Enzyme preparations and reaction mixtures for nucleic acid amplification

Abstract

We provide polymerase preparations and reaction mixtures that mitigate enzyme reaction inhibition caused by residual detergents. [Solution] A formulation and reaction mixture comprising at least one polymerase, α-cyclodextrin, and polysorbate 20 is disclosed. Related methods for preparing the disclosed formulation and reaction mixture, as well as related kits and methods for nucleic acid amplification, are also disclosed. The formulation and reaction mixture are useful, for example, to mitigate the inhibitory effect of sample extraction washing agents on nucleic acid amplification reactions. The formulation may be a lyophilized polymerase formulation. This formulation may further contain 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 Jul. 1, 2021, which is hereby incorporated by reference in its entirety.

[0002] Reference to Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS - Web, which is hereby incorporated by reference in its entirety. The ASCII copy created on Jun. 8, 2022, is named "GPR_7910 PC_20220608_Seq_Listing_ST 25" and is 4,974 bytes in size.

Background Art

[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 use a sample extraction step(s) that leave one or more residual detergents that are often inhibitory to polymerase enzymes in the extracted sample. The inhibitory effect of such residual detergents poses challenges to the polymerase enzyme and the formulation of the amplification reaction mixture.

Summary of the Invention

[0004] Summary In one aspect, the present invention provides an aqueous polymerase formulation. In some embodiments, the formulation generally comprises 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 buffer. 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 present invention provides a method for preparing a lyophilized polymerase formulation. This method generally comprises (a) providing the aqueous formulation described above, and (b) lyophilizing the aqueous formulation to form a lyophilized polymerase formulation. In another aspect, the present invention provides a lyophilized polymerase formulation prepared by the method described above.

[0006] In another aspect, the present invention provides a lyophilized polymerase formulation that enables reconstitution into the above-mentioned aqueous formulation.

[0007] In another aspect, the present invention provides a method for preparing an aqueous polymerase formulation. This method generally includes (a) providing the above-mentioned lyophilized polymerase formulation, and (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation.

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

[0009] In another embodiment, the present invention provides an amplification reaction mixture. The reaction mixture generally comprises 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-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 yet another non-exclusive embodiment, the reaction mixture further comprises the target nucleic acid to be amplified and / or at least one detergent, such as an anionic detergent. Particularly preferred anionic detergents include sodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS).

[0010] In another embodiment, the present invention provides a method for preparing an amplification reaction mixture. In some embodiments, the method generally comprises (a) providing the aqueous polymerase formulation described above, and (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. In some such variations, the method further comprises mixing the mixture produced in step (b) with a sample containing or suspected to contain a target nucleic acid. In other embodiments, the method generally comprises (a) providing the lyophilized polymerase formulation described above, (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation, and (c) mixing 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 method further comprises mixing the mixture produced in step (c) with a sample containing or suspected to contain a target nucleic acid. In some of the above embodiments, which include further mixing with a sample, the sample is an extract sample containing at least one cleaning agent, such as an anionic cleaning agent. Particularly preferred anionic cleaning agents include sodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS). In other non-exclusive embodiments described above, which include further mixtures with the sample, the sample containing or suspected to contain the target nucleic acid constitutes at least about 20% or at least about 40% of the reaction mixture.

[0011] In another aspect, the present invention provides a method for carrying out an amplification reaction. The method generally comprises (a) providing a sample containing or suspected to contain a target nucleic acid; (b) contacting the sample with an aqueous mixture comprising at least one polymerase, α-cyclodextrin, polysorbate 20, at least one buffer, a nucleotide triphosphate 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 α-cyclodextrin is present at a concentration of about 5 mg / mL to about 20 mg / mL and polysorbate 20 is present at a concentration of about 0.001% to about 0.025% (v / v); and (c) carrying out 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 for generating one or more amplicons corresponding to a target region. In some embodiments, the method further includes detecting one or more amplicons (e.g., detecting one or more amplicons in real time), and in some such variations, the detection step includes contacting a nucleic acid amplification reaction product in vitro 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 washing agent, such as an anionic washing agent (e.g., sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS)). In other embodiments, the sample constitutes at least about 20% or at least about 40% of the reaction mixture produced as step (b). [Modes for carrying out the invention]

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

[0013] Embodiment Embodiment 1. An aqueous polymerase preparation, At least one polymerase, α-cyclodextrin at concentrations of approximately 10 mg / mL to approximately 40 mg / mL, Polysorbate 20 with a concentration of approximately 0.002% to approximately 0.05% (v / v), At least one cushioning agent, A water-based polymerase preparation containing [a specific ingredient / method].

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

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

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

[0017] Embodiment 5. A formulation according to any one of Embodiments 1 to 3, wherein the polysorbate 20 concentration is approximately 0.0042% (v / v), approximately 0.0035% (v / v), approximately 0.0026% (v / v), or approximately 0.02% (v / v).

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

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

[0020] Embodiment 8. The formulation according to Embodiment 7, wherein the reverse transcriptase is Moloney mouse leukemia virus (M-MLV) mutant reverse transcriptase.

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

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

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

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

[0025] Embodiment 13. The formulation according to 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 according to Embodiment 12, wherein the EDTA is present at a concentration of about 0.16 mM or about 0.14 mM. and the formulation according to Embodiment 12.

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

[0028] Embodiment 16. The formulation according to Embodiment 15, wherein the cryoprotectant is trehalose.

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

[0030] Embodiment 18. The formulation according to 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) To provide an aqueous formulation according to any one of embodiments 15 to 18, (b) Freeze-dry the aqueous formulation to form the freeze-dried polymerase formulation. Methods that include...

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

[0033] Embodiment 21. A lyophilized polymerase formulation that enables reconstitution into an aqueous formulation described in any one of Embodiments 15 to 18.

[0034] Embodiment 22. A method for preparing an aqueous polymerase preparation, (a) To provide a lyophilized polymerase preparation according to Embodiment 20 or 21, (b) Dissolve the lyophilized polymerase preparation in a diluent to provide a reconstituted preparation. Methods that include...

[0035] Embodiment 23. A kit, A kit comprising a first sealed container containing the lyophilized polymerase preparation described in Embodiment 20 or 21.

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

[0037] Embodiment 25. An amplified reaction mixture, At least one polymerase, Alpha-cyclodextrin at concentrations of approximately 5 mg / mL to approximately 20 mg / mL, Polysorbate 20 at a concentration of approximately 0.001% to approximately 0.025% (v / v), At least one cushioning agent, A mixture of amplified reaction components.

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

[0039] Embodiment 27. The reaction mixture according to Embodiment 25, wherein the α-cyclodextrin concentration is approximately 10 mg / mL.

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

[0041] Embodiment 29. The reaction mixture according to any one of Embodiments 25 to 27, wherein the concentration of the 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 according to any one of Embodiments 25 to 29, wherein the at least one polymerase is a DNA polymerase.

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

[0044] Embodiment 32. The reaction mixture according to Embodiment 31, wherein the reverse transcriptase is Moloney's mouse leukemia virus (M-MLV) mutant reverse transcriptase.

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

[0046] Embodiment 34. The reaction mixture according to 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 according to any one of Embodiments 25 to 34, further comprising nucleotide triphosphates suitable for nucleic acid amplification.

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

[0049] Embodiment 37. The reaction mixture according to 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 according to Embodiment 36, wherein the EDTA is present at a concentration of approximately 0.08 mM.

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

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

[0053] Embodiment 41. The reaction mixture according to 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 according to Embodiment 41, wherein the trehalose is present at a concentration of about 0.15 M.

[0055] Embodiment 43. The reaction mixture according to 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. At least two amplifications configured to amplify the target region of a target nucleic acid. The reaction mixture according to Embodiment 43, comprising an oligomer.

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

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

[0059] Embodiment 47. The reaction mixture according to 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 according to any one of Embodiments 25 to 47, further comprising at least one cofactor.

[0061] Embodiment 49. The reaction mixture according to 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 according to any one of Embodiments 43 to 49, further comprising the target nucleic acid.

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

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

[0065] Embodiment 53. The reaction mixture according to Embodiment 52, wherein at least one of the cleaning agents is an anionic cleaning agent.

[0066] Embodiment 54. The reaction mixture according to 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, (a) To provide an aqueous polymerase preparation 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. Methods that include...

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

[0069] Embodiment 57. The method according to 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 according to Embodiment 57, wherein the at least one detection probe oligomer includes chemiluminescence labeling or fluorescent labeling.

[0071] Embodiment 59. The at least one detection probe oligomer is fluorescently labeled and non-fluorescent. The method according to embodiment 57, including an optical quencher.

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

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

[0074] Embodiment 62. The method according to 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 according to 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 to contain the target nucleic acid.

[0076] Embodiment 64. A method for preparing an amplification reaction mixture, (a) To provide a lyophilized polymerase preparation according to Embodiment 20 or 21, (b) Dissolving the lyophilized polymerase preparation in a diluent to provide a reconstituted preparation, (c) Mixing the reconstituted formulation with a second formulation comprising at least one amplified oligomer configured to amplify a target region of a target nucleic acid, Methods that include...

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

[0078] Embodiment 66. The method according to 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 according to Embodiment 66, wherein the at least one detection probe oligomer includes chemiluminescence labeling or fluorescent labeling.

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

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

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

[0083] Embodiment 71. The method according to 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 according to 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 to contain the target nucleic acid.

[0085] Embodiment 73. The sample is an extraction sample containing at least one cleaning agent. The method described in Forms 63-72.

[0086] Embodiment 74. The method according to Embodiment 73, wherein the at least one cleaning agent is an anionic cleaning agent.

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

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

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

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

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

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

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

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

[0095] Embodiment 83. A method for carrying out an amplification reaction, (a) To provide a sample containing or suspected of containing a target nucleic acid, (b) The sample is At least one polymerase, α-cyclodextrin and Polysorbate 20 and At least one cushioning agent, Nucleotide triphosphates suitable for nucleic acid amplification, At least one cofactor, Contacting an aqueous mixture containing at least one amplification oligomer configured to amplify a target region of the target nucleic acid, By contacting the aforementioned sample with the aqueous mixture, it is determined that the α-cyclodextrin is present at a concentration of approximately 5 mg / mL to approximately 20 mg / mL, and the polysorbate 20 is present at a concentration of approximately 0.001% to approximately 0.025% (v / v). (c) Carrying out an in vitro nucleic acid amplification reaction using the reaction mixture, wherein, if the target nucleic acid is present in the sample, it is used as a template for generating one or more amplicons corresponding to the target region. Methods that include...

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

[0097] Embodiment 85. The method according to 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 according to 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 according to 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 according to any one of Embodiments 83 to 87, wherein the aqueous mixture comprises at least two amplification oligomers configured to amplify the target region of the target nucleic acid.

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

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

[0103] Embodiment 91. The method according to Embodiment 90, wherein the reverse transcriptase is Moloney mouse leukemia virus (M-MLV) mutant reverse transcriptase.

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

[0105] Embodiment 93. The method according to 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 according to any one of Embodiments 83 to 93, wherein the aqueous mixture further comprises EDTA.

[0107] Embodiment 95. The method according to 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 according to Embodiment 94, wherein the EDTA is present in the reaction mixture at a concentration of about 0.08 mM.

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

[0110] Embodiment 98. The method according to Embodiment 97, wherein the cryoprotectant is trehalose.

[0111] Embodiment 99. The method according to 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 according to Embodiment 99, wherein the trehalose is present in the reaction mixture at a concentration of about 0.15 M.

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

[0114] Embodiment 102. The method according to Embodiment 101, wherein the detection step includes contacting the in vitro nucleic acid amplification reaction product with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained in the one or more amplicons.

[0115] Embodiment 103. The method according to Embodiment 102, wherein the at least one detection probe oligomer includes chemiluminescence labeling or fluorescent labeling.

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

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

[0118] Embodiment 106. The method according to any one of Embodiments 83 to 105, wherein the sample is an extraction sample containing at least one cleaning agent.

[0119] Embodiment 107. The method according to Embodiment 106, wherein the at least one cleaning agent is an anionic cleaning agent.

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

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

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

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

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

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

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

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

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

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

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

[0131] These and other aspects of the present invention will become apparent by referring to the following detailed description of the invention.

[0132] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art relating to the methods and compositions described herein. Where used herein, the following terms and phrases have the same meanings unless otherwise specified.

[0133] The terms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise.

[0134] "Sample" includes any specimen that may contain target nucleic acids. "Sample" includes "biological specimens" that contain any tissue or material derived from a living or dead human. Biological specimens 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, washing agents, etc., used to prepare the biological specimen for analysis. Furthermore, "Sample" may include treated specimens, such as specimens in which one or more components have been concentrated or purified. Treated specimens include those obtained, for example, by passing the specimen over or through a filter, after centrifugation, or by adhesion to a culture medium, matrix, or support.

[0135] A "cleaning agent" refers to a substance that can disperse hydrophobic substances (e.g., lipids) in water through emulsification and can be used to dissolve or solubilize biological samples for subsequent analysis. Cleaning agents can be ionic or nonionic.

[0136] As used herein, the term “freeze protectant” refers to molecules that prevent or reduce the chemical and / or physical instability of proteins or other substances during freeze-drying and subsequent storage. Examples of freeze protectants 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 trivalent or higher sugar alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol, propylene glycol, polyethylene glycol, etc.; pluronic acid; and combinations thereof. In some embodiments, the freeze protectant is a non-reducing sugar such as trehalose or sucrose. As used herein in the context of aqueous formulations or reaction mixtures containing α-cyclodextrin and polysorbate 20, the term “freeze protectant” refers to molecules other than α-cyclodextrin, although α-cyclodextrin may still act as a secondary freeze protectant.

[0137] As used herein, the term “polymerase” means any enzyme capable of catalyzing the polymerization of nucleotides (including their analogues) into nucleic acid chains. Typically, such nucleotide polymerization occurs in a template-dependent manner. Examples of such polymerases include, for example, naturally occurring polymerases and any of their subunits and cleavage forms, mutant polymerases, variant polymerases, recombinant, fused or otherwise manipulated polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any of their analogues, homologs, derivatives or fragments that retain the ability to catalyze such polymerization. Where necessary, a polymerase may be a mutant polymerase, including 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 a polymerase, or the linking of two or more polymerase segments, including the linking of two or more segments from polymerases of different species or families. Exemplary polymerases include DNA polymerases, e.g., Taq polymerase or reverse transcriptase. In other variations, the polymerase is RNA polymerase.

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

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

[0140] "Nucleic acid" and "polynucleotide" together refer to multimeric compounds containing nucleotides and / or nucleotide analogs that are linked together to form biopolymers. Biopolymers include nucleotide analogs containing conventional RNA, conventional DNA, mixed RNA-DNA, and their versions. The nucleic acid "backbone" may consist of a variety of linkages, including one or more sugar-phosphodiester links, peptide-nucleic acid links ("peptide nucleic acid" or PNA), phosphorothioate links, methylphosphonate links, or combinations thereof. The sugar portion of the nucleic acid may be ribose, deoxyribose, or similar compounds with substitutions, for example, analogs having a methoxy, fluoro, or halide group at the 2' position of ribose (also referred herein as "2'-O-Me" or "2'-methoxy" or 2'-fluoro or "2'-halide"). Nitrogen bases can be conventional bases, adenine (A), uracil (U), guanine (G), thymine (T), and cytosine (C), as well as their analogues (e.g., inosine, 5-methyl 2'-deoxyxocytosine ("5-Me-dC" or "5MeC"), and isoguanine). Nucleic acids may have a backbone containing one or more "debased" residues that do not contain a nitrogen base at the polymer site(s).

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

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

[0143] As used herein, the terms “target sequence” or “target nucleic acid sequence” refer to a specific nucleotide sequence of the target nucleic acid to be amplified and / or detected. The “target sequence” includes the complex-forming sequence into which oligonucleotides (e.g., priming oligonucleotides and / or promoter oligonucleotides) complex during the amplification process (e.g., PCR, TMA). Unless the context explicitly 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 (+) and antisense (-) strands.

[0144] "Nucleic acid amplification" refers to any well-known in vitro procedure that generates 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) (e.g., U.S. Patents 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. Patent 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Patents 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. Patent 5,422,252).

[0145] An "amplicon" or "amplification product" refers to a nucleic acid molecule produced in a nucleic acid amplification reaction and derived from the target nucleic acid. The amplicon or amplification product contains a target nucleic acid sequence that may be identical or opposite in sense to the target nucleic acid.

[0146] An "amplifying oligonucleotide" or "amplifying oligomer" is an oligonucleotide that hybridizes to a target nucleic acid and participates in nucleic acid amplification reactions, for example, acting as a primer. An amplifying oligomer may have a 3' end that is extended by polymerization as part of the nucleic acid amplification reaction. Alternatively, an amplifying oligomer may have a 3' end that is not extended by polymerization but provides a component that promotes nucleic acid amplification, such as a promoter sequence 5'-bound to the target-specific sequence of the amplifying oligomer. Such an amplifying oligomer is called a promoter provider. An amplifying oligomer that provides both a 3' target-specific sequence that can be extended by polymerization and a 5' promoter sequence is called a promoter primer. An amplifying oligomer may be modified as needed to include a 5' non-target-specific sequence, such as a tag, a promoter (as mentioned), or other sequences used or useful to manipulate or amplify the primer or target oligonucleotide.

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

[0148] "Label" or "detectable label" refers to a moiety or compound directly or indirectly bound to a probe that is detected or gives a detectable signal. Direct binding may be by covalent or non-covalent interactions (e.g., hydrogen bonds, hydrophobic or ionic interactions, and the formation of chelates or coordination complexes), while indirect binding may be by a bridging moiety or linker (e.g., via an antibody or additional oligonucleotide(s) that amplifies the detectable signal). Any detectable moiety can be radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles that impart a detectable color (e.g., latex or metal beads), 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 quencher molecules that absorb light when in proximity to the fluorophore, thereby reducing background fluorescence. Examples of detectably labeled probes include hydrolysis (e.g., TaqMan®) probes, AE-labeled probes, molecular torches, and molecular beacons.

[0149] The term "configured" refers to the actual arrangement of the polynucleotide sequence configuration of the referenced oligonucleotide target hybridization sequence. For example, an amplification oligomer configured to produce a specific amplicon from a target sequence has a polynucleotide sequence that hybridizes to the target sequence and can be used in the amplification reaction to produce 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 appropriate concentrations described herein.

[0151] The term "container" refers to anything that can hold or contain an object or liquid, for example, for storage (e.g., a holder, receptacle, container, etc.).

[0152] References to numerical ranges in this specification (e.g., "X to Y" or "X to Y") include the endpoint defining the range and all values ​​that fall within that range.

[0153] Unless otherwise stated in the context, when a value is expressed as "approximately" X or "about" X, the stated value of X is understood to be accurate to within ±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 enabling a larger 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 second cryoprotectant (e.g., in situations where increasing the concentration of the primary cryoprotectant results in incomplete reconstruction of the lyophilized product).

[0155] In certain embodiments, the polymerase formulation is an aqueous formulation. Such formulations may be, for example, pre-lyophilized formulations or formulations reconstituted from lyophilized forms. 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 buffer. In some embodiments, α-cyclodextrin is present in concentrations of approximately 10 mg / mL to 35 mg / mL, approximately 10 mg / mL to 30 mg / mL, approximately 10 mg / mL to 25 mg / mL, approximately 10 mg / mL to 20 mg / mL, approximately 10 mg / mL to 15 mg / mL, approximately 12 mg / mL to 40 mg / mL, approximately 12 mg / mL to 35 mg / mL, approximately 12 mg / mL to 30 mg / mL, approximately 12 mg / mL to 25 mg / mL, and approximately 12 mg / mL. It is present at concentrations of approximately 20 mg / mL, 12 mg / mL to 15 mg / mL, 16 mg / mL to 40 mg / mL, 16 mg / mL to 35 mg / mL, 16 mg / mL to 30 mg / mL, 16 mg / mL to 25 mg / mL, or 15 mg / mL to 20 mg / mL. In more specific variations, α-cyclodextrin is present at concentrations of approximately 20 mg / mL, 17.5 mg / mL, or 12.5 mg / mL. In some embodiments, polysorbate 20 is present at concentrations of approximately 0.002% to approximately 0.05% (v / v), approximately 0.002% to approximately 0.04% (v / v), approximately 0.002% to approximately 0.03% (v / v), approximately 0.003% to approximately 0.05% (v / v), approximately 0.003% to approximately 0.04% (v / v), or approximately 0.003% to approximately 0.03% (v / v), and in more specific modifications, polysorbate 20 is present at concentrations of approximately 0.0042% (v / v), approximately 0.0035% (v / v), approximately 0.0026% (v / v), or approximately 0.02% (v / v).

[0156] Polymerases suitable for use according to the present invention include DNA-dependent DNA polymerases, RNA-dependent DNA polymerases (reverse transcriptases), RNA polymerases, and enzymes having two or more polymerase activities, the enzymes of which may be thermally unstable or thermally stable. A mixture of two or more enzymes may also be used. Exemplary polymerases include DNA-dependent DNA polymerases, such as DNA polymerase I ("Pol I"), Klenow fragments of Pol I, T4, and 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 Escherichia coli (E. coli), SP6, T3, and T7 RNA polymerases; and reverse transcriptases, such as avian myeloblastosis virus (AMV), Moloney's mouse 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 reverse transcriptase (e.g., MMLV mutant reverse transcriptase) and DNA-dependent DNA polymerase (e.g., Taq DNA polymerase).

[0157] The buffer is typically present at a concentration sufficient to maintain a pH suitable for the use of polymerase in amplification assays. In some embodiments, the buffer is present at a concentration sufficient to maintain a pH in the range of about 6.0–about 9.0, about 6.5–about 8.5, about 6.5–about 8.0, or about 6.5–about 7.5. Suitable buffers include Tris(2-amino-2-(hydroxymethyl)-1,3-propanediol), PIPES(piperazine-N,N'-bis(2-ethanesulfonic acid)), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphates, citrates, succinates, and histidines. In certain embodiments, the Tris buffer is present at a concentration of about 10 mM–about 100 mM or about 20 mM–about 100 mM. Other suitable concentrations of buffers for formulations according to the present invention can be readily determined by those skilled in the art.

[0158] The polymerase formulations described above may further contain 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; as well as / or ATP, CTP, GTP, and UTP) and / or chelating. Suitable chelating agents include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In some embodiments containing 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 modifications, formulations (such as those suitable for lyophilization, those reconstituted from lyophilized forms, or lyophilized formulations for reconstitution into aqueous formulations described herein) may contain cryoprotectants. Exemplary cryoprotectants include non-reducing sugars, e.g., sucrose, raffinose, or trehalose; glycerol; and amino acids, e.g., glycine, arginine, or methionine. The use of cryoprotectants, including the selection of appropriate concentrations to prevent unacceptable degradation and / or aggregation of carrier molecules during lyophilization, is well known in the art. A particularly suitable cryoprotectant is trehalose, which may be present in aqueous formulations at concentrations, for example, about 0.2 M to about 0.35 M or about 0.2 M to about 0.4 M. In some modifications containing trehalose as a cryoprotectant, trehalose is present at a concentration of about 0.26 M or about 0.3 M.

[0160] In some embodiments, the polymerase preparations 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 modifications, the formulation is stable over a long period. For example, the formulation may be stable for at least about two weeks, at least about one month, at least about two months, at least about three months, or at least about six 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 may be stored at temperatures of approximately -80°C to approximately 40°C, approximately -20°C to approximately 25°C, approximately 0°C to approximately 25°C, approximately 0°C to approximately 15°C, approximately 0°C to approximately 10°C, or approximately 2°C to approximately 8°C. In various embodiments, the formulations may be stored at approximately 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 their activity within these ranges. In some modifications, the formulations are stable at approximately -80°C to approximately 25°C or approximately 4°C to approximately 25°C. In more specific modifications, liquid formulations are stable at temperatures of approximately -80°C to approximately -20°C, approximately -80°C to approximately 4°C, or approximately -80°C to approximately 25°C. In other specific modifications, lyophilized formulations are stable at temperatures of approximately 4°C to approximately 25°C or approximately 4°C to approximately 40°C. The intermediate temperature ranges mentioned above, for example, from about 2°C to about 18°C, are also intended to be part of the present invention. For example, this range is intended to include values ​​using any combination of the values ​​listed above as upper and / or lower limits.

[0163] In certain embodiments, for long-term storage, the aqueous formulations described herein may be dispensed, for example, into 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 modifications, the seal allows a diluent to be later injected into the container through the seal. Methods for preparing such lyophilized polymerase formulations from aqueous formulations, as well as the lyophilized formulations prepared by such methods, are additional embodiments of the present invention. In yet another embodiment, the present invention relates to the aqueous far-red dye probe formulations described herein. The present invention provides a stabilized lyophilized polymerase formulation that enables the reconstitution of [the substance].

[0164] Methods for preparing aqueous polymerase formulations from lyophilized formulations described herein are also included in the present invention. 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. A suitable diluent can be readily selected by those skilled in the art and may include, for example, water or an aqueous solution containing a buffer (e.g., Tris).

[0165] In certain embodiments of the present invention, a container comprising the lyophilized polymerase formulation described herein is provided in a kit having a second container containing one or more other components, such as a diluent. The polymerase formulation may be packaged in various different embodiments, and those skilled in the art will understand that the present invention encompasses many different kit configurations. For example, a kit may further include a container containing one or more amplification oligomers for amplifying a target region, and optionally have 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 suitable nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, UTP). In certain embodiments, the kit further includes a set of instructions for carrying out the method according to the present invention, the instructions may be associated with the accompanying documentation and / or packaging of the kit or its components.

[0166] In other embodiments, 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. The reaction mixtures of the present disclosure typically 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 embodiments, α-cyclodextrin is present at concentrations of approximately 5 mg / mL to 17.5 mg / mL, approximately 5 mg / mL to 15 mg / mL, approximately 5 mg / mL to 12.5 mg / mL, approximately 8 mg / mL to 20 mg / mL, approximately 8 mg / mL to 17.5 mg / mL, approximately 8 mg / mL to 15 mg / mL, or approximately 8 mg / mL to 12.5 mg / mL, and in more specific modifications, α-cyclodextrin is present at a concentration of approximately 10 mg / mL. In some embodiments, polysorbate 20 is present at concentrations 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 concentrations of about 0.002% (v / v), about 0.0021% (v / v), or about 0.01% (v / v).

[0167] Polymerases suitable for use in the reaction mixture include those suitable for the polymerase formulations described above. For example, the reaction mixture may contain at least two DNA polymerases, such as reverse transcriptase (e.g., MMLV mutant reverse transcriptase) and 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 the use of polymerase in amplification assays. In some embodiments, the buffer is present at a concentration sufficient to maintain a pH in the range of about 6.0–about 9.0, about 6.5–about 8.5, about 6.5–about 8.0, or about 6.5–about 7.5. Suitable buffers include buffers suitable for polymerase formulations as described above. In certain embodiments, the Tris buffer is present at a concentration of about 5 mM–about 50 mM or about 10 mM–about 50 mM. Other suitable concentrations of buffer for reaction mixtures according to the present invention can be readily determined by those skilled in the art.

[0169] In certain embodiments, the amplification reaction mixture may contain a cryoprotectant (for example, 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, which 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 the reaction mixture containing trehalose, the trehalose is present at a concentration of about 0.15 M.

[0170] The reaction mixture described above may further include one or more additional components for performing an amplification assay. For example, the reaction mixture may further include at least one amplification oligomer configured to amplify a target region of a target nucleic acid. In some such embodiments, the reaction mixture includes 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 the amplification assay. In other non-mutually exclusive modifications, 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 an amplification reaction mixture typically include detectable labels such as chemiluminescent compounds or fluorophores. 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 amplified product in real time, for example, 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 those containing fluorescently labeled and non-fluorescent quenchers (e.g., hydrolysis probes, molecular torches, or molecular beacons).

[0171] In other non-mutually exclusive embodiments, the reaction mixture comprises nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP; as well as / or ATP, CTP, GTP, and UTP), a chelating agent (e.g., EDTA or EGTA), an inorganic salt (e.g., KCl), and one or more additional suitable amplification assay components selected from cofactors. In some variations, the chelating agent is EDTA, which may be present at a concentration of, for example, 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 at a concentration of, for example, 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 to contain a target nucleic acid. For example, the reaction mixture described above, comprising at least one amplified oligomer, may further comprise a target nucleic acid targeted by at least one amplified oligomer, or a sample suspected to contain a 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 may be derived from a pathogen such as a virus, bacteria, protozoa, fungi, or other microorganisms that cause disease in humans or animals.

[0173] In some embodiments, the reaction mixture 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 to contain a target nucleic acid, and the sample contains one or more residual detergents used for sample extraction. In certain modifications, the detergent is an anionic detergent such as dodecyl sulfate (e.g., sodium dodecyl sulfate, potassium dodecyl sulfate, or lithium dodecyl sulfate (also known 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 modifications, the sample containing at least one detergent, It constitutes approximately 40% to 50%, 50% to 70%, 20% to 33%, 42.9%, or 62.5% by volume of the reaction mixture.

[0174] In other embodiments, the present invention provides a method for preparing an amplification reaction mixture. In certain embodiments, the method for preparing the reaction mixture comprises (a) providing the aqueous polymerase formulation described above, and (b) mixing 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 the reaction mixture comprises (a) providing the above lyophilized polymerase formulation, (b) dissolving the lyophilized polymerase formulation in a diluent to provide a reconstituted formulation, and (c) mixing 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 above polymerase formulations or reaction mixtures. In certain modifications, the above method further comprises mixing the aqueous or reconstituted formulation with one or more additional amplification assay components. Such additional components(s) may be contained in a second formulation or in one or more separate formulations mixed with an 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 a target region (e.g., a probe including a label such as a chemiluminescent label or a fluorescent label, or a probe including 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 chelating agent (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 is a formulation further comprising the nucleotide triphosphates and / or chelating agents described herein, wherein one or more additional components mixed with the aqueous or reconstituted formulation comprise (i) at least one detection probe oligomer configured to specifically hybridize to a sequenced target contained within a target region, and / or (ii) at least one cofactor.

[0175] In some embodiments, a method for preparing a reaction mixture further comprises mixing a sample containing or suspected to contain a target nucleic acid with an aqueous or reconstituted formulation, or the above mixture containing an aqueous or reconstituted formulation. In some such embodiments, the sample is an extracted sample obtained by treating a biological sample with one or more detergents, thereby the extracted sample contains one or more detergents as residual components. The residual detergents may be 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 may include the concentrations described herein for the amplified reaction mixture. For example, α-cyclodextrin may be mixed to a concentration of about 5 mg / mL to about 20 mg / mL, polysorbate 20 to a concentration of about 0.001% to about 0.025% (v / v), Tris, if present, to a concentration of about 5 mM to about 50 mM, EDTA, if present, to a concentration of about 0.025 mM to about 0.25 mM, trehalose, if present, to a concentration of about 0.1 M to about 0.2 M, and / or magnesium chloride, if present, to a concentration of about 1 mM to about 5 mM. It is acceptable to mix them.

[0177] In another embodiment, the present invention provides a method for carrying out an amplification reaction. In certain embodiments, the method comprises (a) providing a sample containing or suspected to contain a target nucleic acid, (b) contacting the sample with an aqueous mixture comprising at least one polymerase, α-cyclodextrin, polysorbate 20, at least one buffer (e.g., Tris), nucleotide triphosphates suitable for 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 α-cyclodextrin is present at a concentration of about 5 mg / mL to about 20 mg / mL and polysorbate 20 is present at a concentration of about 0.001% to about 0.025% (v / v), and (c) using the reaction mixture in The method comprises carrying out a vitro nucleic acid amplification reaction using a 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 a target region. Suitable polymerases include those suitable for the polymerase formulation or reaction mixture described above. In certain modifications, the method described above further comprises contacting the sample with one or more additional assay components, e.g., at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within 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 cryoprotective agent (e.g., used in a lyophilized formulation from which the aqueous formulation is reconstituted), e.g., trehalose. The final amplification assay concentrations of the mixture components may include the concentrations described herein for the amplification reaction mixture.Amplification of target regions of target nucleic acids can be achieved using a variety of known nucleic acid amplification reactions, including, for example, transcription-related 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).

[0178] In some embodiments of the method for carrying out the amplification reaction as described above, the sample is an extracted sample obtained by treating a biological sample with one or more detergents, thereby the extracted sample contains one or more detergents as residual components. The residual detergents may be anionic detergents such as dodecyl sulfates (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, the method for carrying out the amplification reaction described above further includes a step of purifying the target nucleic acid from other components in the sample before amplification. Such purification may include a method of separating and / or concentrating organisms contained in the sample from other sample components, or a method of removing or degrading non-nucleic acid sample components, such as proteins, carbohydrates, salts, lipids, etc. In certain embodiments, the target nucleic acid is captured specifically or nonspecifically 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 a target sequence of the target nucleic acid under hybridization conditions. In embodiments including a capture probe tail, the target:capture-probe complex is captured by using hybridization conditions in which the capture probe tail hybridizes to an immobilized probe. In certain embodiments, particulate solid supports, such as paramagnetic beads, are used. The following methods are used. Selective and nonspecific target capture methods are also described, for example, in U.S. Patent No. 6,110,678 and International Patent Application Publication No. 2008 / 016988 (which are incorporated herein by reference, respectively). In some embodiments, the purification step (e.g., a purification step including specific or nonspecific target capture) utilizes one or more detergents, such as 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 the various known techniques for detecting signals specifically associated with the amplified target sequence, for example, by hybridizing the amplified product with a labeled detection probe and detecting the signal resulting from the labeled probe (in some embodiments, including a label released from the probe after hybridization). In some embodiments, the labeled probe includes a second part, such as a quencher or other part, that interacts with the first label. Detection may be performed after the completion of the amplification reaction, or concurrently with the amplification of the target region, for example, in real time. In certain modifications 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 part that is detected when the probe binds to the amplified product (e.g., a double-labeled hairpin probe containing both a fluorescent label and a quenching part). In other embodiments for real-time detection, the detection probe may be a linear oligomer, such as an oligomer labeled with both a fluorophore and a quenching part (e.g., a TaqMan probe). Such probes may include target hybridizing sequences and non-target hybridizing sequences. Various forms of such probes have been previously described (see, for example, U.S. Patents Nos. 5,210,015, 5,487,972; 5,118,801; 5,312,728; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; as well as U.S. Patent Application Publications 20060068417A1 and 20060194240A1, respectively; which are incorporated herein by reference). [Examples]

[0181] The following embodiments are provided to illustrate specific disclosed embodiments and should not be construed as limiting the scope of this disclosure.

[0182] Example 1 Several PCR reaction mixtures were prepared for testing with samples containing a washing agent. An initial master mix was prepared containing 0.46 U / μL of DNA polymerase, 0.5 U / μL of reverse transcriptase, 0.2 U / μL of an RNase inhibitor, inorganic salts including 0.25 mM dNTP, 0.05 mM dUTP, 81 mM and 5.1 mM KCl and MgCl2, respectively, 0.1 mM EDTA, and several primers and probes (SEQ ID NOs. 8-14, 0.64 μM-1.29 μM) for amplification and detection of influenza A target nucleic acids. A second master mix was prepared containing 1.0 U / μL of DNA polymerase, 3.2 U / μL of reverse transcriptase, 0.53 mM of dNTPs, 1.06 mM of dUTPs, inorganic salts including 173 mM and 10.93 mM of KCl and MgCl2, 0.21 mM of 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 acids. The initial master mix was divided into conditions (A) to (F). Conditions (B) and (D) further contained 0.025% (v / v) polysorbate 20, conditions (B), (C), and (F) further contained 12.5 mg / mL of α-cyclodextrin, condition (C) further contained 0.13% (v / v) polysorbate 20, and condition (E) further contained 50 mg / mL of 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 appropriate media, such as Micro Test M4 medium (Remel Inc., catalog no. R12500), Micro Test M5 virus transport medium (Remel, Inc., catalog no. R12515), Micro Test M6 virus transport medium (Remel, Inc., catalog no. R12530), Micro Test M4RT virus transport medium (Remel, Inc., catalog no. R12505), or Copan Universal transport medium (Copan Diagnostics, Inc., catalog no. 330C). The target nucleic acid was the RNA transcript of SEQ ID NO: 7. 360 μL of the prepared sample was incubated separately in a buffer containing 100 μg of poly-T (SEQ ID NO: 2) coated magnetic microparticles and 20 picomoles of target capture oligomer (SEQ ID NO: 1) in a final reaction volume of 936 μL to conjugate the target nucleic acid to a magnetic solid support. 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 the wash buffer. The resuspended particles were subjected to another separation, supernatant removal, and resuspending in the wash buffer. After the second separation and removal of the wash buffer, the particles were incubated in 50 μL of elution buffer (5 mM Tris in water containing a preservative). The magnetic particles were separated by applying a magnetic field, and the eluate containing nucleic acids was recovered. The eluate was divided into separate containers, and 30% (v / v) wash buffer was further spiked into one of the eluate portions to produce an eluate containing 100 mg / mL sodium dodecyl sulfate, simulating carryover of wash buffer from the upstream sample processing step. Washing agents present in the wash buffer are known to inhibit or reduce nucleic acid amplification reactions.

[0184] Each of the above master mix conditions (A) to (F) was added to the wells of a multiwell plate, and combined with aliquots of eluate spiked with wash buffer and eluate without wash buffer (20 μL master mix, 10 μL eluate, 30 μL total reaction volume). Each condition was prepared with 20 copies, except for condition (E), which was prepared with 19 copies. Real-time amplification and detection reactions were set up for each condition with 20 (19) copies, and the reactions were carried out using a thermal cycling instrument (Panther Fusion Instrument, Hologic, Inc., San Diego, California). 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 effect of the washing buffer detergent on the nucleic acid amplification reaction. These data also show that the combination of these two reagents had a synergistic mitigating effect against washing buffer-induced inhibition compared to the mitigating effect observed using either α-cyclodextrin alone or polysorbate 20 alone.

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

[0187] Seasonal coronavirus type OC43 (Zeptometrix catalog number 0810024CF) was spiked into whole blood sample matrix or sample transport medium (STM) and 0.125 TCID was obtained for each sample type. 50 The final viral titer was obtained at 1 / mL. 400 μL of whole blood matrix or STM matrix was separately incubated in a buffer reagent containing 100 μg of poly-T (SEQ ID NO: 2) coated magnetic microparticles and 20 picomoles of target capture oligomer (SEQ ID NO: 3) at a final reaction volume of 976 μL to conjugate the target nucleic acid to a magnetic solid support. The nucleic acid conjugated to the magnetic microparticles was 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) washing buffer to produce an eluate containing 100 mg / mL of sodium dodecyl sulfate, simulating carryover of washing buffer from the upstream sample processing step. Washing agents present in the washing buffer are known to inhibit or reduce the nucleic acid amplification reaction.

[0188] Each of the above master mix conditions (G1) to (G4) was added to the wells of a multiwell plate and combined with either an aliquot of the wash buffer spike eluate or an aliquot of the wash buffer unspiked eluate (except for condition (G4), which consisted of 20 μL master mix, 5 μL eluate, and 25 μL total volume; the other conditions consisted of 15 μL master mix, 25 μL eluate, and 40 μL total reaction volume). Each condition was prepared in 24 copies, except for condition (G1) which included unspiked eluate and was prepared in 20 copies. The RT-PCT reaction was performed in a thermal cycle machine. The procedure was performed using a Panther Fusion Instrument (Hologic, Inc., San Diego, California). Condition (G4) was prepared to have 5.3 times less target nucleic acid than when prepared using 15 μL of master mix and 25 μL of eluate. Therefore, under the same reaction conditions, (G4) has a higher Ct than when prepared using the alternative reaction conditions (theoretically 2.4 Ct). 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, and 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, and 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, and 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, and RFU improvements of +57%, +44%, and +46% for conditions (G1)d, (G2)d, and (G3)d, respectively. These results demonstrate a substantial improvement in Ct when samples containing the inhibitor and treated with various concentrations of α-cyclodextrin and polysorbate 20 are compared to samples containing the inhibitor without such treatment.

[0190] Example 3 Seasonal coronavirus type OC43 (Zeptometrix catalog number 0810024CF) was spiked into several plasma and serum samples, resulting in 70 TCID in a total sample volume of 1.15 mL. 50 The final OC43 virus titer was obtained at 0 / 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 a target capture oligomer (SEQ ID NO: 3). Each sample was shaken in an orbital shaker (approximately 200°F). The mixture was mixed at rpm (1 min). The sample was 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. Magnetic particles were separated by applying 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 inhibitors. One of the RT-PCR reaction mixtures further contained 12.5 mg / mL of polysorbate 20 and 0.0026% (v / v) of α-cyclodextrin. Four conditions were prepared by combining 20 μL of RT-PCR reaction with 5 μL of eluate from each sample type: (A) plasma sample eluate containing RT-PCR reaction containing polysorbate 20 and α-cyclodextrin, (B) plasma sample eluate containing RT-PCR reaction - neat, (C) serum sample eluate containing RT-PCR reaction containing polysorbate 20 and α-cyclodextrin, and (D) serum sample eluate containing RT-PCR reaction - neat. The RT-PCR reaction was prepared in 9 to 11 replications (see Table 3), and an internal control was spiked. The RT-PCR reaction was 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, the samples produced RT-PCR results with higher Ct, lower RFU, and greater inter-sample variability than those produced with reactants containing reagents that mitigate the effects of LLS. This indicates that the addition of α-cyclodextrin and polysorbate 20 significantly improved RNA detection by more than 1 log (more than 4 Ct lower with the additives).

[0192] Example 4 A series of aqueous mixtures containing α-cyclodextrin and polysorbate 20 were prepared. These mixtures were either prepared using a constant concentration of polysorbate 20 and combined with several concentrations of α-cyclodextrin, or prepared using a constant concentration of α-cyclodextrin and combined with several different concentrations of polysorbate 20 (Table 4). Each combination in mixtures 1-7 was effective in mitigating the inhibitory effect of the washing agent in the nucleic acid amplification reaction mixture. Each aqueous mixture was incubated on ice for 1 hour, and then the mixtures were centrifuged to assess the presence of a precipitant. After 1 hour of incubation on ice, varying levels of precipitate were observed in mixtures 1-6. [Table 4]

[0193] Several dry RT-PCR reaction mixtures were prepared to contain various concentrations of α-cyclodextrin and polysorbate 20. An RT-PCR master mixture was prepared to contain 1.0 U / μL of DNA polymerase, 3.2 U / μL of reverse transcriptase, 0.4 M trehalose, 0.53 mM dNTP, 1.06 mM dUTP, 0.21 mM EDTA, 1.6 μM each of SEQ ID NOs. 4 and 5, and 1.07 μM of SEQ ID NO. 6. This master mix was then divided into separate conditions: condition (1) contained 16 mg / mL of α-cyclodextrin and 0.0034% (v / v) polysorbate 20; condition (2) contained 20 mg / mL of α-cyclodextrin and 0.02% (v / v) polysorbate 20; and condition (3) contained neither α-cyclodextrin nor polysorbate 20. Next, each of these conditions was aliquoted at 24 μL into several reaction wells for each condition, and the aliquots were dried using freeze-drying techniques to form single-unit dose freeze-dried 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) for each sample type, resulting in 14.2 TCID 50 The final viral titer was obtained at 1 / mL. 400 μL of whole blood matrix, or 200 μL of plasma or serum matrix, or 360 μL of STM matrix were separately incubated in a buffer containing 100 μg of poly-T (SEQ ID NO: 2) coated magnetic microparticles and 20 picomoles of target capture oligomer (SEQ ID NO: 3) at a final reaction volume of 936 μL to conjugate the target nucleic acid to a magnetic solid support. The nucleic acid conjugated to the magnetic microparticles was 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) washing buffer to produce an eluate containing 100 mg / mL of sodium dodecyl sulfate, simulating the carryover of washing buffer from the upstream sample processing step.

[0195] The freeze-dried pellets under conditions (1) to (3) were rehydrated with 25 μL of HEPES buffer containing KCl and MgCl2, respectively. 15 μL of the rehydrated reaction mixture was combined with 25 μL of eluates from conditions (1) and (2), and 20 μL of the rehydrated reaction mixture (3) was combined with 5 μL of eluate (all in replication 6). The RT-PCT reaction was performed using a thermal cycling instrument. The results are shown in Table 5. [Table 5]

[0196] These data show that reactions containing the combination of α-cyclodextrin and polysorbate 20 exhibit lower Ct values ​​and higher RFU values ​​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 dNTP, 0.7 mM dUTP, inorganic salts containing 81 and 5.1 mM KCl and MgCl2, respectively, 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 to BK virus (BKV) (SEQ ID NOs. 15-17; see Table 6) or primers and probes specific to Epstein-Barr virus (EBV) detection (SEQ ID NOs. 18-20; see Table 6).

[0198] The samples consisted of human whole blood diluted in a 1:4 ratio in blood transport medium, human urine diluted in a 1:2 ratio in urine transport medium, or undiluted human plasma. 400 μL of each of these samples was separately incubated in a 976 μL final reaction volume containing 270 μg of poly-T (SEQ ID NO: 2; 5'-(T)14-3') coated magnetic microparticles and 220 picomoles 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 applying a magnetic field, and the supernatant was removed from the [capture target]:[capture probe]:[magnetic microparticle] combination. The magnetic microparticles were then resuspended in washing buffer. The particles were subjected to a first separation, removal of the supernatant, and resuspension in a washing buffer. After the second separation and removal of the washing buffer, the microparticles were incubated in 50 μL of elution buffer (5 mM Tris in water containing a preservative). Magnetic particles were separated by applying a magnetic field, and the eluate containing nucleic acids was recovered.

[0199] Each of the above master mix conditions was combined in the wells of a multi-well 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 the reactions were carried out using a thermal cycling instrument (Panther Fusion Instrument, Hologic, Inc., San Diego, California).

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

[0201] From the foregoing, it will be understood that while specific embodiments of the present invention are described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of the invention. Therefore, the present invention is not limited except as provided for by the appended claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes. In certain embodiments, for example, the following are provided: (Item 1) A aqueous polymerase preparation, At least one polymerase, α-cyclodextrin at concentrations of approximately 10 mg / mL to approximately 40 mg / mL, Polysorbate 20 with a concentration of approximately 0.002% to approximately 0.05% (v / v), At least one cushioning agent, A water-based polymerase preparation containing [a specific ingredient / method]. (Item 2) The preparation described in item 1, wherein the α-cyclodextrin concentration is approximately 16 mg / mL to approximately 30 mg / mL, approximately 15 mg / mL to approximately 20 mg / mL, or approximately 10 mg / mL to approximately 15 mg / mL. (Item 3) The preparation described in item 1, wherein the α-cyclodextrin concentration is approximately 20 mg / mL, approximately 17.5 mg / mL, or approximately 12.5 mg / mL. (Item 4) The preparation according to any one of items 1 to 3, wherein the concentration of the polysorbate 20 is approximately 0.003% to approximately 0.03% (v / v). (Item 5) The formulation according to any one of items 1 to 3, wherein the polysorbate 20 concentration is approximately 0.0042% (v / v), approximately 0.0035% (v / v), approximately 0.0026% (v / v), or approximately 0.02% (v / v). (Item 5) Item 5a. The preparation according to any one of items 1 to 5, wherein the at least one polymerase is a DNA polymerase. (Item 6) A preparation according to any one of items 1 to 5a, further comprising nucleotide triphosphates suitable for nucleic acid amplification. (Item 7) A preparation according to any one of items 1 to 6, further comprising a cryoprotective agent. (Item 8) The formulation according to item 7, wherein the cryoprotective agent is trehalose. (Item 9) The preparation described in item 8, wherein the aforementioned trehalose is present at a concentration of approximately 0.2 M to approximately 0.4 M. (Item 10) The formulation according to item 9, wherein the trehalose is present at a concentration of approximately 0.26 M or approximately 0.3 M. (Item 11) A method for preparing a freeze-dried polymerase preparation, (a) To provide an aqueous formulation described in any one of items 7 to 10, (b) A method comprising freeze-drying the aqueous formulation to form the freeze-dried polymerase formulation. (Item 12) A lyophilized polymerase preparation prepared by the method described in item 11. (Item 13) A lyophilized polymerase preparation that enables reconstitution into an aqueous formulation as described in any one of items 7-10. (Item 14) A method for preparing an aqueous polymerase preparation, (a) To provide a lyophilized polymerase preparation as described in item 12 or 13, (b) Dissolve the lyophilized polymerase preparation in a diluent to provide a reconstituted preparation. Methods that include... (Item 15) It's a kit, A kit comprising a first sealed container containing a lyophilized polymerase preparation as described in item 12 or 13. (Item 16) The kit according to item 15, further comprising a second sealed container containing a diluent. (Item 17) A mixture of amplified reactions, At least one polymerase, Alpha-cyclodextrin at concentrations of approximately 5 mg / mL to approximately 20 mg / mL, Polysorbate 20 at a concentration of approximately 0.001% to approximately 0.025% (v / v), At least one cushioning agent, A mixture of amplified reaction components. (Item 18) The reaction mixture described in item 17, wherein the α-cyclodextrin concentration is approximately 8 mg / mL to approximately 15 mg / mL. (Item 19) The reaction mixture described in item 17, wherein the α-cyclodextrin concentration is approximately 10 mg / mL. (Item 20) The reaction mixture according to any one of items 17 to 19, wherein the concentration of the polysorbate 20 is approximately 0.0015% to approximately 0.015% (v / v). (Item 21) The reaction mixture according to any one of items 17 to 19, wherein the concentration of the polysorbate 20 is approximately 0.002% (v / v), approximately 0.0021% (v / v), or approximately 0.01% (v / v). (Item 22) The reaction mixture according to any one of items 17 to 21, wherein the at least one polymerase is a DNA polymerase. (Item 23) A reaction mixture according to any one of items 17 to 22, further comprising a cryoprotectant. (Item 24) The reaction mixture according to item 23, wherein the cryoprotectant is trehalose. (Item 25) The reaction mixture described in item 24, wherein the trehalose is present at a concentration of approximately 0.1 M to approximately 0.29 M. (Item 26) The reaction mixture described in item 25, wherein the trehalose is present at a concentration of approximately 0.15 M. (Item 27) A reaction mixture according to any one of items 17 to 26, further comprising nucleotide triphosphates suitable for nucleic acid amplification. (Item 28) A reaction mixture according to 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) The reaction mixture according to item 28, further comprising at least one detection probe oligomer configured to specifically hybridize to a target sequence contained within the target region. (Item 30) The reaction mixture according to item 28 or 29, further comprising the target nucleic acid. (Item 31) The reaction mixture according to item 30, further comprising at least one detergent. (Item 32) The reaction mixture according to item 31, wherein at least one of the detergents is an anionic detergent, and optionally the anionic detergent is sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS). (Item 33) A method for preparing an amplification reaction mixture, (a) To provide an aqueous polymerase preparation described in 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. Methods that include... (Item 34) The method according to item 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) The method according to item 33 or 34, further comprising mixing the mixture produced in step (b) with a sample containing or suspected to contain the target nucleic acid. (Item 36) A method for preparing an amplification reaction mixture, (a) To provide a lyophilized polymerase preparation as described in item 12 or 13, (b) Dissolving the lyophilized polymerase preparation in a diluent to provide a reconstituted preparation, (c) Mixing the reconstituted formulation with a second formulation comprising at least one amplified oligomer configured to amplify a target region of a target nucleic acid, Methods that include... (Item 37) The method according to item 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) 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 to contain the target nucleic acid. (Item 39) The method according to item 35 or 38, wherein the sample is an extract containing at least one cleaning agent. (Item 40) The method according to item 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) The method according to any one of items 35 and 38-40, wherein the sample containing or suspected to contain the target nucleic acid constitutes at least about 40% of the reaction mixture. (Item 42) A method for carrying out an amplification reaction, (a) To provide a sample containing target nucleic acid, (b) The sample is At least one polymerase, α-cyclodextrin and Polysorbate 20 and At least one cushioning agent, Nucleotide triphosphates suitable for nucleic acid amplification, At least one cofactor, Contacting an aqueous mixture containing at least one amplification oligomer configured to amplify a target region of the target nucleic acid, By contacting the aforementioned sample with the aqueous mixture, a reaction mixture is provided in which the α-cyclodextrin is present at a concentration of approximately 5 mg / mL to approximately 20 mg / mL and the polysorbate 20 is present at a concentration of approximately 0.001% to approximately 0.025% (v / v). (c) Carrying out an in vitro nucleic acid amplification reaction using the reaction mixture, wherein, if the target nucleic acid is present in the sample, it is used as a template for generating one or more amplicons corresponding to the target region. Methods that include... (Item 43) The method according to item 42, further comprising detecting one or more amplicons. (Item 44) The method according to item 43, wherein the detection step includes contacting the in vitro nucleic acid amplification reaction product with at least one detection probe oligomer configured to specifically hybridize to a target sequence contained in the one or more amplicons. (Item 45) The method according to item 43 or 44, wherein the detection step is performed in real time. (Item 46) The method according to any one of items 42 to 45, wherein the sample is an extract containing at least one cleaning agent. (Item 47) The method according to 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

[Claim 1] The invention described herein.

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