a DNA polymerase.

[0019] Embodiment 7. The formulation of embodiment 6, wherein the formulation comprises at least two DNA polymerases, wherein a first DNA polymerase is a reverse transcriptase and a second DNA polymerase is a DNA-dependent DNA polymerase.

[0020] Embodiment 8. The formulation of embodiment 7, wherein the reverse transcriptase is a Moloney murine leukemia virus (M-MLV) mutant reverse transcriptase.

[0021] Embodiment 9. The formulation of any one of embodiments 1 to 8, wherein the at least one buffering agent is Tris.

[0022] Embodiment 10. The formulation of embodiment 9, wherein the Tris buffer is present at a concentration of about 10 mM to about 100 mM.

[0023] Embodiment 11. The formulation of any one of embodiments 1 to 10, further comprising nucleotide triphosphates suitable for performing nucleic acid amplification.

[0024] Embodiment 12. A formulation according to any one of embodiments 1 to 11, further comprising EDTA.

[0025] Embodiment 13. The formulation of embodiment 12, wherein the EDTA is present at a concentration of about 0.05 mM to about 0.5 mM.

[0026] Embodiment 14. The formulation of embodiment 12, wherein the EDTA is present at a concentration of about 0.16 mM or about 0.14 mM.

[0027] Embodiment 15. The formulation of any one of embodiments 1 to 14, further comprising a cryoprotectant.

[0028] Embodiment 16. The formulation of embodiment 15, wherein the cryoprotectant is trehalose.

[0029] Embodiment 17. The formulation of embodiment 16, wherein the trehalose is present at a concentration of about 0.2M to about 0.35M or about 0.2M to about 0.4M.

[0030] Embodiment 18. The formulation of embodiment 17, wherein the trehalose is present at a concentration of about 0.26 M or about 0.3 M.

[0031] Embodiment 19. A method for preparing a lyophilized polymerase formulation, comprising: (a) providing an aqueous formulation according to any one of embodiments 15 to 18; (b) lyophilizing the aqueous formulation to form the lyophilized polymerase formulation; A method comprising:

[0032] Embodiment 20. A lyophilized polymerase formulation prepared by the method of embodiment 19.

[0033] Embodiment 21. A lyophilized polymerase formulation that allows for reconstitution into an aqueous formulation according to any one of embodiments 15 to 18.

[0034] Embodiment 22. A method for preparing an aqueous polymerase formulation, comprising: (a) providing a lyophilized polymerase formulation according to embodiment 20 or 21; (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation; A method comprising:

[0035] Embodiment 23. A kit comprising: 22. A kit comprising a first sealed container containing the lyophilized polymerase formulation of embodiment 20 or 21.

[0036] Embodiment 24. The kit of embodiment 23, further comprising a second sealed container containing a diluent.

[0037] Embodiment 25. An amplification reaction mixture comprising: at least one polymerase; α-cyclodextrin at a concentration of about 5 mg / mL to about 20 mg / mL; Polysorbate 20 at a concentration of about 0.001% to about 0.025% (v / v), at least one buffer; and an amplification reaction mixture comprising:

[0038] Embodiment 26. The reaction mixture of embodiment 25, wherein the α-cyclodextrin concentration is from about 8 mg / mL to about 15 mg / mL.

[0039] Embodiment 27. The reaction mixture of embodiment 25, wherein the α-cyclodextrin concentration is about 10 mg / mL.

[0040] Embodiment 28. The reaction mixture of any one of embodiments 25 to 27, wherein the polysorbate 20 concentration is from about 0.0015% to about 0.015% (v / v).

[0041] Embodiment 29. The reaction mixture of any one of embodiments 25 to 27, wherein the concentration of polysorbate 20 is about 0.002% (v / v), about 0.0021% (v / v), or about 0.01% (v / v).

[0042] Embodiment 30. The reaction mixture of any one of embodiments 25 to 29, wherein the at least one polymerase is a DNA polymerase.

[0043] Embodiment 31. The reaction mixture of embodiment 30, wherein the reaction mixture comprises at least two DNA polymerases, a first DNA polymerase being a reverse transcriptase and a second DNA polymerase being a DNA-dependent DNA polymerase.

[0044] Embodiment 32. The reaction mixture of embodiment 31, wherein the reverse transcriptase is a Moloney murine leukemia virus (M-MLV) mutant reverse transcriptase.

[0045] Embodiment 33. The reaction mixture of any one of embodiments 25 to 32, wherein the at least one buffer is Tris.

[0046] Embodiment 34. The reaction mixture of embodiment 33, wherein the Tris buffer is present at a concentration of about 5 mM to about 50 mM.

[0047] Embodiment 35. The reaction mixture of any one of embodiments 25 to 34, further comprising nucleotide triphosphates suitable for carrying out nucleic acid amplification.

[0048] Embodiment 36. The reaction mixture of any one of embodiments 25 to 35, further comprising EDTA.

[0049] Embodiment 37. The reaction mixture of embodiment 36, wherein the EDTA is present at a concentration of about 0.025 mM to about 0.25 mM.

[0050] Embodiment 38. The reaction mixture of embodiment 36, wherein the EDTA is present at a concentration of about 0.08 mM.

[0051] Embodiment 39. The reaction mixture of any one of embodiments 25 to 38, further comprising a cryoprotectant.

[0052] Embodiment 40. The reaction mixture of embodiment 39, wherein the cryoprotectant is trehalose.

[0053] Embodiment 41. The reaction mixture of embodiment 40, wherein the trehalose is present at a concentration of about 0.1 M to about 0.2 M.

[0054] Embodiment 42. The reaction mixture of embodiment 41, wherein the trehalose is present at a concentration of about 0.15M.

[0055] Embodiment 43. The reaction mixture of any one of embodiments 25 to 42, further comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid.

[0056] Embodiment 44. The reaction mixture of embodiment 43, comprising at least two amplification oligomers configured to amplify a target region of a target nucleic acid.

[0057] Embodiment 45. The reaction mixture of embodiment 43 or 44, further comprising at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within said target region.

[0058] Embodiment 46. The reaction mixture of embodiment 45, wherein the at least one detection probe oligomer comprises a chemiluminescent or fluorescent label.

[0059] Embodiment 47. The reaction mixture of embodiment 45, wherein the at least one detection probe oligomer comprises a fluorescent label and a non-fluorescent quencher.

[0060] Embodiment 48. The reaction mixture of any one of embodiments 25 to 47, further comprising at least one cofactor.

[0061] Embodiment 49. The reaction mixture of embodiment 48, wherein the at least one cofactor is magnesium chloride at a concentration of about 1 mM to about 5 mM.

[0062] Embodiment 50. The reaction mixture of any one of embodiments 43 to 49, further comprising the target nucleic acid.

[0063] Embodiment 51. The reaction mixture of embodiment 50, wherein the target nucleic acid is RNA.

[0064] Embodiment 52. The reaction mixture of embodiment 50 or 51, further comprising at least one detergent.

[0065] Embodiment 53. The reaction mixture of embodiment 52, wherein the at least one detergent is an anionic detergent.

[0066] Embodiment 54. The reaction mixture of embodiment 53, wherein the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS).

[0067] Embodiment 55. A method for preparing an amplification reaction mixture, comprising: (a) providing an aqueous polymerase formulation according to any one of embodiments 1 to 18; (b) mixing the aqueous formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid; A method comprising:

[0068] Embodiment 56. The method of embodiment 55, wherein the second formulation comprises at least two amplification oligomers configured to amplify a target region of the target nucleic acid.

[0069] Embodiment 57. The method of embodiment 55 or 56, further comprising mixing the aqueous formulation with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region.

[0070] Embodiment 58. The method of embodiment 57, wherein the at least one detection probe oligomer comprises a chemiluminescent or fluorescent label.

[0071] Embodiment 59. The method of embodiment 57, wherein the at least one detection probe oligomer comprises a fluorescent label and a non-fluorescent quencher.

[0072] Embodiment 60. The method of any one of embodiments 55 to 59, further comprising mixing the aqueous formulation with at least one cofactor.

[0073] Embodiment 61. The method of embodiment 60, wherein the at least one cofactor is magnesium chloride.

[0074] Embodiment 62. The method of embodiment 61, wherein the magnesium chloride is mixed to a final concentration of about 1 mM to about 5 mM.

[0075] Embodiment 63. The method of any one of embodiments 55 to 62, further comprising the step of mixing the mixture produced in step (b) with a sample containing or suspected of containing the target nucleic acid.

[0076] Embodiment 64. A method for preparing an amplification reaction mixture, comprising: (a) providing a lyophilized polymerase formulation according to embodiment 20 or 21; (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation; (c) combining the reconstituted formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid; A method comprising:

[0077] Embodiment 65. The method of embodiment 64, wherein the second formulation comprises at least two amplification oligomers configured to amplify a target region of the target nucleic acid.

[0078] Embodiment 66 The method of embodiment 64 or 65, further comprising mixing the reconstituted formulation with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region.

[0079] Embodiment 67. The method of embodiment 66, wherein the at least one detection probe oligomer comprises a chemiluminescent or fluorescent label.

[0080] Embodiment 68. The method of embodiment 66, wherein the at least one detection probe oligomer comprises a fluorescent label and a non-fluorescent quencher.

[0081] Embodiment 69. The method of any one of embodiments 64 to 68, further comprising mixing the reconstituted formulation with at least one cofactor.

[0082] Embodiment 70. The method of embodiment 69, wherein the at least one cofactor is magnesium chloride.

[0083] Embodiment 71. The method of embodiment 70, wherein the magnesium chloride is mixed to a final concentration of about 1 mM to about 5 mM.

[0084] Embodiment 72. The method of any one of embodiments 64 to 71, further comprising the step of mixing the mixture produced in step (c) with a sample containing or suspected of containing the target nucleic acid.

[0085] Embodiment 73. The method of any one of embodiments 63 to 72, wherein the sample is an extracted sample containing at least one detergent.

[0086] Embodiment 74. The method of embodiment 73, wherein the at least one detergent is an anionic detergent.

[0087] Embodiment 75. The method of embodiment 74, wherein the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS).

[0088] Embodiment 76. The method of any one of embodiments 63 and 72 to 75, wherein the sample containing or suspected of containing the target nucleic acid constitutes at least about 20% of the reaction mixture.

[0089] Embodiment 77. The method of embodiment 76, wherein the sample containing or suspected of containing the target nucleic acid comprises at least about 40% of the reaction mixture.

[0090] Embodiment 78. The method of embodiment 77, wherein the sample containing or suspected of containing the target nucleic acid comprises about 40% to about 50% of the reaction mixture.

[0091] Embodiment 79. The method of embodiment 78, wherein the sample containing or suspected of containing the target nucleic acid comprises about 42.9% of the reaction mixture.

[0092] Embodiment 80. The method of embodiment 77, wherein the sample containing or suspected of containing the target nucleic acid comprises about 50% to about 70% of the reaction mixture.

[0093] Embodiment 81. The method of embodiment 80, wherein the sample containing or suspected of containing the target nucleic acid comprises about 62.5% of the reaction mixture.

[0094] Embodiment 82. The method of embodiment 76, wherein the sample containing or suspected of containing the target nucleic acid comprises about 20% to about 33% of the reaction mixture.

[0095] Embodiment 83. A method for performing an amplification reaction, comprising: (a) providing a sample containing or suspected of containing a target nucleic acid; (b) subjecting the sample to at least one polymerase; α-cyclodextrin, Polysorbate 20 and at least one buffer; and nucleotide triphosphates suitable for carrying out nucleic acid amplification; at least one cofactor; at least one amplification oligomer configured to amplify a target region of the target nucleic acid; contacting the sample with the aqueous mixture so that the α-cyclodextrin is present at a concentration of about 5 mg / mL to about 20 mg / mL and the polysorbate 20 is present at a concentration of about 0.001% to about 0.025% (v / v); (c) performing an in vitro nucleic acid amplification reaction using the reaction mixture, wherein the target nucleic acid, if present in the sample, is used as a template to generate one or more amplicons corresponding to the target region; A method comprising:

[0096] Embodiment 84. The method of embodiment 83, wherein the α-cyclodextrin is present in the reaction mixture at a concentration of from about 8 mg / mL to about 15 mg / mL.

[0097] Embodiment 85. The method of embodiment 83, wherein the α-cyclodextrin is present in the reaction mixture at a concentration of about 10 mg / mL.

[0098] Embodiment 86. The method of any one of embodiments 83 to 85, wherein the polysorbate 20 is present in the reaction mixture at a concentration of about 0.0015% to about 0.015% (v / v).

[0099] Embodiment 87. The method of any one of embodiments 83 to 85, wherein the polysorbate 20 is present in the reaction mixture at a concentration of about 0.002% (v / v), about 0.0021% (v / v), or about 0.01% (v / v).

[0100] Embodiment 88. The method of any one of embodiments 83 to 87, wherein the aqueous mixture comprises at least two amplification oligomers configured to amplify a target region of the target nucleic acid.

[0101] Embodiment 89. The method of any one of embodiments 83 to 88, wherein the at least one polymerase is a DNA polymerase.

[0102] Embodiment 90. The method of embodiment 89, wherein the aqueous mixture comprises at least two DNA polymerases, a first DNA polymerase being a reverse transcriptase and a second DNA polymerase being a DNA-dependent DNA polymerase.

[0103] Embodiment 91. The method of embodiment 90, wherein the reverse transcriptase is a Moloney murine leukemia virus (M-MLV) mutant reverse transcriptase.

[0104] Embodiment 92. The method of any one of embodiments 83 to 91, wherein the at least one buffer is Tris.

[0105] Embodiment 93. The method of embodiment 92, wherein the Tris buffer is present in the reaction mixture at a concentration of about 5 mM to about 50 mM.

[0106] Embodiment 94. The method of any one of embodiments 83 to 93, wherein the aqueous mixture further comprises EDTA.

[0107] Embodiment 95. The method of embodiment 94, wherein the EDTA is present in the reaction mixture at a concentration of about 0.025 mM to about 0.25 mM.

[0108] Embodiment 96. The method of embodiment 94, wherein the EDTA is present in the reaction mixture at a concentration of about 0.08 mM.

[0109] Embodiment 97. The method of any one of embodiments 83 to 96, wherein the aqueous mixture further comprises a cryoprotectant.

[0110] Embodiment 98. The method of embodiment 97, wherein the cryoprotectant is trehalose.

[0111] Embodiment 99. The method of embodiment 98, wherein the trehalose is present in the reaction mixture at a concentration of about 0.1 M to about 0.2 M.

[0112] Embodiment 100. The method of embodiment 99, wherein the trehalose is present in the reaction mixture at a concentration of about 0.15 M.

[0113] Embodiment 101. The method of any one of embodiments 83 to 100, further comprising detecting said one or more amplicons.

[0114] Embodiment 102. The method of embodiment 101, wherein said detecting step comprises contacting said in vitro nucleic acid amplification reaction with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within said one or more amplicons.

[0115] Embodiment 103. The method of embodiment 102, wherein the at least one detection probe oligomer comprises a chemiluminescent or fluorescent label.

[0116] Embodiment 104. The method of embodiment 102, wherein the at least one detection probe oligomer comprises a fluorescent label and a non-fluorescent quencher.

[0117] Embodiment 105. The method of any one of embodiments 101 to 104, wherein the detecting step is performed in real time.

[0118] Embodiment 106. The method of any one of embodiments 83 to 105, wherein the sample is an extracted sample containing at least one detergent.

[0119] Embodiment 107. The method of embodiment 106, wherein the at least one detergent is an anionic detergent.

[0120] Embodiment 108. The method of embodiment 107, wherein the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS).

[0121] Embodiment 109. The method of any one of embodiments 83 to 108, wherein the sample containing or suspected of containing the target nucleic acid constitutes at least about 20% of the reaction mixture.

[0122] Embodiment 110. The method of embodiment 109, wherein the sample containing or suspected of containing the target nucleic acid constitutes at least about 40% of the reaction mixture.

[0123] Embodiment 111. The method of embodiment 110, wherein the sample containing or suspected of containing the target nucleic acid comprises about 40% to about 50% of the reaction mixture.

[0124] Embodiment 112. The method of embodiment 111, wherein the sample containing or suspected of containing the target nucleic acid comprises about 42.9% of the reaction mixture.

[0125] Embodiment 113. The method of embodiment 110, wherein the sample containing or suspected of containing the target nucleic acid comprises about 50% to about 70% of the reaction mixture.

[0126] Embodiment 114. The method of embodiment 113, wherein the sample containing or suspected of containing the target nucleic acid comprises about 62.5% of the reaction mixture.

[0127] Embodiment 115. The method of embodiment 109, wherein the sample containing or suspected of containing the target nucleic acid comprises about 20% to about 33% of the reaction mixture.

[0128] Embodiment 116. The α-cyclodextrin concentration is about 17.5 mg / mL; The formulation of embodiment 1, wherein the polysorbate 20 concentration is about 0.0035% (v / v).

[0129] Embodiment 117. The α-cyclodextrin concentration is about 10 mg / mL; 26. The reaction mixture of embodiment 25, wherein the polysorbate 20 concentration is about 0.002% (v / v).

[0130] Embodiment 118. The α-cyclodextrin concentration is about 10 mg / mL; 84. The method of embodiment 83, wherein the polysorbate 20 concentration is about 0.002% (v / v).

[0131] These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention.

[0132] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the methods and compositions being described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0133] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0134] A "sample" includes any specimen that may contain a target nucleic acid. Samples include "biological samples," including any tissue or material derived from a living or dead human. Biological samples may be treated to physically or mechanically disrupt tissue or cellular structures, thus releasing intracellular components into a solution that may further contain enzymes, buffers, salts, detergents, etc., used to prepare the biological sample for analysis. Samples may also include treated samples, such as samples in which one or more components have been enriched or purified. Treated samples include those obtained, for example, by passing the sample over or through a filtration device, or after centrifugation, or by adherence to a medium, matrix, or support.

[0135] "Detergent" refers to a substance that can disperse hydrophobic substances (e.g., lipids) in water by emulsification and can be used to dissolve or solubilize biological samples for subsequent analysis. Detergents can be ionic or non-ionic.

[0136] As used herein, the term "cryoprotectant" refers to a molecule that prevents or reduces chemical and / or physical instability of proteins or other substances during lyophilization and subsequent storage. Exemplary cryoprotectants include sugars such as sucrose or trehalose; amino acids such as sodium glutamate and histidine; methylamines such as betaine; lyotropic salts such as magnesium sulfate; polyols such as trihydric or higher sugar alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol, propylene glycol, polyethylene glycol, etc.; Pluronics; and combinations thereof. In some embodiments, the cryoprotectant is a non-reducing sugar such as trehalose or sucrose. When used in the context of an aqueous formulation or reaction mixture containing α-cyclodextrin and polysorbate 20 described herein, the term "cryoprotectant" refers to a molecule other than α-cyclodextrin, although α-cyclodextrin may still act as a secondary cryoprotectant.

[0137] As used herein, the term "polymerase" refers to any enzyme capable of catalyzing the polymerization of nucleotides (including analogs thereof) into nucleic acid chains. Typically, such nucleotide polymerization occurs in a template-dependent manner. Such polymerases can include, for example, naturally occurring polymerases and any subunits and truncated forms thereof, mutant polymerases, variant polymerases, recombinant, fusion, or other engineered polymerases, chemically modified polymerases, synthetic molecules, or assemblies, as well as any analogs, homologs, derivatives, or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase can be a mutant polymerase containing one or more mutations, including the substitution of one or more amino acids with other amino acids, the insertion or deletion of one or more amino acids from the polymerase, or the linkage of two or more polymerase portions, including the linkage of two or more portions from polymerases of different species or families. Exemplary polymerases include DNA polymerases, such as Taq polymerase or reverse transcriptase. In other variations, the polymerase is an RNA polymerase.

[0138] The term "cofactor" refers to a divalent cation or its salt required for polymerase enzymatic activity. Suitable cofactors for use with polymerases are generally known in the art and include, for example, magnesium (Mg 2+ ) and manganese (Mn 2+ Typical divalent cation salts for use in polymerase reaction mixtures include chloride salts (e.g., magnesium chloride, manganese chloride).

[0139] As used herein, a "nucleotide" is a subunit of a nucleic acid consisting of a phosphate group, a five-carbon sugar, and a nitrogenous base (also referred to herein as a "nucleobase"). The five-carbon sugar found in RNA is ribose. In DNA, the five-carbon sugar is 2'-deoxyribose.

[0140] "Nucleic acid" and "polynucleotide" refer to polymeric compounds containing nucleotides and / or nucleotide analogs linked together to form biopolymers. Biopolymers include traditional RNA, traditional DNA, mixed RNA-DNA, and nucleotide analog-containing versions thereof. The nucleic acid "backbone" can be composed of various linkages, including one or more sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acids" or PNA), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, such as analogs with a methoxy, fluoro, or halide group at the 2' position of the ribose (also referred to herein as "2'-O-Me" or "2'-methoxy" or 2'-fluoro or "2'-halide"). The nitrogenous bases can be the conventional bases adenine (A), uracil (U), guanine (G), thymine (T), and cytosine (C), as well as their analogs (e.g., inosine, 5-methyl-2'-deoxyxocytosine ("5-Me-dC" or "5MeC"), and isoguanine). Nucleic acids can contain one or more "abasic" residues in which the backbone does not contain a nitrogenous base for a position or positions in the polymer.

[0141] "Oligomer," "oligonucleotide," or "oligo" generally refers to a nucleic acid less than 1,000 nucleotides (nt), including those with a size range having a lower limit of about 5 nt and an upper limit of about 900 nt. The term oligonucleotide does not indicate any specific function for the reagent, but is used generically to encompass all such reagents described herein. Although oligomers can be referred to by functional names (e.g., capture probe, detection probe, primer, or promoter primer), those skilled in the art will understand that these terms refer to oligomers.

[0142] As used herein, a "target nucleic acid" is a nucleic acid containing a target sequence to be amplified. The target nucleic acid may be DNA or RNA, and may be either single-stranded or double-stranded. The target nucleic acid may contain other sequences other than the target sequence that may not be amplified.

[0143] As used herein, the term "target sequence" or "target nucleic acid sequence" refers to a specific nucleotide sequence of a target nucleic acid to be amplified and / or detected. A "target sequence" includes a complexing sequence to which an oligonucleotide (e.g., a priming oligonucleotide and / or a promoter oligonucleotide) complexes during an amplification process (e.g., PCR, TMA). Unless the context clearly indicates otherwise, if the target nucleic acid is originally single-stranded, the term "target sequence" also refers to a sequence complementary to the "target sequence" present in the target nucleic acid; if the target nucleic acid is originally double-stranded, the term "target sequence" refers to both the sense (+) strand and the antisense (-) strand.

[0144] "Nucleic acid amplification" refers to any well-known in vitro procedure for generating multiple copies of a target nucleic acid sequence. Examples of such procedures include transcription-related methods such as transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), etc. (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase-mediated amplification (e.g., U.S. Pat. No. 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), ligase chain reaction (LCR) (e.g., European Patent No. 0320308), and strand displacement amplification (SDA) (e.g., U.S. Pat. No. 5,422,252).

[0145] "Amplicon" or "amplification product" means a nucleic acid molecule produced in a nucleic acid amplification reaction and derived from a target nucleic acid. The amplicon or amplification product contains a target nucleic acid sequence that can be of the same or opposite sense as the target nucleic acid.

[0146] An "amplification oligonucleotide" or "amplification oligomer" is an oligonucleotide that hybridizes to a target nucleic acid and participates in a nucleic acid amplification reaction, e.g., serves as a primer. An amplification oligomer can have a 3' end that is extended by polymerization as part of a nucleic acid amplification reaction. Alternatively, an amplification oligomer can have a 3' end that is not extended by polymerization but provides a moiety that facilitates nucleic acid amplification, such as a promoter sequence attached 5' to the target-specific sequence of the amplification oligomer. Such an amplification oligomer is referred to as a promoter-provider. An amplification oligomer that provides both a 3' target-specific sequence and a 5' promoter sequence that can be extended by polymerization is referred to as a promoter-primer. Amplification oligomers may be optionally modified to include 5' non-target-specific sequences, such as tags, (referred to as) promoters, or other sequences used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0147] "Detection probe oligomer," "detection probe," or "probe" refers to an oligomer that specifically hybridizes to a target sequence, including an amplification product, under conditions that promote nucleic acid hybridization, for detection of the target nucleic acid. Detection can be either direct (i.e., a probe hybridized directly to the target) or indirect (i.e., a probe hybridized to an intermediate structure that links the probe to the target). The target-specific sequence of a probe generally refers to the specific sequence within a larger sequence to which the probe specifically hybridizes. A detection probe can contain target-specific sequence(s) and non-target-specific sequence(s). Such non-target-specific sequences can include sequences that impart desired secondary or tertiary structures, such as hairpin structures, that can be used to facilitate detection and / or amplification.

[0148] A "label" or "detectable label" refers to a moiety or compound attached, directly or indirectly, to a probe that is detected or provides a detectable signal. Direct attachment may use covalent or non-covalent interactions (e.g., hydrogen bonding, hydrophobic or ionic interactions, and the formation of chelate or coordination complexes), while indirect attachment may use a bridging moiety or linker (e.g., via an antibody or additional oligonucleotide(s) that amplify the detectable signal). Any detectable moiety can be used, such as radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles (e.g., latex or metal beads) that impart a detectable color, luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent compounds such as acridinium ester ("AE") compounds), and fluorescent compounds (i.e., fluorophores). Fluorophores may be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore, thereby reducing background fluorescence. Detectably labeled probes include, for example, hydrolysis (eg, TaqMan™) probes, AE-labeled probes, molecular torches, and molecular beacons.

[0149] The term "composed of" refers to the actual arrangement of the polynucleotide sequence composition of the referenced oligonucleotide target hybridization sequence. For example, an amplification oligomer configured to generate a specific amplicon from a target sequence has a polynucleotide sequence that hybridizes to the target sequence and can be used in an amplification reaction to generate the amplicon. Also, as an example, an oligonucleotide configured to specifically hybridize to a target sequence has a polynucleotide sequence that specifically hybridizes to a reference sequence under stringent hybridization conditions.

[0150] As used herein, the term "diluent" refers to a solution suitable for modifying or achieving the exemplary or suitable concentrations described herein.

[0151] The term "container" refers to anything in which an object or liquid can be placed or contained, for example, for storage (eg, a holder, receptacle, vessel, etc.).

[0152] References herein to numerical ranges (eg, "X to Y" or "X to Y") include the endpoints defining the range, and all values ​​subsumed within the range.

[0153] Unless otherwise clear from the context, when a value is expressed as "about" X or "approximately" X, the stated value of X is understood to be accurate to ±10%.

[0154] explanation The present invention provides aqueous polymerase formulations and reaction mixtures (including related methods for preparing and using such formulations and reaction mixtures) comprising a combination of α-cyclodextrin and polysorbate 20. The formulations and reaction mixtures are based, in part, on the surprising observation that α-cyclodextrin and polysorbate 20 provide a synergistic effect in mitigating the inhibition of polymerase activity observed in the presence of detergents often used in sample extraction buffers (e.g., detergents such as sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS)). In certain embodiments, this mitigating effect on polymerase inhibition provides advantages, such as increased sensitivity in amplification and detection assays and allowing a greater proportion of extracted samples containing residual detergent(s) used in amplification reaction mixtures. In other embodiments, the present invention helps stabilize lyophilized polymerase formulations due to α-cyclodextrin acting as a secondary cryoprotectant (e.g., in situations where increasing the concentration of the primary cryoprotectant results in incomplete reconstitution of the lyophilized product).

[0155] In certain embodiments, the polymerase formulation is an aqueous formulation. Such a formulation may be, for example, a pre-lyophilized formulation or a formulation reconstituted from a lyophilized form. In some variations, the formulation is provided as an aqueous polymerase formulation comprising at least one polymerase, α-cyclodextrin at a concentration of about 10 mg / mL to about 40 mg / mL, polysorbate 20 at a concentration of about 0.002% to about 0.05% (v / v), and at least one buffering agent. In some embodiments, α-cyclodextrin is from about 10 mg / mL to about 35 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 25 mg / mL, from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 15 mg / mL, from about 12 mg / mL to about 40 mg / mL, from about 12 mg / mL to about 35 mg / mL, from about 12 mg / mL to about 30 mg / mL, from about 12 mg / mL to about 25 mg / mL, or from about 12 mg / mL to about 35 mg / mL. In more specific variations, the α-cyclodextrin is present at a concentration of about 20 mg / mL, about 17.5 mg / mL, or about 12.5 mg / mL. In some embodiments, polysorbate 20 is present at a concentration of about 0.002% to about 0.05% (v / v), about 0.002% to about 0.04% (v / v), about 0.002% to about 0.03% (v / v), about 0.003% to about 0.05% (v / v), about 0.003% to about 0.04% (v / v), or about 0.003% to about 0.03% (v / v), and in more specific variations, polysorbate 20 is present at a concentration of about 0.0042% (v / v), about 0.0035% (v / v), about 0.0026% (v / v), or about 0.02% (v / v).

[0156] Polymerases suitable for use in accordance with the present invention include DNA-dependent DNA polymerases, RNA-dependent DNA polymerases (reverse transcriptases), RNA polymerases, and enzymes with two or more polymerase activities, which may be thermolabile or thermostable. Mixtures of two or more enzymes may also be used. Exemplary polymerases include DNA-dependent DNA polymerases, such as DNA polymerase I ("Pol I"), Pol I, T4, Klenow fragment of T7, Sequenase® T7, Sequenase® version 2.0 T7, Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli, and Pyrococcus sp. GB-D DNA polymerase; RNA polymerases, such as E. coli, SP6, T3, and T7 RNA polymerases; and reverse transcriptases, such as avian myeloblastosis virus (AMV), Moloney murine leukemia virus (MMLV), RNAse H-MMLV (SuperScript®), SuperScript® II, ThermoScript®, HIV-1, and RAV2 reverse transcriptases. In some embodiments, the formulation comprises at least two DNA polymerases, such as, for example, a reverse transcriptase (eg, MMLV mutant reverse transcriptase) and a DNA-dependent DNA polymerase (eg, Taq DNA polymerase).

[0157] The buffering agent is typically present at a concentration sufficient to maintain a pH suitable for use of the polymerase in an amplification assay. In some embodiments, the buffering agent is present at a concentration sufficient to maintain a pH in the range of about 6.0 to about 9.0, about 6.5 to about 8.5, about 6.5 to about 8.0, or about 6.5 to about 7.5. Suitable buffering agents include Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphate, citrate, succinate, and histidine. In certain embodiments, the Tris buffer is present at a concentration of about 10 mM to about 100 mM or about 20 mM to about 100 mM. Other suitable concentrations of buffers for formulations according to the present invention can be readily determined by one of ordinary skill in the art.

[0158] The polymerase formulations described above may further comprise one or more additional components. For example, the formulation may further contain nucleotide triphosphates suitable for nucleic acid amplification (e.g., dATP, dCTP, dGTP, and dTTP; and / or ATP, CTP, GTP, and UTP) and / or chelating agents. Suitable chelating agents include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In some embodiments that include EDTA as a chelating agent, EDTA is present at a concentration of about 0.05 mM to about 0.5 mM (e.g., about 0.16 mM or about 0.14 mM).

[0159] In certain variations, formulations (such as those suitable for lyophilization, those reconstituted from lyophilized forms, or lyophilized formulations for reconstitution into aqueous formulations described herein) may contain a cryoprotectant. Exemplary cryoprotectants include non-reducing sugars such as sucrose, raffinose, or trehalose; glycerol; and amino acids such as glycine, arginine, or methionine. The use of cryoprotectants, including the selection of an appropriate concentration to prevent unacceptable amounts of degradation and / or aggregation of the carrier molecule upon lyophilization, is generally well known in the art. A particularly suitable cryoprotectant is trehalose, which may be present in the aqueous formulation at a concentration of, for example, about 0.2 M to about 0.35 M or about 0.2 M to about 0.4 M. In some variations including trehalose as the cryoprotectant, the trehalose is present at a concentration of about 0.26 M or about 0.3 M.

[0160] In some embodiments, the polymerase formulations described herein are concentrated preparations of one or more polymerases that can be used as bulk products for the preparation of amplification reaction mixtures.

[0161] In typical variations, the formulation is stable for an extended period of time. For example, the formulation may be stable for at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, or at least about 6 months. In some embodiments, the formulation is stable for at least about 12 months, at least about 14 months, at least about 18 months, at least about 24 months, or at least about 30 months.

[0162] The polymerase formulations described herein can be stored at temperatures of about -80°C to about 40°C, about -20°C to about 25°C, about 0°C to about 25°C, about 0°C to about 15°C, about 0°C to about 10°C, or about 2°C to about 8°C. In various embodiments, the formulations can be stored at about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. Generally, the formulations are stable and retain activity within these ranges. In some variations, the formulations are stable at about -80°C to about 25°C or about 4°C to about 25°C. In more specific variations, liquid formulations are stable at temperatures of about -80°C to about -20°C, about -80°C to about 4°C, or about -80°C to about 25°C. In other specific variations, lyophilized formulations are stable at temperatures of about 4°C to about 25°C or about 4°C to about 40°C. Intermediate ranges of temperatures above are also intended to be part of the invention, e.g., from about 2° C. to about 18° C. For example, ranges of values ​​using any combination of the above-listed values ​​as upper and / or lower limits are intended to be included.

[0163] In certain embodiments, for long-term storage, the aqueous formulations described herein may be dispensed into, for example, vials, ampoules, or other containers and lyophilized according to procedures known in the art. The lyophilized product is typically a powder or cake. The container is then sealed, and in some such variations, the seal allows for subsequent injection of a diluent into the container through the seal. Methods for preparing such lyophilized polymerase formulations from aqueous formulations, as well as lyophilized formulations prepared by such methods, are additional aspects of the present invention. In yet another aspect, the present invention provides stabilized lyophilized polymerase formulations that allow for reconstitution into the aqueous far-red dye probe formulations described herein.

[0164] Also encompassed by the present invention are methods for preparing aqueous polymerase formulations from the lyophilized formulations described herein. Such methods generally include (a) providing a lyophilized polymerase formulation described herein and (b) dissolving the lyophilized polymerase formulation in a suitable diluent to provide a reconstituted formulation. Suitable diluents can be readily selected by those skilled in the art and can include, for example, water or an aqueous solution containing a buffer (e.g., Tris).

[0165] In certain aspects of the invention, a container containing a lyophilized polymerase formulation described herein is provided in a kit with a second container containing one or more other components, such as a diluent. The polymerase formulation may be packaged in a variety of different embodiments, and those skilled in the art will understand that the invention encompasses many different kit configurations. For example, the kit may further include a container containing one or more amplification oligomers for amplifying a target region, and optionally, one or more detection probes for detecting the amplified target region. The kit may contain other reagents suitable for performing in vitro amplification, such as buffers, salt solutions, and / or appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, UTP). In certain embodiments, the kit further includes a set of instructions for practicing a method according to the invention, which instructions may be associated with the package insert and / or packaging of the kit or its components.

[0166] In another aspect, the present invention provides amplification reaction mixtures. Such reaction mixtures can be prepared, for example, using the aqueous formulations described herein as concentrated bulk products. Reaction mixtures of the present disclosure typically include at least one polymerase, α-cyclodextrin at a concentration of about 5 mg / mL to about 20 mg / mL, polysorbate 20 at a concentration of about 0.001% to about 0.025% (v / v), and at least one buffer. In some embodiments, α-cyclodextrin is present at a concentration of about 5 mg / mL to about 17.5 mg / mL, about 5 mg / mL to about 15 mg / mL, about 5 mg / mL to about 12.5 mg / mL, about 8 mg / mL to about 20 mg / mL, about 8 mg / mL to about 17.5 mg / mL, about 8 mg / mL to about 15 mg / mL, or about 8 mg / mL to about 12.5 mg / mL, and in more particular variations, α-cyclodextrin is present at a concentration of about 10 mg / mL. In some embodiments, polysorbate 20 is present at a concentration of about 0.001% to about 0.02% (v / v), about 0.001% to about 0.015% (v / v), about 0.0015% to about 0.025% (v / v), about 0.0015% to about 0.02% (v / v), or about 0.0015% to about 0.015% (v / v), and in more specific variations, polysorbate 20 is present at a concentration of about 0.002% (v / v), about 0.0021% (v / v), or about 0.01% (v / v).

[0167] Suitable polymerases for use in the reaction mixture include those suitable for the polymerase formulations described above. For example, the reaction mixture can include at least two DNA polymerases, such as a reverse transcriptase (e.g., MMLV mutant reverse transcriptase) and a DNA-dependent DNA polymerase (e.g., Taq DNA polymerase).

[0168] The buffer is typically present in the reaction mixture at a concentration sufficient to maintain a pH suitable for use of the polymerase in the amplification assay. In some embodiments, the buffer is present at a concentration sufficient to maintain a pH in the range of about 6.0 to about 9.0, about 6.5 to about 8.5, about 6.5 to about 8.0, or about 6.5 to about 7.5. Suitable buffers include those suitable for polymerase formulations, as described above. In certain embodiments, Tris buffer is present at a concentration of about 5 mM to about 50 mM or about 10 mM to about 50 mM. Other suitable concentrations of buffer for reaction mixtures according to the present invention can be readily determined by one of ordinary skill in the art.

[0169] In certain embodiments, the amplification reaction mixture may contain a cryoprotectant (e.g., when the amplification reaction mixture is prepared using an aqueous polymerase formulation reconstituted from a lyophilized form). In some such embodiments, the cryoprotectant is trehalose, and the trehalose may be present in the reaction mixture at a concentration of, for example, about 0.1 M to about 0.2 M. In some variations of reaction mixtures that include trehalose, the trehalose is present at a concentration of about 0.15 M.

[0170] The reaction mixture described above may further comprise one or more additional components for conducting an amplification assay. For example, the reaction mixture may further comprise at least one amplification oligomer configured to amplify a target region of a target nucleic acid. In some such embodiments, the reaction mixture contains at least two amplification oligomers (e.g., a first amplification oligomer and a second amplification oligomer) configured to amplify the target region over multiple cycles of an amplification assay. In other, non-mutually exclusive variations, the reaction mixture includes at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region to be amplified. Detection probe oligomers for use in amplification reaction mixtures typically comprise a detectable label, such as a chemiluminescent compound or a fluorophore. In certain embodiments, the at least two amplification oligomers and at least one detection probe oligomer in the reaction mixture are configured to amplify the target region and detect the amplification product in real time, such as in a real-time PCR assay or a real-time transcription-mediated amplification (TMA) assay. Particularly suitable detection probe oligomers for real-time detection include oligomers containing a fluorescent label and a non-fluorescent quencher (eg, a hydrolysis probe, a molecular torch, or a molecular beacon).

[0171] In other, non-mutually exclusive embodiments, the reaction mixture includes one or more additional suitable amplification assay components selected from nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP; and / or ATP, CTP, GTP, and UTP), a chelating agent (e.g., EDTA or EGTA), an inorganic salt (e.g., KCl), and a cofactor. In some variations, the chelating agent is EDTA, which may be present, for example, at a concentration of about 0.025 mM to about 0.25 mM (e.g., at a concentration of about 0.08 mM). A particularly suitable cofactor is magnesium chloride, which may be present, for example, at a concentration of about 1 mM to about 5 mM.

[0172] In some embodiments, the reaction mixture described above further comprises a target nucleic acid or a sample suspected of containing the target nucleic acid. For example, a reaction mixture described above containing at least one amplification oligomer may further comprise a target nucleic acid targeted by the at least one amplification oligomer, or a sample suspected of containing the target nucleic acid. The target nucleic acid may be any target nucleic acid of interest, and may be RNA or DNA. In certain embodiments, the target nucleic acid is derived from a pathogen, such as a virus, bacterium, protozoan, fungus, or other microorganism that causes human or animal disease.

[0173] In some embodiments, the reaction mixture described above further comprises at least one detergent. For example, the reaction mixture may contain at least one detergent introduced into the reaction mixture from a sample containing or suspected of containing the target nucleic acid, the sample containing one or more residual detergents used in sample extraction. In certain variations, the detergent is an anionic detergent, such as dodecyl sulfate (e.g., sodium dodecyl sulfate, potassium dodecyl sulfate, or lithium dodecyl sulfate (also referred to herein as lithium lauryl sulfate)) or N-lauroyl sarcosine. In some embodiments, the sample containing at least one detergent constitutes at least about 20% or at least about 40% by volume of the reaction mixture. In more specific variations, the sample containing at least one detergent constitutes about 40% to about 50%, about 50% to about 70%, about 20% to about 33%, about 42.9%, or about 62.5% by volume of the reaction mixture.

[0174] In another aspect, the present invention provides methods for preparing an amplification reaction mixture. In certain embodiments, the method for preparing a reaction mixture comprises (a) providing an aqueous polymerase formulation as described above; and (b) combining the aqueous polymerase formulation with a second formulation containing at least one amplification oligomer (e.g., at least two amplification oligomers) configured to amplify a target region of a target nucleic acid. In other embodiments, the method for preparing a reaction mixture comprises (a) providing a lyophilized polymerase formulation as described above; (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation; and (c) combining the reconstituted formulation with a second formulation containing at least one amplification oligomer (e.g., at least two amplification oligomers) configured to amplify a target region of a target nucleic acid. Suitable polymerases include those suitable for the polymerase formulations or reaction mixtures described above. In certain variations, the above methods further comprise combining the aqueous or reconstituted formulation with one or more additional amplification assay components. Such additional component(s) may be contained within a second formulation or may be contained in one or more separate formulations mixed with the aqueous or reconstituted polymerase formulation. In some embodiments, such one or more additional components include (i) at least one detection probe oligomer configured to specifically hybridize to a sequenced target contained within the target region (e.g., a probe comprising a label, such as a chemiluminescent or fluorescent label, or comprising a fluorescent label and a non-fluorescent quencher), (ii) nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP; and / or ATP, CTP, GTP, and UTP), (iii) at least one chelator (e.g., EDTA or EGTA), and / or (iv) at least one cofactor (e.g., magnesium chloride).In some embodiments, the aqueous or reconstituted polymerase formulation further comprises a nucleotide triphosphate and / or a chelating agent as described herein, and the one or more additional components admixed with the aqueous or reconstituted formulation comprise (i) at least one detection probe oligomer configured to specifically hybridize to a sequenced target contained within the target region, and / or (ii) at least one cofactor.

[0175] In some embodiments, the method for preparing a reaction mixture further comprises mixing a sample containing or suspected of containing a target nucleic acid with an aqueous or reconstituted formulation, or a mixture as described above containing an aqueous or reconstituted formulation. In some such embodiments, the sample is an extracted sample derived from treating a biological sample with one or more detergents, whereby the extracted sample contains one or more detergents as residual components. The residual detergent may be, for example, an anionic detergent such as dodecyl sulfate (e.g., sodium dodecyl sulfate, potassium dodecyl sulfate, or lithium dodecyl sulfate) or N-lauroyl sarcosine. In some embodiments, the extracted sample containing at least one detergent constitutes at least about 20% or at least about 40% by volume of the reaction mixture (e.g., about 40% to about 50%, about 50% to about 70%, about 20% to about 33%, about 42.9%, or about 62.5% by volume of the reaction mixture).

[0176] In certain embodiments of the method for preparing the reaction mixture as described above, the final assay concentrations of the mixture components can include those described above for the amplification reaction mixture. For example, α-cyclodextrin can be mixed to a concentration of about 5 mg / mL to about 20 mg / mL, polysorbate 20 can be mixed to a concentration of about 0.001% to about 0.025% (v / v), Tris, if present, can be mixed to a concentration of about 5 mM to about 50 mM, EDTA, if present, can be mixed to a concentration of about 0.025 mM to about 0.25 mM, trehalose, if present, can be mixed to a concentration of about 0.1 M to about 0.2 M, and / or magnesium chloride, if present, can be mixed to a concentration of about 1 mM to about 5 mM.

[0177] In another aspect, the invention provides a method for performing an amplification reaction. In certain embodiments, the method includes: (a) providing a sample containing or suspected of containing a target nucleic acid; (b) contacting the sample with an aqueous mixture containing at least one polymerase, α-cyclodextrin, polysorbate 20, at least one buffer (e.g., Tris), nucleotide triphosphates suitable for performing nucleic acid amplification (e.g., dATP, dCTP, dGTP, and dTTP; and / or ATP, CTP, GTP, and UTP), at least one cofactor (e.g., magnesium chloride), and at least one amplification oligomer (e.g., at least two amplification oligomers) configured to amplify a target region of the target nucleic acid; wherein contacting the sample with the aqueous mixture provides a reaction mixture in which the α-cyclodextrin is present at a concentration of about 5 mg / mL to about 20 mg / mL and the polysorbate 20 is present at a concentration of about 0.001% to about 0.025% (v / v); and (c) using the reaction mixture to perform an in The method includes performing an in vitro nucleic acid amplification reaction using a reaction mixture in which a target nucleic acid, if present in the sample, is used as a template to generate one or more amplicons corresponding to the target region. Suitable polymerases include those suitable for the polymerase formulations or reaction mixtures described above. In certain variations, the method further includes contacting the sample with one or more additional assay components, such as at least one detection probe oligomer configured to specifically hybridize to the target sequence contained in the one or more amplicons, and / or (iii) at least one chelating agent (e.g., EDTA or EGTA). The aqueous mixture containing the polymerase may further contain a cryoprotectant, such as trehalose (e.g., used in lyophilized formulations to which the aqueous formulation is reconstituted). The final amplification assay concentrations of the mixture components may include those described herein above for the amplification reaction mixture.Amplification of a target region of a target nucleic acid can be achieved using a variety of known nucleic acid amplification reactions, including, for example, transcription-associated amplification (e.g., transcription-mediated amplification (TMA) or nucleic acid sequence-based amplification (NASBA)), polymerase chain reaction (PCR), replicase-mediated amplification, and ligase chain reaction (LCR), to name a few.

[0178] In some embodiments of the methods for performing an amplification reaction as described above, the sample is an extracted sample derived from treating a biological sample with one or more detergents, such that the extracted sample contains one or more detergents as residual components. The residual detergent may be, for example, an anionic detergent such as dodecyl sulfate (e.g., sodium dodecyl sulfate, potassium dodecyl sulfate, or lithium dodecyl sulfate) or N-lauroyl sarcosine. In some embodiments, the extracted sample containing at least one detergent constitutes at least about 20% or at least about 40% by volume of the reaction mixture (e.g., about 40% to about 50%, about 50% to about 70%, about 20% to about 33%, about 42.9%, or about 62.5% by volume of the reaction mixture).

[0179] In certain embodiments, methods for performing amplification reactions such as those described above further include purifying the target nucleic acid from other components in the sample prior to amplification. Such purification may include methods for separating and / or concentrating organisms contained in the sample from other sample components, or methods for removing or degrading non-nucleic acid sample components, such as proteins, carbohydrates, salts, lipids, etc. In certain embodiments, the target nucleic acid is specifically or nonspecifically captured and separated from other sample components. Target capture typically occurs in a solution-phase mixture containing one or more capture probe oligomers that hybridize to the target sequence of the target nucleic acid under hybridization conditions. In embodiments that include a capture probe tail, the target:capture-probe complex is captured by using hybridization conditions under which the capture probe tail hybridizes to the immobilized probe. Certain embodiments use particulate solid supports, such as paramagnetic beads. Selective and nonspecific target capture methods are also described, for example, in U.S. Pat. No. 6,110,678 and International Patent Application Publication No. 2008 / 016988, each of which is incorporated herein by reference. In some embodiments, a purification step (e.g., a purification step involving specific or non-specific target capture) utilizes one or more detergents, such as, for example, anionic detergents (e.g., sodium dodecyl sulfate or lithium lauryl sulfate).

[0180] Methods for amplifying target nucleic acids as described above may further include detecting one or more amplicons. The detection step may be performed using any of a variety of known techniques for detecting a signal specifically associated with the amplified target sequence, such as by hybridizing the amplification product with a labeled detection probe and detecting a signal resulting from the labeled probe (which, in some embodiments, includes a label released from the probe after hybridization). In some embodiments, the labeled probe includes a second moiety, such as a quencher or other moiety, that interacts with the first label. Detection may occur after the amplification reaction is complete, or may occur simultaneously with amplification of the target region, e.g., in real time. In certain variations using real-time detection, the detection probe may be a hairpin probe, such as a molecular beacon, a molecular torch, or a hybridization switch probe labeled with a reporter moiety that is detected when the probe binds to the amplification product (e.g., a dual-labeled hairpin probe containing both a fluorescent label and a quencher moiety). In other embodiments for real-time detection, the detection probe is a linear oligomer, such as an oligomer labeled with both a fluorophore and a quencher moiety (e.g., a TaqMan probe). Such probe can comprise target hybridization sequence and non-target hybridization sequence.Various forms of such probe have been previously described (see, for example, U.S. Patent No. 5,210,015, U.S. Patent No. 5,487,972; U.S. Patent No. 5,118,801; U.S. Patent No. 5,312,728; U.S. Patent No. 5,925,517; U.S. Patent No. 6,150,097; U.S. Patent No. 6,849,412; U.S. Patent No. 6,835,542; U.S. Patent No. 6,534,274; and U.S. Patent No. 6,361,945; and U.S. Patent Application Publication No. 20060068417A1 and U.S. Patent Application Publication No. 20060194240A1; each of which is incorporated herein by reference). [Example]

[0181] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure.

[0182] Example 1 To test samples containing detergent, several PCR reaction mixtures were prepared. The initial master mix contained 0.46 U / μL DNA polymerase, 0.5 U / μL reverse transcriptase, 0.2 U / μL RNase inhibitor, 0.25 mM dNTPs, 0.05 mM dUTP, 81 mM and 5.1 mM inorganic salts including KCl and MgCl, 0.1 mM EDTA, and several primers and probes (SEQ ID NOs: 8-14, 0.64 μM to 1.29 μM) for amplification and detection of influenza A target nucleic acids. A second master mix was prepared to contain 1.0 U / µL DNA polymerase, 3.2 U / µL reverse transcriptase, 0.53 mM dNTPs, 1.06 mM dUTP, inorganic salts including 173 mM and 10.93 mM KCl and MgCl, 0.21 mM EDTA, and several primers and probes (SEQ ID NOs: 8-14, 0.64 µM-1.29 µM) for amplification and detection of the same target nucleic acid. The initial master mix was divided into conditions (A) to (F), with conditions (B) and (D) further containing 0.025% (v / v) polysorbate 20, conditions (B), (C), and (F) further containing 12.5 mg / mL α-cyclodextrin, condition (C) further containing 0.13% (v / v) polysorbate 20, and condition (E) further containing 50 mg / mL α-cyclodextrin.

[0183] Samples were prepared by spiking 8.3 copies / mL of influenza A in vitro transcript target nucleic acid (SEQ ID NO: 7) into an appropriate medium, such as MicroTest M4 medium (Remel Inc. Catalog No. R12500), MicroTest M5 viral transport medium (Remel, Inc. Catalog No. R12515), MicroTest M6 viral transport medium (Remel, Inc. Catalog No. R12530), MicroTest M4RT viral transport medium (Remel, Inc. Catalog No. R12505), or Copan Universal transport medium (Copan Diagnostics, Inc., Catalog No. 330C). The target nucleic acid was an RNA transcript of SEQ ID NO: 7. 360 μL of the prepared sample was separately incubated in a buffer reagent containing 100 μg of polyT (SEQ ID NO: 2)-coated magnetic microparticles and 20 pmoles of target capture oligomer (SEQ ID NO: 1) in a final reaction volume of 936 μL to bind the target nucleic acid to the magnetic solid support. The magnetic particles and bound nucleic acids were separated from the solution by applying a magnetic field, and the supernatant was removed from the [capture target]:[capture probe]:[magnetic particles] combination. The magnetic particles were then resuspended in wash buffer. The resuspended particles were subjected to another round of separation, supernatant removal, and resuspension in wash buffer. After a second round of separation and removal of the wash buffer, the particles were incubated in 50 μL of elution buffer (5 mM Tris in water with preservative). The magnetic particles were separated by applying a magnetic field, and the eluate containing the nucleic acids was collected. The eluate was divided into separate containers, and one aliquot was further spiked with 30% (v / v) wash buffer to generate an eluate containing 100 mg / mL sodium dodecyl sulfate, simulating carryover of wash buffer from upstream sample processing steps. Detergents present in wash buffers are known to inhibit or reduce nucleic acid amplification reactions.

[0184] Each of the master mix conditions (A) through (F) above was added to a well of a multiwell plate and combined with an aliquot of the eluate spiked with wash buffer and the eluate without wash buffer (20 μL master mix, 10 μL eluate, 30 μL total reaction volume). Each condition was prepared in 20 replicates, except for condition (E), which was prepared in 19 replicates. Real-time amplification and detection reactions were set up for 20 (19) replicates per condition, and the reactions were run using a thermal cycler (Panther Fusion Instrument, Hologic, Inc., San Diego, CA). The results are shown in Table 1 below. [Table 1-1] [Table 1-2]

[0185] These data indicate that the addition of α-cyclodextrin and polysorbate 20 to the reaction mixture mitigated the negative / inhibitory effects of wash buffer detergents on nucleic acid amplification reactions. These data also indicate that the combination of these two reagents had a synergistic mitigating effect on wash buffer-induced inhibition compared to the mitigating effect observed using either α-cyclodextrin or polysorbate 20 alone.

[0186] Example 2 A PCR master mix was prepared substantially as described in Example 1 and designated the second master mix. In this master mix, the primers and probes were SEQ ID NOS: 4-6, and the master mix further contained 0.4 M trehalose. The master mix was separated into separate conditions: condition (G1) further contained 26.7 mg / mL α-cyclodextrin and 0.0057% (v / v) polysorbate 20; condition (G2) further contained 28.0 mg / mL α-cyclodextrin and 0.0063% (v / v) polysorbate 20; condition (G3) further contained 25.4 mg / mL α-cyclodextrin and 0.0051% (v / v) polysorbate 20; and condition (G4) contained no additional additives.

[0187] Seasonal coronavirus type OC43 (Zeptometrix catalog number 0810024CF) was spiked into whole blood sample matrix or sample transport medium (STM) to achieve 0.125 TCID in each sample type. 50 A final virus titer of 100 mg / mL was obtained. 400 μL of whole blood matrix or STM matrix was separately incubated in a buffer reagent containing 100 μg of polyT (SEQ ID NO: 2)-coated magnetic microparticles and 20 pmoles of target capture oligomer (SEQ ID NO: 3) in a final reaction volume of 976 μL to bind the target nucleic acid to the magnetic solid support. The magnetic microparticles and bound nucleic acids were separated as outlined in Example 1, and the eluate containing the target nucleic acid was collected. The eluate was then divided into two equal parts, and one part was further spiked with 30% (v / v) wash buffer to generate an eluate containing 100 mg / mL sodium dodecyl sulfate, simulating carryover of wash buffer from upstream sample processing steps. Detergents present in wash buffers are known to inhibit or reduce nucleic acid amplification reactions.

[0188] Each of the master mix conditions (G1) through (G4) was added to a well of a multiwell plate and combined with an aliquot of either the wash buffer-spiked eluate or the eluate that had not received the wash buffer spike (15 μL master mix, 25 μL eluate, 40 μL total reaction volume, except for the (G4) condition, which consisted of 20 μL master mix, 5 μL eluate, and 25 μL total volume). Each condition was prepared in 24 replicates, except for the condition (G1) containing the unspiked eluate, which was prepared in 20 replicates. RT-PCR reactions were performed using a thermal cycler (Panther Fusion Instrument, Hologic, Inc., San Diego, CA). Condition (G4) was prepared with 5.3-fold less target nucleic acid than when prepared using 15 μL master mix and 25 μL eluate. Therefore, under identical reaction conditions, (G4) has a higher Ct than when prepared using the alternative reaction conditions (theoretically a Ct of 2.4). The results are shown in Table 2 below. [Table 2]

[0189] These data show lower Ct values ​​and higher RFU values ​​for conditions (G1), (G2), and (G3) compared to condition (G4). Compared to condition (G4)a, these data show Ct improvements of -5.1, -5.3, and -4.7, representing RFU improvements of +112%, +127%, and +102% for conditions (G1)a, (G2)a, and (G3)a, respectively. Compared to (G4)b, these data show Ct improvements of -3.2, -3.4, and -3.0, representing RFU improvements of +61%, +72%, and +56% for conditions (G1)b, (G2)b, and (G3)b, respectively. Compared to (G4)c, these data show Ct improvements of -5.5, -5.5, and -6.4, representing RFU improvements of +72%, +35%, and +67% for conditions (G1)c, (G2)c, and (G3)c, respectively. Compared to (G4)d, these data show Ct improvements of -3.2, -4.1, and -3.2, representing RFU improvements of +57%, +44%, and +46% for conditions (G1)d, (G2)d, and (G3)d, respectively. These results show substantial improvements in Ct when samples containing inhibitors and treated with various concentrations of α-cyclodextrin and polysorbate 20 are compared to samples containing inhibitors without such treatment.

[0190] Example 3 Seasonal coronavirus type OC43 (Zeptometrix catalog number 0810024CF) was spiked into some plasma and serum samples to produce 70 TCID in a total sample volume of 1.15 mL. 50The final OC43 virus titer was obtained at 1000 / mL. The spiked samples were then combined with a reagent containing 10% (v / v) lithium lauryl sulfate. Other components of this reagent included buffer, salt, magnetic particles coated with SEQ ID NO:2, SEQ ID NO:1, and target capture oligomer (SEQ ID NO:3). Each sample was mixed on an orbital shaker (approximately 200 rpm, 1 min). The samples were then incubated in a 43°C water bath for 4 minutes, then at 63°C for 29 minutes, and then at 43°C for 15 minutes. The magnetic particles were separated by application of a magnetic field, and the eluate containing nucleic acids was collected. Two reverse transcription PCR reaction mixtures were prepared, each containing SEQ ID NOs:4, 5, and 6, DNA polymerase, reverse transcriptase, dNTPs, inorganic salts, BSA, and RNase inhibitor. One of the RT-PCR reaction mixtures further contained 12.5 mg / mL polysorbate 20 and 0.0026% (v / v) α-cyclodextrin. 20 µL of RT-PCR reaction mixture was combined with 5 µL of eluate from each sample type to prepare the following four conditions: (A) plasma sample eluate with RT-PCR reaction mixture containing polysorbate 20 and α-cyclodextrin, (B) plasma sample eluate with RT-PCR reaction mixture - neat, (C) serum sample eluate with RT-PCR reaction mixture containing polysorbate 20 and α-cyclodextrin, and (D) serum sample eluate with RT-PCR reaction mixture - neat. RT-PCR reactions were prepared in 9-11 replicates (see Table 3) and spiked with an internal control. RT-PCR reactions were performed, and the data are shown in Table 3. [Table 3]

[0191] The data in Table 3 show that in the presence of residual LLS, samples produced RT-PCR results with higher Ct, lower RFU, and higher sample-to-sample variability than those produced for reactions containing reagents that mitigated the effects of LLS, indicating that the addition of α-cyclodextrin and polysorbate 20 significantly aided in the detection of RNA by more than 1 log (more than 4 Ct lower with the additive).

[0192] Example 4 A series of aqueous mixtures containing α-cyclodextrin and polysorbate 20 were prepared. The mixtures were set up using a fixed concentration of polysorbate 20 combined with several concentrations of α-cyclodextrin, or a fixed concentration of α-cyclodextrin combined with several different concentrations of polysorbate 20 (Table 4). Each combination in mixtures 1 through 7 was effective in mitigating the inhibitory effects of detergents in nucleic acid amplification reaction mixtures. Each aqueous mixture was kept on ice for 1 hour, and then the mixtures were centrifuged to assess the presence of precipitants. After 1 hour of incubation on ice, various levels of precipitate were observed in mixtures 1 through 6. [Table 4]

[0193] Several dried RT-PCR reaction mixtures were prepared containing various concentrations of α-cyclodextrin and polysorbate 20. An RT-PCR master mix was prepared containing 1.0 U / μL DNA polymerase, 3.2 U / μL reverse transcriptase, 0.4 M trehalose, 0.53 mM dNTPs, 1.06 mM dUTP, 0.21 mM EDTA, 1.6 μM each of SEQ ID NOs: 4 and 5, and 1.07 μM SEQ ID NO: 6. This master mix was then divided into separate conditions: condition (1) contained 16 mg / mL α-cyclodextrin and 0.0034% (v / v) polysorbate 20; condition (2) contained 20 mg / mL α-cyclodextrin and 0.02% (v / v) polysorbate 20; and condition (3) contained neither α-cyclodextrin nor polysorbate 20. Each of these conditions was then aliquoted at 24 μL into several reaction wells per condition, and the aliquots were dried using lyophilization techniques to form single unit dose lyophilized pellets.

[0194] Seasonal coronavirus type OC43 (Zeptometrix catalog number 0810024CF) was spiked into whole blood sample matrix, plasma sample matrix, serum sample matrix, or sample transport medium (STM) to produce 14.2 TCID in each sample type. 50 A final virus titer of 100 mg / mL was obtained. 400 μL of whole blood matrix, 200 μL of plasma or serum matrix, or 360 μL of STM matrix were separately incubated in a buffer reagent containing 100 μg of polyT (SEQ ID NO: 2)-coated magnetic microparticles and 20 pmoles of target capture oligomer (SEQ ID NO: 3) in a final reaction volume of 936 μL to bind the target nucleic acid to the magnetic solid support. The magnetic microparticles and bound nucleic acids were separated as outlined in Example 1, and an eluate containing the target nucleic acid was collected. The eluate was then divided into two equal parts, and one part was further spiked with 30% (v / v) wash buffer to generate an eluate containing 100 mg / mL sodium dodecyl sulfate, simulating carryover of wash buffer from upstream sample processing steps.

[0195] The lyophilized pellets from conditions (1) to (3) were each rehydrated with 25 μL of HEPES buffer containing KCl and MgCl. 15 μL of the rehydrated reaction mixture was combined with 25 μL of the eluate from conditions (1) and (2), and 20 μL of the rehydrated reaction mixture (3) was combined with 5 μL of the eluate (both in replicate 6). RT-PCT reactions were performed using a thermal cycling instrument. The results are shown in Table 5. [Table 5]

[0196] These data show lower Ct values ​​and higher RFU values ​​for reactions containing a combination of α-cyclodextrin and polysorbate 20 compared to equivalent reactions without these additives.

[0197] Example 5 An amplification reagent mixture was prepared consisting of 0.65 U / µL DNA polymerase, 0.35 mM dNTPs, 0.7 mM dUTP, inorganic salts including 81 and 5.1 mM KCl and MgCl, 0.26 M trehalose, 0.14 mM ethylenediaminetetraacetic acid, 17.5 mg / mL α-cyclodextrin, and 0.0035% polysorbate 20. The reagent mixture contained either primers and probes specific for BK virus (BKV) (SEQ ID NOs: 15–17; see Table 6) or primers and probes specific for Epstein-Barr virus (EBV) detection (SEQ ID NOs: 18–20; see Table 6).

[0198] The samples consisted of human whole blood diluted 1:4 in blood transport medium, human urine diluted 1:2 in urine transport medium, or undiluted human plasma. 400 μL of each of these samples was separately incubated in a final reaction volume of 976 μL containing 270 μg of polyT (SEQ ID NO: 2; 5'-(T)14-3')-coated magnetic microparticles and 220 pmoles of target capture oligomer (SEQ ID NO: 1; 5'-(K)18(T)3(A)30-3') to bind the target nucleic acid to the magnetic solid support. The magnetic microparticles and bound nucleic acid were separated from the solution by application of a magnetic field, and the supernatant was removed from the [capture target]:[capture probe]:[magnetic microparticle] combination. The magnetic microparticles were then resuspended in wash buffer. The resuspended microparticles were subjected to another round of separation, supernatant removal, and resuspension in wash buffer. After separation and removal of the second wash buffer, the microparticles were incubated in 50 μL of elution buffer (5 mM Tris in water with preservatives). The magnetic particles were separated by application of a magnetic field, and the eluate containing the nucleic acids was collected.

[0199] Each of the above master mix conditions was combined in a well of a multiwell plate (20 μL master mix, 15 μL eluate, 35 μL total reaction volume). Real-time amplification and detection reactions were set up for the extracted samples and run using a thermal cycling instrument (Panther Fusion Instrument, Hologic, Inc., San Diego, CA).

[0200] The linearity of EBV detection was evaluated over a range of 2.45 log IU / mL to 9.86 log IU / mL for human whole blood diluted with blood transport medium and over a range of 1.98 log IU / mL to 9.26 log IU / mL for undiluted human plasma. The linearity of BKV detection was evaluated over a range of 2.11 log IU / mL to 9.38 log IU / mL for human urine diluted with urine transport medium and over a range of 1.80 log IU / mL to 9.08 log IU / mL for undiluted human plasma. Linearity was demonstrated across the range tested for all conditions. [Table 6-1] [Table 6-2]

[0201] From the foregoing, it will be understood that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. In certain embodiments, for example, the following are provided: (Item 1) 1. An aqueous polymerase formulation comprising: at least one polymerase; α-cyclodextrin at a concentration of about 10 mg / mL to about 40 mg / mL; Polysorbate 20 at a concentration of about 0.002% to about 0.05% (v / v), at least one buffer; and 1. An aqueous polymerase formulation comprising: (Item 2) 2. The formulation according to item 1, wherein the α-cyclodextrin concentration is about 16 mg / mL to about 30 mg / mL, about 15 mg / mL to about 20 mg / mL, or about 10 mg / mL to about 15 mg / mL. (Item 3) 2. The formulation of item 1, wherein the α-cyclodextrin concentration is about 20 mg / mL, about 17.5 mg / mL, or about 12.5 mg / mL. (Item 4) 4. The formulation according to any one of items 1 to 3, wherein the concentration of the polysorbate 20 is about 0.003% to about 0.03% (v / v). (Item 5) 4. The formulation of any one of items 1 to 3, wherein the polysorbate 20 concentration is about 0.0042% (v / v), about 0.0035% (v / v), about 0.0026% (v / v), or about 0.02% (v / v). (Item 5) Item 5a. The formulation of any one of items 1 to 5, wherein the at least one polymerase is a DNA polymerase. (Item 6) The formulation according to any one of items 1 to 5a, further comprising nucleotide triphosphates suitable for performing nucleic acid amplification. (Item 7) 7. The formulation according to any one of items 1 to 6, further comprising a cryoprotectant. (Item 8) 8. The formulation of item 7, wherein the cryoprotectant is trehalose. (Item 9) Item 9. The formulation according to item 8, wherein the trehalose is present at a concentration of about 0.2 M to about 0.4 M. (Item 10) 10. The formulation of item 9, wherein the trehalose is present at a concentration of about 0.26 M or about 0.3 M. (Item 11) 1. A method for preparing a lyophilized polymerase formulation, comprising: (a) providing the aqueous formulation according to any one of items 7 to 10; (b) lyophilizing the aqueous formulation to form the lyophilized polymerase formulation. (Item 12) Item 12. A lyophilized polymerase formulation prepared by the method described in Item 11. (Item 13) A lyophilized polymerase formulation that can be reconstituted into the aqueous formulation according to any one of items 7 to 10. (Item 14) 1. A method for preparing an aqueous polymerase formulation, comprising: (a) providing a lyophilized polymerase formulation according to item 12 or 13; (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation; A method comprising: (Item 15) A kit comprising: 14. A kit comprising a first sealed container containing the lyophilized polymerase formulation of item 12 or 13. (Item 16) 16. The kit of item 15, further comprising a second sealed container containing a diluent. (Item 17) 1. An amplification reaction mixture comprising: at least one polymerase; α-cyclodextrin at a concentration of about 5 mg / mL to about 20 mg / mL; Polysorbate 20 at a concentration of about 0.001% to about 0.025% (v / v); at least one buffer; and an amplification reaction mixture comprising: (Item 18) Item 18. The reaction mixture according to Item 17, wherein the α-cyclodextrin concentration is about 8 mg / mL to about 15 mg / mL. (Item 19) 18. The reaction mixture according to item 17, wherein the α-cyclodextrin concentration is about 10 mg / mL. (Item 20) 20. The reaction mixture according to any one of items 17 to 19, wherein the polysorbate 20 concentration is about 0.0015% to about 0.015% (v / v). (Item 21) 20. The reaction mixture of any one of items 17 to 19, wherein the polysorbate 20 concentration is about 0.002% (v / v), about 0.0021% (v / v), or about 0.01% (v / v). (Item 22) 22. The reaction mixture of any one of items 17 to 21, wherein the at least one polymerase is a DNA polymerase. (Item 23) 23. The reaction mixture according to any one of items 17 to 22, further comprising a cryoprotectant. (Item 24) 24. The reaction mixture of claim 23, wherein the cryoprotectant is trehalose. (Item 25) 25. The reaction mixture of item 24, wherein the trehalose is present at a concentration of about 0.1 M to about 0.29 M. (Item 26) 26. The reaction mixture of claim 25, wherein the trehalose is present at a concentration of about 0.15M. (Item 27) 27. The reaction mixture according to any one of items 17 to 26, further comprising nucleotide triphosphates suitable for carrying out nucleic acid amplification. (Item 28) 28. The reaction mixture of any one of items 17 to 27, further comprising at least two amplification oligomers configured to amplify a target region of a target nucleic acid. (Item 29) 29. The reaction mixture of claim 28, further comprising at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region. (Item 30) 30. The reaction mixture of item 28 or 29, further comprising the target nucleic acid. (Item 31) 31. The reaction mixture of item 30, further comprising at least one detergent. (Item 32) 32. The reaction mixture of claim 31, wherein the at least one detergent is an anionic detergent, and optionally the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS). (Item 33) 1. A method for preparing an amplification reaction mixture, comprising: (a) providing an aqueous polymerase formulation according to any one of items 1 to 10; (b) mixing the aqueous formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid; A method comprising: (Item 34) 34. The method of claim 33, further comprising mixing the aqueous formulation with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region. (Item 35) 35. The method of claim 33 or 34, further comprising mixing the mixture produced in step (b) with a sample containing or suspected of containing the target nucleic acid. (Item 36) 1. A method for preparing an amplification reaction mixture, comprising: (a) providing a lyophilized polymerase formulation according to item 12 or 13; (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation; (c) combining the reconstituted formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid; A method comprising: (Item 37) 37. The method of claim 36, further comprising mixing the reconstituted formulation with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region. (Item 38) 38. The method according to item 36 or 37, further comprising the step of mixing the mixture produced in step (c) with a sample containing or suspected of containing the target nucleic acid. (Item 39) 39. The method according to item 35 or 38, wherein the sample is an extracted sample containing at least one detergent. (Item 40) 40. The method of claim 39, wherein the at least one detergent is an anionic detergent, and optionally the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS). (Item 41) 41. The method of any one of items 35 and 38 to 40, wherein the sample containing or suspected of containing the target nucleic acid constitutes at least about 40% of the reaction mixture. (Item 42) 1. A method for performing an amplification reaction, comprising: (a) providing a sample containing a target nucleic acid; (b) subjecting the sample to at least one polymerase; α-cyclodextrin, Polysorbate 20 and at least one buffer; and nucleotide triphosphates suitable for carrying out nucleic acid amplification; at least one cofactor; at least one amplification oligomer configured to amplify a target region of the target nucleic acid; contacting the sample with the aqueous mixture to provide a reaction mixture in which the α-cyclodextrin is present at a concentration of about 5 mg / mL to about 20 mg / mL and the polysorbate 20 is present at a concentration of about 0.001% to about 0.025% (v / v); (c) performing an in vitro nucleic acid amplification reaction using the reaction mixture, wherein the target nucleic acid, if present in the sample, is used as a template to generate one or more amplicons corresponding to the target region; A method comprising: (Item 43) 43. The method of claim 42, further comprising detecting the one or more amplicons. (Item 44) 44. The method of claim 43, wherein the detecting step comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the one or more amplicons. (Item 45) 45. The method of claim 43 or 44, wherein the detecting step is performed in real time. (Item 46) 46. ​​The method according to any one of items 42 to 45, wherein the sample is an extracted sample containing at least one detergent. (Item 47) Item 47. The method of item 46, wherein the at least one detergent is an anionic detergent, and optionally the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS).

Claims

1. 1. A method for preparing an amplification reaction mixture, said method comprising: (a) at least one polymerase; α-cyclodextrin at a concentration of about 10 mg / mL to about 40 mg / mL; Polysorbate 20 at a concentration of about 0.002% to about 0.05% (v / v); at least one buffer; and A cryoprotectant, wherein "about" refers to ±10% of the given value; (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation; (c) combining the reconstituted formulation with a second formulation comprising at least one amplification oligomer configured to amplify a target region of a target nucleic acid; and (d) mixing the mixture produced in step (c) with a sample containing or suspected of containing the target nucleic acid, wherein the sample is an extracted sample containing at least one detergent. A method comprising:

2. 10. The method of claim 1, further comprising mixing the reconstituted formulation with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region.

3. 3. The method of claim 1, wherein the at least one detergent is an anionic detergent.

4. The method described in claim 3, wherein the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS).

5. 2. The method of claim 1, wherein the sample containing or suspected of containing the target nucleic acid comprises at least about 40% of the reaction mixture, where "about" refers to ±10% of a given value.

6. 10. The method of claim 1, wherein the concentration of the α-cyclodextrin in the aqueous formulation is from about 16 mg / mL to about 30 mg / mL, from about 15 mg / mL to about 20 mg / mL, or from about 10 mg / mL to about 15 mg / mL, where "about" refers to ±10% of the given value.

7. The method described in claim 6, wherein the concentration of the α-cyclodextrin in the aqueous formulation is about 20 mg / mL, about 17.5 mg / mL, or about 12.5 mg / mL, where "about" refers to ±10% of the given value.

8. 2. The method of claim 1, wherein the concentration of the polysorbate 20 in the aqueous formulation is from about 0.003% to about 0.03% (v / v), where "about" refers to ±10% of the given value.

9. The method of claim 8, wherein the concentration of the polysorbate 20 in the aqueous formulation is about 0.0042% (v / v), about 0.0035% (v / v), about 0.0026% (v / v), or about 0.02% (v / v), where "about" refers to ±10% of the given value.

10. the at least one polymerase is a DNA polymerase, and / or 10. The method of claim 1, wherein the aqueous formulation further comprises nucleotide triphosphates for performing nucleic acid amplification.

11. 10. The method of claim 1, wherein the cryoprotectant is trehalose.

12. The method of claim 11, wherein the trehalose is present in the aqueous formulation at a concentration of about 0.2 M to about 0.4 M, where "about" refers to ±10% of the given value.

13. The method of claim 12, wherein the trehalose is present in the aqueous formulation at a concentration of about 0.26 M or about 0.3 M, where "about" refers to ±10% of the given value.

14. 1. A method for performing an amplification reaction, said method comprising: (a) providing a sample containing a target nucleic acid, said sample being an extracted sample containing at least one detergent; (b) subjecting the sample to at least one polymerase; α-cyclodextrin, Polysorbate 20, at least one buffer; and Cryoprotectant and an aqueous mixture comprising: nucleotide triphosphates for performing nucleic acid amplification; at least one cofactor; with at least one amplification oligomer configured to amplify a target region of the target nucleic acid; The aqueous mixture is reconstituted from a lyophilized polymerase formulation according to the method of claim 1, contacting the sample with the aqueous mixture to provide a reaction mixture in which the α-cyclodextrin is present at a concentration of about 5 mg / mL to about 20 mg / mL and the polysorbate 20 is present at a concentration of about 0.001% to about 0.025% (v / v), "about" referring to ±10% of the given value; (c) performing an in vitro nucleic acid amplification reaction using the reaction mixture, wherein the target nucleic acid, if present in the sample, is used as a template to generate one or more amplicons corresponding to the target region. A method comprising:

15. 15. The method of claim 14, further comprising detecting the one or more amplicons.

16. 16. The method of claim 15, wherein the detecting step comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the one or more amplicons.

17. 17. The method of claim 15 or 16, wherein the detecting step is performed in real time.

18. 15. The method of claim 14, wherein the at least one detergent is an anionic detergent.

19. The method of claim 18, wherein the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS).

20. 15. The method of claim 14, wherein the sample containing the target nucleic acid comprises at least about 40% of the reaction mixture produced in step (b), where "about" refers to ±10% of a given value.

21. 15. The method of claim 14, wherein the concentration of the α-cyclodextrin in the reaction mixture is from about 8 mg / mL to about 15 mg / mL, where "about" refers to ±10% of the given value.

22. The concentration of the α-cyclodextrin in the reaction mixture is about 10 mg / mL, and / or the concentration of the polysorbate 20 in the reaction mixture is about 0.0015% to about 0.015% (v / v); 22. The method of claim 21, wherein "about" refers to ±10% of a given value.

23. The method of claim 22, wherein the concentration of polysorbate 20 in the reaction mixture is about 0.002% (v / v), about 0.0021% (v / v), or about 0.01% (v / v), where "about" refers to ±10% of the given value.

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