Dried composition containing flap endonuclease

Abstract

An aqueous solution composition is disclosed, comprising, consisting of, or consisting essentially of a flap endonuclease, a swelling agent, and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 5 mM or less, and wherein the composition is substantially free of glycerol.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of each of the following provisional applications: 62 / 508,975 filed May 19, 2017, 62 / 508,990 filed May 19, 2017, 62 / 540,478 filed August 2, 2017, each of which is incorporated by reference in its entirety for all purposes. Background Art

[0003] Commercial kits for performing nucleic acid-based assays typically contain reagents such as enzymes, nucleotides, detergents, buffers, primers, probes, and inorganic salts, including MnCl2, MgCl2, NaCl, and KCl (Innis et al., (1990) PCR Protocols: A Guide to Methods and Applications, Chapter 1, Optimizations of PCRs).

[0004] Nucleic acid-based assays can be "nucleic acid amplification-based assays", i.e., assays that utilize one or more steps to amplify a nucleic acid target sequence. Various amplification methods for nucleic acid-based assays are known in the art. Alternatively, nucleic acid-based assays can be "non-nucleic acid amplification-based assays", i.e., assays that do not rely on any steps to amplify a nucleic acid target sequence. An exemplary non-nucleic acid amplification-based assay is a "cutting-based assay", which is an assay that relies on specific cutting of a linear duplex cleavage structure by a flap endonuclease, which is formed by specific hybridization of overlapping oligonucleotides with a target nucleic acid. In these assays, an intruder oligonucleotide (also referred to as an "intruder probe") is designed to stably anneal to a target nucleic acid at an assay reaction temperature. A signal probe oligonucleotide containing a target hybridization sequence and a non-target hybridization flap region overlapping with the intruder oligonucleotide is designed to have a melting temperature of approximately the assay temperature. As a result, the signal probe is in a state of annealing / dissociation with the target nucleic acid under reaction conditions. When the signal probe is annealed to the target nucleic acid in the presence of a flap endonuclease, the non-target hybridized flap region is cut by the flap endonuclease in an overlap-dependent manner to release the cut product. The cut flap region is then annealed with a probe of a hairpin structure including a signaling portion and a quenching portion (commonly referred to as a "FRET probe") to form an overlap between a portion of the cut flap portion and a portion of the FRET probe connected to one of the signaling portion or the quenching portion. In the presence of a flap endonuclease, the FRET probe is cut in an overlap-dependent manner to release one of the signaling portion or the quenching portion from the FRET probe, thereby generating a detectable signal. Because an excess of signal probes and FRET probes relative to the invader probes are used in these assays, they are often referred to as signal amplification assays. The principles of cleavage-based assays are well known in the art, and exemplary assays are described, for example, in Lyamichev et al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al. (Mol. Diagn. 4:135-144, 1999), Allaw et al. (J. Clin. Microbiol. 44:3443-3447, 2006), U.S. Pat. Nos. 5,846,717 and 6,706,471 to Brow et al., and U.S. Pat. No. 5,614,402 to Dahlberg et al. Cleavage-based assays include, for example, commercially available Assay (Hologic, Inc., Marlborough, MA).

[0005] In some cases, it may be desirable to perform both an "amplification-based assay" (e.g., PCR) and a "non-amplification-based assay" (e.g., using enzyme assay). The enzyme is a thermostable structure-specific endonuclease that cleaves at the junction between single-stranded and double-stranded DNA.

[0006] Magnesium ions are reported to increase the activity of polymerases and other enzymes. Potassium chloride is reported to promote nucleic acid hybridization. Many inorganic salts are reported to protect proteins under various stress conditions including heating, chaotropic agent exposure, and lyophilization (Liu et al. (2007) FEBS Letters 581:1047; Kanaya et al. (1996) J. Biol. Chem. 271:327-29; Innis et al. (1990) PCR Protocols: A Guide to Methods and Applications, Chapter 1, Optimization of PCR; Menendez et al. (1998) J. Biol. Chem. 273:167; Janeway et al. (1993) Biochemistry 32:1601; Fo et al. (1994) PCR Protocols: A Guide to Methods and Applications, Chapter 1, Optimization of PCR; Menendez et al. (1998) J. Biol. Chem. 273:167; Janeway et al. (1993) Biochemistry 32:1601; Fo et al. (1994) PCR Protocols: A Guide to Methods and Applications, Chapter 1, Optimization of PCR). X et al. (1971) Journal of Biochemistry 246:5739; Chang et al. (2002) Journal of Biochemistry 277:277:4663; Rutter et al. (1958) Journal of Biochemistry 233:374; Huszar et al. (1981) Journal of Virology (J.Virol.) 37:580-588; Wang (2000) International Journal of Pharmaceutical Sciences (Int.J.Pharmaceutics) 203:1-60). Salts are also used in "amplification-based assays" and "non-amplification-based assays". By way of example, Typical reaction conditions for the assay include potassium chloride in the enzyme stock solution and magnesium chloride in the reaction buffer.

[0007] However, the stability of the lyophilized material is affected by the hygroscopicity of any salt present in the lyophilized cake. The hygroscopicity of the lyophilized material in turn affects the time that can be used to package the lyophilized material, and affects the duration and conditions under which the lyophilized material can be stored and transported. Undesirable rehydration of the lyophilized material can adversely affect the activity of the lyophilized components. In order to minimize the adverse effects of undesirable rehydration from the lyophilized material, such materials are usually stored for a long time under refrigeration.

[0008] The present invention seeks to provide improvements in lyophilized compositions, particularly when the lyophilized compositions are used in nucleic acid based assays, such as non-amplification based assays or non-amplification based assays in combination with amplification based assays. Summary of the invention

[0009] An aqueous solution composition is disclosed, comprising, consisting of, or consisting essentially of a flap endonuclease, a swelling agent, and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 6 mM or less, and wherein the composition is substantially free of glycerol.

[0010] Suitably, the aqueous solution further comprises at least one oligonucleotide useful for performing a molecular assay.

[0011] Suitably, the aqueous solution further comprises at least one oligonucleotide useful for performing a nucleic acid based assay.

[0012] Suitably, the flap endonuclease is Enzyme.

[0013] Suitably, the composition further comprises at least one polymerase.

[0014] Suitably, the at least one polymerase in the aqueous solution comprises a polymerase present in the aqueous solution at a concentration of about 0.10 U / μl to about 0.25 U / μl.

[0015] Suitably, the at least one polymerase comprises a polymerase present in the aqueous solution at a concentration selected from the group consisting of 0.11 U / μl, 0.12 U / μl, 0.14 U / μl, 0.146 U / μl, 0.1687 U / μl, 0.2 U / μl and 0.022 U / μl.

[0016] Suitably, the at least one polymerase comprises a polymerase that is a hot start polymerase.

[0017] Suitably, the hot-start polymerase is a recombinant Taq DNA polymerase, which is bound by an antibody that specifically blocks the polymerase activity of the polymerase.

[0018] Suitably, the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase, wherein the chemical modification inhibits the polymerase activity of the polymerase.

[0019] Suitably, said at least one polymerase comprises a reverse transcriptase enzyme suitably present in said aqueous solution at a concentration of about 0.1 U / μl to about 4.0 U / μl.

[0020] Suitably, the reverse transcriptase is AMV reverse transcriptase.

[0021] Suitably, the reverse transcriptase is MMLV reverse transcriptase.

[0022] Suitably, said at least one oligonucleotide comprises an invader probe.

[0023] Suitably, the sequence of the invader probe is partially complementary or fully complementary to the target nucleic acid sequence.

[0024] Suitably, said at least one oligonucleotide comprises a signalling probe.

[0025] Suitably, the sequence of the signalling probe is partially complementary to the target nucleic acid sequence.

[0026] Suitably, said sequence of said signalling probe comprises a flap region.

[0027] Suitably, the petal at least partially overlaps the invasive probe.

[0028] Suitably, said at least one oligonucleotide comprises a FRET probe.

[0029] Suitably, the sequence of said FRET probe is complementary to said flap portion of the signalling probe.

[0030] Suitably, the FRET probe comprises a label covalently attached thereto.

[0031] Suitably, the label is a fluorescent molecule.

[0032] Suitably, the label is located at the 5' end of the FRET probe.

[0033] Suitably, the FRET probe comprises a quencher molecule covalently attached to the FRET probe, within the quenching proximity of the fluorescent molecule and capable of at least partially quenching fluorescence from the fluorescent molecule.

[0034] Suitably, the at least one oligonucleotide comprises a target capture probe.

[0035] Suitably, the target capture probe has a target hybridising portion that hybridises specifically or non-specifically to a target nucleic acid under stringent conditions.

[0036] Suitably, the target capture probe has a target hybridising portion which hybridises non-specifically to the target nucleic acid under stringent conditions.

[0037] Suitably, the non-specific target hybridising portion of the target capture probe comprises K randomly arranged nucleotides or R randomly arranged nucleotides (IUPAB-IUB ambiguity code).

[0038] Suitably, the aqueous solution comprises two or more oligonucleotides for performing a multiplex molecular assay.

[0039] Suitably, the bulking agent is trehalose.

[0040] Suitably, the swelling agent is present in a concentration of about 0.2M to about 0.5M, suitably about 0.36M.

[0041] Suitably, the aqueous solution comprises an inorganic salt concentration of 6 mM or less. Suitably, the aqueous solution comprises an inorganic salt concentration of between about 6 mM and about 0.5 mM.

[0042] Suitably, the inorganic salt is present in a mass per microliter of about 0.373 μg / μl to about 0.029 μg / μl.

[0043] Suitably, the inorganic salt is sodium chloride, suitably wherein the sodium chloride is present at a mass per microliter of about 0.35 μg / μl to about 0.029 μg / μl, suitably about 0.32 μg / μl.

[0044] Suitably, the inorganic salt is potassium chloride, suitably wherein the potassium chloride is present in a mass per microliter of from about 0.373 μg / μl potassium chloride to about 0.019 μg / μl potassium chloride, suitably about 0.03 μg / μl.

[0045] Suitably, the aqueous solution contains from about 0.135 μg / μl sodium ions to about 0.006 μg / μl sodium ions, suitably about 0.127 μg / μl.

[0046] Suitably, the aqueous solution contains from about 0.196 μg / μl potassium ions to about 0.010 μg / μl potassium ions, suitably about 0.016 μg / μl.

[0047] Suitably, the aqueous solution contains from about 0.355 μg / μl chloride ions to about 0.009 μg / μl chloride ions, suitably about 0.337 μg / μl.

[0048] Suitably, the aqueous solution comprises an inorganic salt concentration of 4 mM or less.

[0049] Suitably, the aqueous solution comprises an inorganic salt in an amount per microliter of about 0.298 μg / μl to about 0.234 μg / μl.

[0050] Suitably, the aqueous solution comprises chloride ions in an amount per microliter of about 0.284 μg / μl to about 0.071 μg / μl.

[0051] Suitably, the aqueous solution comprises an inorganic salt concentration of 3 mM or less.

[0052] Suitably, the aqueous solution comprises an inorganic salt in an amount per microliter of about 0.224 μg / μl to about 0.175 μg / μl.

[0053] Suitably, the aqueous solution comprises chloride ions in an amount per microliter of about 0.213 μg / μl to about 0.053 μg / μl.

[0054] Suitably, the aqueous solution comprises an inorganic salt concentration of 2 mM or less.

[0055] Suitably, the aqueous solution comprises an inorganic salt in an amount per microliter of about 0.149 μg / μl to about 0.117 μg / μl.

[0056] Suitably, the aqueous solution comprises chloride ions in an amount per microliter of about 0.142 μg / μl to about 0.036 μg / μl.

[0057] Suitably, the aqueous solution comprises an inorganic salt concentration of 1 mM or less.

[0058] Suitably, the aqueous solution comprises an inorganic salt in an amount per microliter of about 0.075 μg / μl to about 0.058 μg / μl.

[0059] Suitably, the aqueous solution comprises chloride ions in an amount per microliter of about 0.071 μg / μl to about 0.018 μg / μl.

[0060] Suitably, the aqueous solution comprises an inorganic salt concentration of 500 μM or less.

[0061] Suitably, the aqueous solution comprises an inorganic salt in an amount per microliter of about 0.037 μg / μl to about 0.029 μg / μl.

[0062] Suitably, the aqueous solution comprises chloride ions in an amount per microliter of about 0.036 μg / μl to about 0.009 μg / μl.

[0063] Suitably, said inorganic salt concentration of said aqueous solution is less than 1 mM sodium chloride.

[0064] Suitably, the aqueous solution contains no sodium chloride.

[0065] Suitably, the aqueous solution contains less than 1 mM magnesium ions.

[0066] Suitably, the aqueous solution contains less than 0.1 mM magnesium ions.

[0067] Suitably, the aqueous solution further comprises deoxynucleotide triphosphates (dNTPs).

[0068] Suitably, said dNTPs comprise dATP at a concentration of 0.1 mM to 0.5 mM, suitably about 0.28 mM to about 0.46 mM in said aqueous solution.

[0069] Suitably, the concentration of said dATP in said aqueous solution is 0.3 mM to 0.4 mM, such as 0.375 mM.

[0070] Suitably, said dNTPs comprise dGTP at a concentration of 0.1 mM to 0.4 mM in said aqueous solution.

[0071] Suitably, the concentration of said dGTP in said aqueous solution is 0.3 mM to 0.4 mM, suitably 0.29 mM to 0.46 mM, for example 0.375 mM.

[0072] Suitably, said dNTPs comprise dCTP at a concentration of 0.1 mM to 0.4 mM, suitably 0.29 mM to 0.46 mM in said aqueous solution.

[0073] Suitably, the concentration of said dCTP in said aqueous solution is 0.3 mM to 0.4 mM, such as 0.375 mM.

[0074] Suitably, said dNTPs comprise dTTP at a concentration of 0.1 mM to 0.4 mM, suitably 0.2 mM to 0.37 mM (eg 0.284 mM) in said aqueous solution.

[0075] Suitably, said dNTPs comprise dUTP at a concentration of 0.1 mM to 0.4 mM, suitably 0.125 mM to 0.234 mM (eg 0.182 mM) in said aqueous solution.

[0076] Suitably, the flap endonuclease is present in the aqueous solution at about 0.010 μg / μl to about 0.050 μg / μl, suitably at about 0.12 μg / μl to 0.047 μg / μl.

[0077] Suitably, the flap endonuclease is present in the aqueous solution at about 0.030 μg / μl to about 0.04 μg / μl, suitably, wherein the flap endonuclease is present in the aqueous solution at about 0.030 μg / μl to about 0.035 μg / μl.

[0078] Suitably, the organic buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

[0079] Suitably, said MOPS buffer is present in said aqueous solution at a concentration of 10 mM to 20 mM, suitably at a concentration of 12.5 mM to about 20 mM.

[0080] Suitably, the organic buffer is Tris buffer.

[0081] Suitably, said Tris buffer is present in said aqueous solution at a concentration of 40 mM to 60 mM, suitably at a concentration of 50 mM.

[0082] Suitably, the composition contains globular protein.

[0083] Suitably, the globular protein is bovine serum albumin (BSA).

[0084] Suitably, said bovine serum albumin (BSA) is non-acetylated BSA, suitably ultrapure non-acetylated BSA.

[0085] Suitably, the globular protein is present in an amount of 0.40 μg / μl to 0.60 μg / μl, suitably 0.50 μg / μl.

[0086] Suitably, the composition comprises, consists of or consists essentially of: enzymes, trehalose, MOPS buffer, dNTPs, inorganic salts at a concentration of 5 mM or less, and wherein the composition is substantially free of glycerol.

[0087] Suitably, the composition comprises, consists of or consists essentially of: enzyme, trehalose present at a concentration of about 0.3 M, MOPS buffer at a concentration of about 12.5 mM, dNTPs at a concentration of about 0.3 mM each (optionally, dATP, dGTP and dCTP at about 0.375 mM, dTTP at about 0.284 mM and dUTP at about 0.182 mM), an inorganic salt at a concentration of 5 mM or less, and wherein the composition is substantially free of glycerol.

[0088] Suitably, the composition comprises, consists of or consists essentially of: enzyme, trehalose, Tris buffer, dNTP, bovine serum albumin, inorganic salts at a concentration of 5 mM or less, and wherein the composition is substantially free of glycerol.

[0089] Suitably, the composition comprises, consists of or consists essentially of: enzyme, trehalose at a concentration of about 0.3 M, Tris buffer at a concentration of about 50 mM, dNTPs at a concentration of about 0.3 mM each (optionally, dATP, dGTP and dCTP at about 0.375 mM, dTTP at about 0.284 mM and dUTP at about 0.182 mM), about 0.5 μg / μl bovine serum albumin, an inorganic salt at a concentration of 5 mM or less, and wherein the composition is substantially free of glycerol.

[0090] Suitably, the composition comprises, consists of or consists essentially of: Trehalose, MOPS buffer, dNTPs, bovine serum albumin, inorganic salts at a concentration of 5 mM or less, and wherein the composition is substantially free of glycerol.

[0091] Suitably, the composition comprises, consists of, or consists essentially of: Trehalose present at a concentration of about 0.36M, MOPS buffer at a concentration of about 15mM, dNTPs at a concentration of about 0.38mM each, bovine serum albumin at about 0.5μg / μl, inorganic salts at a concentration of 5mM or less, and wherein the composition is substantially free of glycerol.

[0092] Suitably, the composition comprises alpha-cyclodextrin, for example at a concentration of 0.1-0.5 μg / ml.

[0093] A kind of dry form according to the composition of the present disclosure is also disclosed.By example, dry 24 μ l aqueous solution as described herein is obtained dry composition, and the mass range of described composition is about 0.003g to about 0.004g, about 0.0032g to about 0.0037g, 0.0033g, 0.0034g, 0.0035g or 0.0036g.It should be understood that the multiple dry compositions made by the single bulk aqueous solution of aliquot will change in the mass aspect of every kind of dry composition.By example, the bulk aqueous solution of two or more 24 μ l aliquots will be dried separately to provide the dry composition of mass range of about 0.003g to about 0.004g separately. Therefore, the average weight range of each dry aggregate in the multiple dry aggregates is about 0.003g to about 0.004g, about 0.0032g to about 0.0037g, 0.0033g, 0.0034g, 0.0035g or 0.0036g.Changing the concentration of the component in the aqueous solution will cause the mass of the dry composition to change.For example, changing the concentration of the enzyme to optimize the enzyme activity for a specific reaction may cause the mass of the dry composition to change.Similarly, the change in the type of components used may cause the mass of the dry composition to change.For example, using different flap-shaped endonucleases may require concentration changes to provide the desired enzyme activity level.Changing the concentration and / or type of one or more components in the various components should be understood to fall within the scope of the present disclosure, and therefore the corresponding changes to the mass of the dry composition should be understood to fall within the scope of the present disclosure.

[0094] Also disclosed is a dry composition comprising, consisting of, or consisting essentially of a flap endonuclease, a swelling agent, and an organic buffer, wherein one or more inorganic salts are present in the dry composition in a mass that is 0.350% or less of the total mass of the dry composition, and wherein the dry composition is essentially free of glycerol.

[0095] The dried composition can be used in nucleic acid-based assays after reconstitution. Surprisingly, the dried composition provides robust results after long-term exposure to a humid environment. The dried form of the composition can be used in nucleic acid-based assays after exposure to a humid environment, wherein the absolute humidity level of the humid environment is greater than 2.3 grams of water per cubic meter of air, and the composition is exposed for a period of up to 3 hours; preferably a period of 90 minutes to 180 minutes; preferably about 90 minutes; preferably about 180 minutes. Surprisingly, the dried composition provides robust results after long-term incubation of the pre-dried aqueous solution.

[0096] Suitably, one or more inorganic salts are present in the dry composition in an amount of from about 0.311% to about 0.024% of the total mass of the dry composition.

[0097] Suitably, the one or more inorganic salts are selected from the group consisting of sodium chloride, potassium chloride and both sodium chloride and potassium chloride.

[0098] Suitably, the dried composition further comprises at least one oligonucleotide useful for performing a molecular assay.

[0099] Suitably, the dried composition further comprises at least one oligonucleotide useful for performing a nucleic acid based assay.

[0100] Suitably, the at least one oligonucleotide comprises a probe oligonucleotide, suitably at least two probe oligonucleotides.

[0101] Suitably, the one or more probe oligonucleotides are partially complementary or fully complementary to the target nucleic acid sequence.

[0102] Suitably, the flap endonuclease is Enzyme.

[0103] Suitably, the dry composition further comprises at least two probe oligonucleotides capable of annealing to a target nucleic acid to form a three-dimensional structure that can be recognized by the flap endonuclease.

[0104] Suitably, the dry composition further comprises at least one polymerase.

[0105] Suitably, the at least one polymerase in the aqueous solution comprises a polymerase present in the aqueous solution at a concentration of about 0.10 U / μl to about 0.2 U / μl.

[0106] Suitably, the at least one polymerase comprises a polymerase present in the aqueous solution at a concentration selected from about 0.1 U / μl to about 0.25 U / μl.

[0107] Suitably, the at least one polymerase comprises a polymerase that is a hot start polymerase.

[0108] Suitably, the hot-start polymerase is a recombinant Taq DNA polymerase, which is bound by an antibody that specifically blocks the polymerase activity of the polymerase.

[0109] Suitably, the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase, wherein the chemical modification inhibits the polymerase activity of the polymerase.

[0110] Suitably, said at least one polymerase comprises a reverse transcriptase enzyme suitably present in said aqueous solution at a concentration of about 0.1 U / μl to about 0.6 U / μl.

[0111] Suitably, the reverse transcriptase is AMV reverse transcriptase.

[0112] Suitably, the reverse transcriptase is MMLV reverse transcriptase.

[0113] Suitably, said at least one oligonucleotide comprises a detection probe.

[0114] Suitably, said at least one oligonucleotide comprises a detection probe.

[0115] Suitably, the sequence of the detection probe is partially complementary or fully complementary to the target nucleic acid sequence.

[0116] Suitably, the detection probe comprises a label covalently attached thereto.

[0117] Suitably, the label is a fluorescent molecule or a chemiluminescent molecule.

[0118] Suitably, the label is located at the 5' end of the detection probe and internal quencher molecule.

[0119] Suitably, said at least one oligonucleotide comprises an invader probe.

[0120] Suitably, the sequence of the invader probe is partially complementary or fully complementary to the target nucleic acid sequence.

[0121] Suitably, said at least one oligonucleotide comprises a signalling probe.

[0122] Suitably, the sequence of the signalling probe is partially complementary to the target nucleic acid sequence.

[0123] Suitably, said sequence of said signalling probe comprises a flap region.

[0124] Suitably, the petal at least partially overlaps the invasive probe.

[0125] Suitably, said at least one oligonucleotide comprises a FRET probe.

[0126] Suitably, the sequence of said FRET probe is complementary to said flap portion of the signalling probe.

[0127] Suitably, the FRET probe comprises a label covalently attached thereto.

[0128] Suitably, the label is a fluorescent molecule.

[0129] Suitably, the label is located at the 5' end of the FRET probe.Suitably, the FRET probe comprises a quencher molecule covalently attached to the FRET probe, within the quenching proximity of the fluorescent molecule and capable of at least partially quenching fluorescence from the fluorescent molecule.

[0130] Suitably, the at least one oligonucleotide comprises a target capture probe.

[0131] Suitably, the target capture probe has a target hybridising portion that hybridises specifically or non-specifically to a target nucleic acid under stringent conditions.

[0132] Suitably, the target capture probe has a target hybridising portion which hybridises non-specifically to the target nucleic acid under stringent conditions.

[0133] Suitably, the non-specific target hybridising portion of the target capture probe comprises K randomly arranged nucleotides or R randomly arranged nucleotides.

[0134] Suitably, the composition comprises oligonucleotides for performing a multiplex molecular assay.

[0135] Suitably, the bulking agent is trehalose.

[0136] Suitably, the dry composition further comprises deoxynucleotide triphosphates (dNTPs).

[0137] Suitably, the organic buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

[0138] Suitably, the organic buffer is Tris buffer.

[0139] Suitably, the dry composition contains globular protein.

[0140] Suitably, the globular protein is bovine serum albumin (BSA).

[0141] Suitably, said bovine serum albumin (BSA) is non-acetylated BSA, suitably ultrapure non-acetylated BSA.

[0142] Also disclosed is a method of forming a mixture for performing a nucleic acid-based assay, the method comprising combining a reconstitution solution with a dry composition described herein, wherein the reconstitution solution comprises at least one inorganic salt.

[0143] Suitably, the reconstitution solution comprises an inorganic salt concentration of less than 1 mM.

[0144] Suitably, the reconstitution solution comprises magnesium ions.

[0145] Suitably, the reconstitution solution comprises MgCl2 at a concentration of about 5mM to about 15mM, suitably 9mM to 10mM or 9-12mM, optionally 11.25mM.

[0146] Suitably, the reconstitution solution is selected from the group consisting of: methylparaben at a concentration of about 0.012% w / v to about 0.020% w / v, propylparaben at a concentration of about 0.006% w / v to about 0.010% w / v, anhydrous ethanol at a concentration of about 0.20% v / v to about 0.30% v / v, or a combination thereof.

[0147] Suitably, the concentration of said methylparaben in said reconstituted solution is 0.016% w / v.

[0148] Suitably, the concentration of said propylparaben in said reconstituted solution is 0.008% w / v.

[0149] Suitably, said absolute ethanol is present in said reconstitution solution at a concentration of about 0.26% v / v.

[0150] Also disclosed is a method for preparing a dry composition for performing a nucleic acid-based assay, the method comprising the steps of: (i) freezing an aqueous solution of the present disclosure, thereby forming a frozen form of the aqueous solution; and (ii) exposing the frozen form from step (i) to lyophilization conditions, thereby forming a dry composition.

[0151] Suitably, the dry composition is exposed to a humid environment, wherein the humid environment has an absolute humidity level of greater than 2.3 grams of water per cubic meter of air at 30°C.

[0152] Suitably, said dry composition is exposed to said moist environment for up to 3 hours.

[0153] Suitably, the method comprises the step of storing the dry composition in a sealed container.

[0154] Also disclosed is a method for preparing a dry composition for performing a nucleic acid-based assay, the method comprising the steps of drying the aqueous solution described herein using a drying method selected from the group consisting of dehydration, dehumidification, freeze drying, and spray drying, thereby forming a dry composition.

[0155] Suitably, the drying method is freeze-drying and the dried composition is a freeze-dried composition.

[0156] Suitably, the method further comprises the step of storing the dry composition in a sealed container.

[0157] Also disclosed is a kit for performing a nucleic acid-based assay, the kit comprising a first vessel containing a dried composition as described herein and a second vessel containing a reconstitution solution, the reconstitution solution comprising MgCl2 at a concentration of about 3.8 mM to about 14.4 mM, suitably about 9.4 mM or about 11.25 mM.

[0158] Suitably, the first vessel is a multiwell plate comprising one or more wells.

[0159] Suitably, each of the one or more wells contains a dry single unit dose granule containing an inorganic salt by mass to mass of the granule of 0.311% or less.

[0160] Suitably, the first vessel and the second vessel are combined within a device adapted to automatically transfer the reconstitution solution from the second vessel into the first vessel.

[0161] Suitably, each of the one or more wells contains a dry single unit dose pellet having a weight of about 0.000125 g to about 0.000667 g per microliter of aqueous solution, the aqueous solution being dried to form the dry single unit dose pellet in each well.

[0162] Suitably, each of the one or more wells contains a dry single unit dose granule having a weight range of about 0.003g to about 0.004g, about 0.0032g to about 0.0037g, 0.0033g, 0.0034g, 0.0035g or 0.0036g.

[0163] Also disclosed is a dialysis composition comprising, consisting of, or consisting essentially of an aqueous solution containing an organic buffer, a swelling agent, chloride ions, and a chelating agent.

[0164] Suitably, the bulking agent is trehalose.

[0165] Suitably, the bulking agent is present at a concentration of about 100 mM to 300 mM, suitably 200 mM.

[0166] Suitably, the organic buffer is Tris buffer.

[0167] Suitably, the Tris buffer is present in the aqueous solution at a concentration of 10 mM to 30 mM, suitably at a concentration of 20 mM. Suitably, the pH is about 8.0.

[0168] Suitably, the chloride ion is KCl.

[0169] Suitably, said KCI is present at a concentration of about 40 mM to 60 mM, suitably 50 mM.

[0170] Suitably, the chelating agent is EDTA.

[0171] Suitably, said EDTA is present in said aqueous solution at a concentration of 0.05 mM to 0.2 mM, suitably 0.1 mM.

[0172] Suitably, the composition comprises a flap endonuclease, suitably Enzyme.

[0173] Also disclosed is a method for preparing a flap-shaped nuclease composition that is substantially free of glycerol, the method comprising: (i) providing an aqueous solution comprising, consisting of, or consisting essentially of a flap-shaped nuclease and glycerol; (ii) dialyzing the aqueous solution into a dialysis composition as described herein; and (iii) obtaining a flap-shaped nuclease composition that is substantially free of glycerol.

[0174] Suitably, the aqueous solution in step (i) contains about 0.0 to about 0.5% (w / v) glycerol, suitably about 0.35% (w / v) glycerol, more suitably less than 0.2% (w / v) glycerol.

[0175] Also disclosed is a use of the dry composition described herein for preparing a composition containing a dry flap-shaped endonuclease.

[0176] Also disclosed is a use of a dry composition described herein in combination with a reconstitution solution for performing a nucleic acid-based assay.

[0177] Also disclosed is a use of the dialysis composition described herein for preparing a flap endonuclease composition that is substantially free of glycerol.

[0178] definition

[0179] The term "about" indicates that insubstantial variations in the amount of a component of the composition do not have any significant effect on the activity or stability of the composition.

[0180] "Bulking agents" provide a matrix for the deposition of proteins and other agents during drying and storage (Carpenter et al. (2002) Rational design of stable lyophilized protein formulations, Kluwer Academic / Plenum, New York, pp. 109-133). Bulking agents can be used to form a product "cake" or other structure, and can prevent protein loss from vials during drying and increase protein stability.

[0181] "Chelating agents" are chelating Mg required for enzyme activity 2+ or Mn 2+ Drugs that produce divalent ions.

[0182] The terms "lyophilization," "freeze-dried," and "freeze-dried" refer to a process in which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. "Lyophilate" refers to a freeze-dried substance.

[0183] The term "stringent" with respect to nucleic acid hybridization (including "stringent hybridization conditions" or "stringent conditions") refers to conditions under which a specific oligonucleotide can hybridize to a target nucleic acid superior to other nucleic acids present in a test sample. It should be understood that these conditions can vary according to factors including GC content and oligonucleotide length, hybridization temperature, the composition of hybridization reagents or solutions, and the degree of hybridization specificity sought. Appropriate hybridization conditions for probes, oligonucleotides, target capture oligonucleotides, blockers, and other oligonucleotides are well known in the art, can be predicted based on sequence composition, or can be determined using routine testing procedures (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor, New York, 1989), at §§1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57, in particular §§9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57).

[0184] The term "cleavage structure" refers to a structure formed by allowing a number of nucleic acids to interact to form a configuration, wherein two of the nucleic acids each contain a nucleobase that is configured to hybridize at a single nucleobase position on a third nucleic acid, such that an overlap is formed between the first two nucleic acids, wherein the resulting non-hybridized flap region (e.g., a cleavage structure) can be cleaved by a flap endonuclease. Compared to nucleic acid molecules that are substrates for non-specific cleavage by agents such as phosphodiesterases, which cleave nucleic acid molecules without regard to secondary structure (i.e., without the need to form a duplex structure), the cleavage structure is a substrate that is specifically cleaved by a flap endonuclease. For a discussion of cleavage structures and other aspects of flap endonuclease detection assays, see U.S. Patent No. 5,846,717.

[0185] As used herein, "flap endonuclease" refers to a class of nucleases that act as structure-specific 5' endonucleases on nucleic acid structures, wherein the duplex contains a single-stranded 5' overhang or flap on one of the strands displaced by the other strand of the nucleic acid (i.e., such that there are overlapping nucleotides where adjacent first and second probes hybridize to the target). Flap endonucleases may also be referred to as "5' endonucleases" or abbreviated by the acronym "FEN". ​​FEN catalyzes the hydrolytic cleavage of the phosphodiester bond at the junction of a single-stranded nucleic acid and a double-stranded nucleic acid, thereby releasing the overhang or flap. Ceska and Savers (Trends Biochem. Sci. 23:331-336, 1998) and Liu et al. (Annu. Rev. Biochem. 73:589-615, 2004) reviewed FEN.

[0186] The "overlap region" or "flap region" consists of one or more bases of the first probe oligonucleotide (e.g., signal probe) that hybridize to the target and overlap with the second probe oligonucleotide (e.g., invader probe). The bases on the 3' end of the second probe oligonucleotide determine the ends of the overlap region and may or may not hybridize to the target.

[0187] With regard to the determination based on cutting, "first probe oligonucleotide" refers to an oligonucleotide that interacts with the target nucleic acid to form a cutting structure (e.g., a signal probe) in the presence of a "second probe oligonucleotide" that hybridizes to a region upstream of the first probe oligonucleotide (e.g., an intruder probe). When annealed to the target nucleic acid, the first probe oligonucleotide and the target form a cutting structure, and the cutting performed by the flap endonuclease can occur within the first probe oligonucleotide. In the presence of a second probe oligonucleotide that overlaps along the target nucleic acid upstream of the first probe oligonucleotide, the cutting site within the first probe oligonucleotide will occur after the last overlapping base (cutting depends on at least one overlapping base of the target hybridization base of the second probe and the first probe). In addition to the target hybridization region that hybridizes with the target sequence in the target nucleic acid, the first probe oligonucleotide contains a non-target hybridization region (also referred to as a "flap region") at the 5' end. When the first probe oligonucleotide and the second probe oligonucleotide are annealed to the target nucleic acid, the site-specific cutting performed by the flap endonuclease occurs to generate a cutting product containing the flap region and the overlapping region of the first probe oligonucleotide.

[0188] With respect to cleavage-based assays, a "second probe oligonucleotide" refers to an oligonucleotide containing a sequence at its 3' end that, when annealed to a target nucleic acid, overlaps the 5' end of a target hybridization sequence within the downstream of the first probe oligonucleotide; typically, these regions will compete for hybridization to the same segment along a complementary target nucleic acid. In some cases, the second probe can be used as a primer as well as an invader probe. The 3' terminal nucleotide of the second probe oligonucleotide may or may not base pair with a nucleotide in the target nucleic acid. In some variations, only the 3' terminal nucleotide overlaps with the 5' end of the target hybridization sequence of the first probe oligonucleotide. The cleaved second probe oligonucleotide can participate in a secondary reaction in which it acts as a probe on a fluorescence resonance energy transfer box (e.g., a FRET probe or FRET box), resulting in the formation of a second probe oligonucleotide. When the FRET cassette is cleaved, the fluorophore is released from the quencher on the FRET cassette, generating a fluorescent signal.

[0189] As used herein, the term "FRET cassette" refers to a hairpin oligonucleotide containing a fluorophore portion and a nearby quencher portion that quenches the fluorophore. Hybridization of the cleavage product to the FRET cassette generates a second substrate for the flap endonuclease. Once this substrate is formed, the base containing the 5' fluorophore is cleaved from the cassette, thereby generating a fluorescent signal.

[0190] An "amplification oligomer" is a primer or promoter primer that can support template-dependent replication. An amplification oligomer pair is a pair of such oligomers that support template-dependent replication of opposing strands of a template. Multiplex amplification is an amplification performed simultaneously with multiple amplification oligomer pairs.

[0191] A "detection probe" is an oligonucleotide that can hybridize to an amplification product or initial target nucleic acid to reveal the presence or amount of the amplification product. Such a detection probe is usually combined with a molecule that produces a fluorescent or other detectable signal, in which case the probe is referred to as a detectably labeled probe.

[0192] A "primer-probe set" is a combination of primers and detection probes that are configured to generate an amplification product from a template nucleic acid.

[0193] "Reconstitution time" is the time required to rehydrate a dry formulation with a solution to produce a solution that is free of particles or haze to the naked eye.

[0194] "Relative Fluorescence Units (RFU)" is a measure of the unquenched fluorophore. In a nucleic acid-based amplification reaction, the presence of product is determined by measuring RFU at multiple cycle times (Ct).

[0195] "Ct" refers to the number of cycles required to reach the exponential phase in real-time PCR. Ct is inversely correlated with the amount of analyte in the sample.

[0196] When referring to the results of an assay reaction, "positive" refers to the percentage of samples that exceed the index threshold in a test involving multiple samples. For example, when the multiple samples are twelve samples, and when the sample exceeding number is determined to be six, the positive is "50%".

[0197] "Single unit dose" or "SUD" refers to the volume of the reaction mixture used to perform an assay on a single sample. A single unit dose can be in liquid form or dry form. By way of example, a single unit dose can be a dry pellet containing reagents that can be used to test a single sample in a single vessel.

[0198] "LOD" is the limit of detection of the analyte. LOD+1 is the LOD that the user has detected plus one logarithm. In other words, LOD+1 is ten times the number of analytes at the LOD.

[0199] Compositions disclosed as comprising dTTP and dUTP may comprise dTTP or dUTP or both at the indicated concentrations. Likewise, when a composition is disclosed as containing dTTP or dUTP, the disclosure should be understood to alternatively encompass compositions comprising both dTTP and dUTP at the specified concentrations. DETAILED DESCRIPTION

[0200] I. Overview

[0201] The present disclosure is based at least in part on the following discovery: the instability of the prior art freeze-dried test kits for carrying out nucleic acid-based assays is due to the presence of inorganic salts. These salts may produce undesirable hybridization products or other byproducts before, during and after drying. These salts also make the dry composition hygroscopic, so that the dry composition absorbs water, and therefore requires limited exposure to moisture, refrigeration or deep freezing storage and / or storage in the presence of a desiccant. The presence of water and salts prematurely inactivates the enzyme components of such test kits and can also promote the nucleic acids in such test kits to hybridize with each other. The present disclosure overcomes these problems by drying reagents from bulk reagents that are substantially free of inorganic salts to carry out nucleic acid-based assays. This salt is provided when the dried composition is reconstituted. Contrary to the expectation that inorganic salts are required for the stability of enzymes for nucleic acid-based assays, it has been found that a reaction mixture that is substantially free of inorganic salts and dried can be stored above freezing for a long time, retaining activity completely or substantially during reconstitution.

[0202] II. Bulk Reagents and Dry Pellets

[0203] According to the bulk reagent of the present disclosure (sometimes referred to as pre-lyophilized mixture, solution, aqueous solution or composition), it is typical to include flap endonuclease, swelling agent, organic buffer. The bulk reagent may or may not include one or more nucleic acids and / or dNTPs. The bulk reagent may include chelating agents and RNase inhibitors.

[0204] Such bulk reagents are substantially free of inorganic salts, which means that the concentration of inorganic salts is individually and collectively less than 5 mM and preferably less than 1 mM. Preferably, the concentration of Mg2+ is less than 1 mM, less than 0.5 mM, less than 0.1 mM or less than 0.05 mM. Preferably, the concentration of Na+ is less than 1 mM, less than 0.5 mM, less than 0.1 mM or less than 0.05 mM. Preferably, the concentration of K+ is less than 1 mM, less than 0.5 mM, less than 0.1 mM or less than 0.05 mM. Preferably, the concentration of Cl- is less than 1 mM, less than 0.5 mM, less than 0.1 mM or less than 0.05 mM.

[0205] This bulk reagent is substantially free of glycerol. As used herein, the term "substantially free of" refers to that glycerol is not present in an amount commonly used for enzyme preparations. Suitably, the amount of glycerol is less than 5% (w / v), less than 4% (w / v), less than 3% (w / v), less than 2% (w / v), less than 1% (w / v), less than 0.5% (w / v), less than 0.1% (w / v) or less than 0.01% (w / v). Suitably, the amount of glycerol is undetectable using conventional methods known in the art. For example, Abcam (Cambridge, England) glycerol assay kit can be used to measure free glycerol concentration by enzymatic oxidation of glycerol to generate a product reacted with a probe producing color and fluorescence. The kit can detect 50pmol-10nmol of glycerol in various samples. Suitably, glycerol is present in a concentration less than 50pmol.

[0206] Nucleotides for incorporation into the reaction mixture are typically provided as dNTPs. Exemplary concentrations of dNTPs are 0.1 mM to 0.4 mM dATP, suitably 0.3 mM to 0.4 mM dATP, suitably 0.29 mM to 0.46 mM dATP; 0.1 mM to 0.4 mM dGTP, suitably 0.3 mM to 0.4 mM dGTP, suitably 0.29 mM to 0.4 mM dGTP; 0.1 mM to 0.4 mM dCTP, suitably 0.3 mM to 0.4 mM dCTP, suitably 0.29 mM to 0.4 mM dCTP; 0.1 mM to 0.4 mM dTTP, suitably 0.2 mM to 0.3 mM dATP; and 0.1 mM to 0.4 mM dUTP, suitably 0.125 mM to 0.234 mM dUTP. Exemplary concentrations in a 1.5x lyophilized mix or final reaction (ie, after reconstitution in 1.5X volume) are as follows.

[0207] In lyophilized mix (mM) In reaction (mM) dATP .375 .25 dGTP .375 .25 dCTP .375 .25 dTTP .284 .189 dUTP .182 .122

[0208] This mixture can be customized for any type of reaction using a flap endonuclease. This mixture can be customized for any type of reaction using a flap endonuclease and a polymerase.

[0209] The flap endonuclease is commercially available or can be prepared by the user. The flap endonuclease is not limited to an enzyme having only 5' nuclease activity. For example, the flap endonuclease can be a natural DNA polymerase having 5' nuclease activity (e.g., Taq DNA polymerase, E. coli DNA polymerase I) or a modified DNA polymerase having 5' nuclease activity due to the lack of synthetic activity (e.g., enzyme).

[0210] By choosing the polymerase and other components, this mixture can be tailored for different types of amplification including PCR, RT-PCR, and transcription-mediated amplification.

[0211] DNA polymerases are commercially available or can be prepared by the user. An example of a polymerase is Taq polymerase, which is commercially available from Qiagen (Germantown, MD, catalog number 201203). Another example of a Taq polymerase is G2Flexi DNA polymerase (Promega, Madison, Wisconsin, catalog number M7801) is commercially available. Other commercially available DNA polymerases include, but are not limited to, Tth DNA polymerase (e.g., Sigma-Aldrich, St. Louis, Missouri, catalog number 11480022001) and Chimeric DNA polymerases such as TAQ DNA polymerase (NEB, Ipswich, MA, catalog number M0530S) are also commercially available. Hot start DNA polymerases are also commercially available. For example, Taq polymerase can be used as Hot start polymerase (Promega, catalog number M5001) was purchased commercially. Hot start polymerase is an antibody-mediated hot start enzyme, in which Taq polymerase is combined with an antibody that blocks polymerase activity. High temperature is used to denature the blocking antibody, so during the initial heating step of the PCR reaction, the antibody is denatured and the polymerase activity is restored. Various antibodies can be used together with the hot start method, for example, TAQSTART antibody (Clontech Laboratories, Mountain View, California, catalog number R028A). Similarly, other hot start polymerases can be obtained, including chemically mediated hot start polymerases. Equivalent polymerases and antibodies can be obtained from various commercial sources and can be prepared by users alternatively.

[0212] Reverse transcriptases are commercially available or can be prepared by the user. Examples of commercially available reverse transcriptases include, but are not limited to, MMLV (Maloney murine leukemia virus) reverse transcriptase and III reverse transcriptase (e.g., Thermo Fisher Scientific, Carlsbad, CA, catalog numbers 28025-013 and 18080-044), MMLV RT (Sigma-Aldrich, catalog number M1302), AMV reverse transcriptase (NEB, Ipswich, MA, catalog number M0277S), and GoScript TMReverse transcriptase (Promega, catalog number A50003). GoScript reverse transcriptase contains a reverse transcriptase and a set of reagents for synthesizing first-strand cDNA optimized for quantitative PCR amplification. Equivalent reverse transcriptases and reagents are available from various commercial sources and can alternatively be prepared by the user.

[0213] An exemplary concentration of a single unit dose of flap endonuclease is between about 0.01 μg / μl and about 1.0 μg / μl, suitably between about 0.01 μg / μl and about 0.8 μg / μl, or between about 0.01 μg / μl and about 0.6 μg / μl, or between about 0.01 μg / μl and about 0.4 μg / μl, or between about 0.01 μg / μl and about 0.2 μg / μl, or between about 0.01 μg / μl and about 0.1 μg / μl. More suitably, the concentration of a single unit dose of flap endonuclease is between about 0.02 μg / μl and about 0.04 μg / μl. More suitably, the concentration of the flap endonuclease in a single unit dose is between about 0.030 μg / μl and about 0.04 μg / μl, more suitably between about 0.030 μg / μl and about 0.035 μg / μl. Suitably, the flap endonuclease is present in the aqueous solution at about 0.010 μg / μl to about 0.050 μg / μl, suitably at about 0.012 μg / μl to 0.047 μg / μl.

[0214] Exemplary concentrations of a single unit dose of DNA polymerase are 0.1-0.2 U / μl (including all whole and partial numbers therein), for example, 0.14 U / μl, 0.146 U / μl, and 0.1687 U / μl.

[0215] One unit of DNA polymerase is defined as the amount of enzyme required to catalyze the incorporation of 10 nanomoles of dNTPs into an acid-insoluble substance at 74°C in 30 minutes. Exemplary concentrations of a single unit dose of reverse transcriptase are 0.01 U / μl-1.0 U / μl (including all whole and partial numbers therein). One unit of reverse transcriptase is defined as the amount of enzyme required to catalyze the transfer of 1 nmol of deoxynucleotides into an acid-precipitable substance at 37°C in 10 minutes.

[0216] A preferred organic buffer is MOPS or MOPS.

[0217] Suitably, the MOPS buffer is present in said aqueous solution at a concentration of about 10 mM to about 20 mM, suitably at a concentration of about 12.5 mM to about 15 mM.

[0218] Suitably, Tris buffer is present in the aqueous solution at a concentration of about 5 mM to about 60 mM, suitably at a concentration of about 40 mM to 60 mM, suitably at a concentration of about 50 mM, suitably at a concentration of about 10 mM.

[0219] Alternative organic buffers that can be incorporated into the bulk reagent include phosphate, citrate, acetate, CHES, histidine, and Good's buffers, such as HEPES, MES, zwitterionic buffers, and glycinamide, and buffer combinations. Other organic buffers include succinate, citrate, gluconate, phosphate, and the like. Preferred buffers are effective in the pH range of about 6.0 to about 10.0 or about 7.0 to about 9.0; suitably, the pH used is about 7.5, or 8.0 or 8.5.

[0220] A preferred bulking agent is trehalose. Other contemplated bulking agents include raffinose, sucrose, mannitol, trehalose plus mannitol, sucrose plus mannitol, sucrose plus glycine, and hydroxyethyl starch. See, Cleland et al. (2001) Journal of Pharmaceutical Sciences (J. Pharm. Sci.) 90:310; Meyer et al. (2009) Eur. J. Pharm. Sci. 38:29; Webb et al. (2003) Journal of Pharmaceutical Sciences 92:715; Garzon Rodrigues et al. (2004) Journal of Pharmaceutical Sciences 93:684; Qiu et al. (2012) International Journal of Pharmaceutics 437:51); Van Dijk-Wolthuis et al. (1997) Polymer 38:6235-6242. Hydroxyethyl starch is classified as hydroxyethyl starch, hexastarch, pentastarch and tetrastarch (see, e.g., WO 2014 / 099198 to Chow). The bulking agent is preferably present in a concentration of about 0.2M to about 0.5M, suitably about 0.2M to about 0.4M, suitably about 0.3M to about 0.4M, suitably about 0.47M, suitably about 0.36M.

[0221] The bulk reagent may include one or more nucleic acids such as probe oligomers, amplification oligomers, capture probes, positive control templates, and negative control templates. The bulk reagent may include one or more nucleic acids for performing non-amplification-based assays, the nucleic acids comprising one or more probe oligonucleotides. The bulk reagent may include one or more nucleic acids for performing non-amplification-based assays and amplification-based assays, the nucleic acids comprising one or more probe oligonucleotides and / or one or more amplification oligomers and / or one or more amplification oligomer pairs and / or multiple amplification oligomer pairs.

[0222] The optional additional components of the bulk reagent include RNase inhibitors, detergents, zwitterionic detergents, anionic detergents, cationic detergents, non-ionic detergents, surfactants, primers, probes, templates, polymers, biopolymers, oligosaccharides, polysaccharides, polyglucose, amylose, chelating agents, methylparabens and propylparabens. The exemplary concentration of methylparabens is 0.01%-0.024% by weight, such as about 0.016%, or alternatively about 0.010%, about 0.014%, about 0.016%, about 0.020%, about 0.024% and any ranges adjacent to these values. Exemplary concentration ranges for propylparaben are 0.002% to 0.016% or 0.008%, or alternatively about 0.002%, about 0.004%, about 0.006%, about 0.008%, about 0.010%, about 0.012%, about 0.014%, about 0.016%, or any range adjacent to these values. One unit is defined as the amount required to inhibit 5 ng of ribonuclease A activity by 50%. Amount of ribonuclease inhibitor. Activity is measured by ribonuclease A inhibition of the hydrolysis of cytidine 2',3'-cyclic monophosphate.

[0223] The chelating agent comprises one or more of EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-tetraacetic acid), EDDS (ethylenediamine-N,N'-disuccinic acid), MGDA (methylglycine diacetic acid) and DTPA (diethylenetriamine pentaacetic acid). An exemplary concentration of the chelating agent is 1.0 mM-2.5 mM. In one embodiment, EDTA is preferably used. Suitably, EDTA is present in a concentration of about 0.05 mM to about 0.2 mM, suitably about 0.1 mM.

[0224] RNase inhibitory proteins are natural and recombinant 50 kDa proteins (Promega, Madison, Wisconsin) that inhibit RNase A family and human placental RNase by non-covalently binding to RNases in a 1:1 ratio. See Botella-Estrada et al. (2001) Cancer Gene Ther. 8:278; Polakowski et al. (1992) EXS. 61:428. Exemplary concentrations of RNase inhibitors are about 0.04 U / μl to about 0.4 U / μl.

[0225] The bulk reagent may contain a low concentration of detergent. Detergents include ionic (cationic or anionic), nonionic, and zwitterionic detergents available from a number of commercial suppliers (e.g., Geno Technology, Inc., St. Louis, MO). Examples include, but are not limited to, lithium lauryl sulfate, amprolium hydrochloride, benzalkonium chloride, choline p-toluenesulfonate, dodecyltrimethylammonium chloride, 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, hexadecyldimethylethylammonium bromide, hexadecylpyridinium chloride, hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, hexadecyltrimethyl p-toluenesulfonate, luviquat TM , methylbenzethonium chloride, myristyl trimethyl ammonium bromide, N,N′,N′-polyoxyethylene (10)-N-tallow-1,3-diaminopropane liquid, oxyphenonium bromide, tetraheptyl ammonium bromide, tetra(decyl) ammonium bromide, methyl trioctyl ammonium chloride, aminosulfonyl betaine-16, tridodecyl methyl ammonium chloride, octadecyl trimethyl ammonium bromide Nonidet Triton

[0226] Exemplary volumes of bulk reagents include about 1 μl, about 5 μl, about 10 μl, about 20 μl, about 24 μl, about 50 μl, about 100 μl, about 200 μl, about 300 μl, about 400 μl, about 500 μl, 600 μl, about 700 μl, about 800 μl, about 900 μl, about 1,000 μl (1 mL), about 2 mL, about 5 mL, about 10 mL, about 20 mL, about 50 mL, etc. The recombined composition can be formed with the same volume as the bulk reagent, a smaller volume or a larger volume than the bulk reagent. Relative to the bulk reagent, the lower volume can be about 90%, about 80%, about 60%, about 40%, about 20%, about 10% or about 5%. The larger volume can be about 120%, 140%, 160%, 180%, 200% (2-fold), about 4-fold, about 6-fold, about 8-fold, about 10-fold, about 20-fold the volume of the bulk reagent.

[0227] The sample to be analyzed can be added to the bulk reagent before, during or after reconstitution. In a preferred embodiment, the entire dry composition after reconstitution is used to combine with the sample, and the relative volumes of the reconstitution solution / sample can be, for example, about 9.9 / 0.1, 9.8 / 0.2, 9.5 / 0.5, 9 / 1, 8 / 2, 7 / 3, 6 / 4, 5 / 5, etc.

[0228] Unless otherwise indicated, the concentration of an agent in a bulk agent can be, for example, 0.0% (neglecting agent), 0.001%, 0.004%, 0.008%, 0.0012%, 0.0016%, 0.0020%, 0.0030%, 0.0040%, 0.0050%, 0.0060%, 0.0080%, 0.01%, 0.02%, 0.04%, 0.06%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 2%, 3%, 4%, 5%, etc. Also provided are agents that are "about" the above concentrations, less than the above concentrations, greater than the above concentrations, or ranges involving any two of the above concentrations.

[0229] Exemplary bulk reagent compositions are substantially free of glycerol, have an inorganic salt concentration of 5 mM or less, and have 0.1-0.4 mM and suitably about 0.3 mM of each of dATP, dGTP, dCTP, and dUTP or dTTP and 0.02 μg / μl-0.40 μg / μL of flap endonuclease, suitably about 0.03 μg / μl of flap endonuclease. Some compositions contain a swelling agent (suitably trehalose) of about 0.2 M to about 0.4 M (suitably about 0.3 M). Some compositions contain a buffer (suitably MOPS buffer), suitably having a pH of about 7.5.

[0230] Another exemplary bulk reagent composition is substantially free of glycerol, has an inorganic salt concentration of 5 mM or less, and has 0.1-0.4 mM and suitably about 0.375 mM of each of dATP, dGTP, dCTP and about 0.182 mM dUTP or about 0.284 mM dTTP, 0.02 μg / μl-0.40 μg / μL flap endonuclease (suitably about 0.03 μg / μl flap endonuclease) and 0.1 U / μl to 0.2 U / μl (suitably 0.146 U / μl) of polymerase. Some compositions include about 0.2 M to about 0.4 M (suitably about 0.3 M) of swelling agent (suitably trehalose). Some compositions include a buffer (suitably MOPS buffer), suitably having a pH of about 7.5.

[0231] Another exemplary bulk reagent composition is substantially free of glycerol, has an inorganic salt concentration of 5 mM or less, and has 0.1-0.4 mM and suitably about 0.375 mM each of dATP, dGTP, dCTP and about 0.182 mM dUTP or about 0.284 mM dTTP, 0.02 μg / μl-0.40 μg / μL flap endonuclease (suitably about 0.035 μg / μl flap endonuclease), 0.1 U / μl to 0.2 U / μl (suitably 0.146 U / μl) polymerase, about 0.2 M to about 0.4 M (suitably about 0.3 M) of a swelling agent (suitably trehalose) and suitably a buffer having a pH of about 7.5 (suitably MOPS buffer).

[0232] Another exemplary bulk reagent composition is substantially free of glycerol, has an inorganic salt concentration of 5 mM or less, and has 0.1-0.4 mM and suitably about 0.375 mM of each of dATP, dGTP, dCTP and about 0.182 mM of dUTP or about 0.284 mM of dTTP and 0.02 μg / μl-0.40 μg / μL of flap endonuclease, suitably about 0.03 μg / μl of flap endonuclease. Some compositions contain about 0.2M to about 0.4M (suitably about 0.3M) of a swelling agent (suitably trehalose). Some compositions contain globular proteins, suitably bovine serum albumin (BSA), more suitably ultrapure non-acetylated BSA. Globular proteins can be present in an amount of 0.40 μg / μl to 0.60 μg / μl (suitably 0.50 μg / μl). Some compositions comprise a buffer suitably at a pH of about 8.5 (suitably Tris buffer).

[0233] Another exemplary bulk reagent composition is substantially free of glycerol, has an inorganic salt concentration of 5 mM or less, and has 0.1-0.4 mM and suitably about 0.375 mM each of dATP, dGTP, dCTP and about 0.182 mM dUTP or about 0.284 mM dTTP and 0.02 μg / μl-0.40 μg / μL flap endonuclease (suitably about 0.03 μg / μl flap endonuclease) and 0.1 U / μl to 0.2 U / μl (suitably 0.146 U / μl) of polymerase. Some compositions contain about 0.2 M to about 0.4 M (suitably about 0.3 M) of a swelling agent (suitably trehalose). Some compositions contain a globular protein, suitably bovine serum albumin (BSA), more suitably ultrapure non-acetylated BSA. The globular protein may be present in an amount of 0.40 μg / μl to 0.60 μg / μl (suitably 0.50 μg / μl).Some compositions comprise a buffer (suitably Tris buffer) suitably at a pH of about 8.5.

[0234] Another exemplary bulk reagent composition is substantially free of glycerol, has an inorganic salt concentration of 5 mM or less, and has 0.1-0.4 mM and suitably 0.3 mM each of dATP, dGTP, dCTP and dUTP or dTTP and 0.02 μg / μl-0.40 μg / μl flap endonuclease (suitably about 0.03 μg / μl flap endonuclease) and 0.1 U / μl to 0.2 U / μl (suitably The invention relates to a method for preparing an aqueous solution of at least 200 μg of the present invention and a method for preparing the aqueous solution of the aqueous solution of at least 200 μg of the aqueous solution. The method comprises the following steps: comprising: preparing the aqueous solution of at least 200 μg of the aqueous solution and the aqueous solution of at least 200 μg of the aqueous solution. The method comprises the step of: preparing the aqueous solution of at least 200 μg of the aqueous solution and the aqueous solution of at least 200 μg of the aqueous solution. The method comprises the step of: preparing the aqueous solution of at least 200 μg of the aqueous solution and the aqueous solution of at least 200 μg of the aqueous solution.

[0235] Exemplary bulk reagent compositions are substantially free of glycerol, have an inorganic salt concentration of 5 mM or less, and have 0.1-0.4 mM and suitably about 0.375 mM each of dATP, dGTP, dCTP, and dUTP or dTTP and 0.02 μg / μl-0.40 μg / μl of flap endonuclease, suitably about 0.035 μg / μl of flap endonuclease. In some compositions, the flap endonuclease is enzyme. Some compositions comprise a bulking agent (suitably trehalose) of about 0.2M to about 0.4M (suitably about 0.36M). Some compositions comprise a globular protein, suitably bovine serum albumin (BSA), more suitably ultrapure non-acetylated BSA. The globular protein may be present in an amount of 0.40 μg / μl to 0.60 μg / μl (suitably 0.50 μg / μl). Some compositions comprise a buffer (suitably MOPS buffer) suitably having a pH of about 7.5.

[0236] Another exemplary bulk reagent composition is substantially free of glycerol, has an inorganic salt concentration of 5 mM or less, and has 0.1-0.4 mM and suitably 0.375 mM each of dATP, dGTP, dCTP, and dUTP or dTTP and 0.02 μg / μl-0.40 μg / μL flap endonuclease (suitably about 0.035 μg / μl flap endonuclease) and 0.1 U / μl to 0.2 U / μl (suitably 0.167 U / μl) polymerase. Some compositions contain about 0.2 M to about 0.4 M (suitably about 0.36 M) of a swelling agent (suitably trehalose). Some compositions contain a globular protein, suitably bovine serum albumin (BSA), more suitably ultrapure non-acetylated BSA. The globular protein may be present in an amount of 0.40 μg / μl to 0.60 μg / μl (suitably 0.50 μg / μl).Some compositions comprise a buffer (suitably a MOPS buffer) suitably at a pH of about 7.5.

[0237] Another exemplary bulk reagent composition is substantially free of glycerol, has an inorganic salt concentration of 5 mM or less, and has 0.1-0.4 mM and suitably 0.375 mM each of dATP, dGTP, dCTP and dUTP or dTTP and 0.02 μg / μl-0.40 μg / μL flap endonuclease (suitably about 0.035 μg / μl flap endonuclease) and 0.1 U / μl to 0.2 U / μl (suitably 0.167 U / μl) of polymerase, about 0.2M to about 0.4M (suitably about 0.36M) of a swelling agent (suitably trehalose), a globular protein (suitably bovine serum albumin (BSA), more suitably ultrapure non-acetylated BSA) and a buffer (suitably MOPS buffer) having a pH of about 7.5 (such as 15mM MOPS buffer), wherein the globular protein is present in an amount of 0.40μg / μl to 0.60μg / μl (suitably 0.50μg / μl).

[0238] Another exemplary bulk reagent composition is substantially free of glycerol, has an inorganic salt concentration of 5 mM or less, and has 0.1-0.4 mM and suitably 0.375 mM each of dATP, dGTP, dCTP and dUTP or dTTP and 0.02 μg / μl-0.40 μg / μL of flap endonuclease (suitably about 0.035 μg / μl of flap endonuclease) and 0.1 U / μl to 0.2 U / μl (suitably 0.169 U / μl) of polymerase, about 0.2 M to about 0.4 M ( Suitably about 0.36M) of a swelling agent (suitably trehalose), a globular protein (suitably bovine serum albumin (BSA), more suitably ultrapure non-acetylated BSA), a buffer (suitably MOPS buffer) having a pH value of about 7.5 (such as 15mM MOPS buffer) and a chelating agent (suitably EDTA) having a concentration of about 0.05mM to 0.2mM (suitably about 0.15mM), the globular protein being present in an amount of 0.40μg / μl to 0.60μg / μl (suitably 0.51μg / μl).

[0239] After the body reagent composition forms, can be placed in room temperature and continue quite long time period before drying.Before drying step starts, described time period can be up to 8 hours or alternately up to 1 hour, up to 2 hours, up to 4 hours, up to 6 hours, up to 10 hours, up to 12 hours, up to 14 hours before drying step starts.During this incubation period, comprising salt in the body reagent causes undesirable hybridization product and other by product.Reduce or eliminate this undesirable hybridization product and by product by forming the body reagent composition that is substantially free of inorganic salts.

[0240] The presence of inorganic salts in the bulk reagent may result in one or more of the following undesirable characteristics. Nucleic acids may hybridize together, and hybridization is stimulated by the presence of inorganic salts such as potassium, sodium, manganese, magnesium and / or chloride. Also in the presence of inorganic salts such as manganese and magnesium, undesirable enzyme activity may occur. Due to the nucleic acid hybridization and enzyme activity that occurs in the presence of salts, undesirable by-products may begin to form. In addition, inorganic salts are hygroscopic and will absorb moisture into the dry pellets. Rehydration of the dry pellets reduces storage stability, enzyme stability, and allows the formation of additional parasitic byproducts.

[0241] The dried pellets may contain reagents to provide a single unit dose (SUD) or optionally two or more SUDs. A single unit dose is a collection of reagents required for a nucleic acid-based assay for no more than a single sample. A single unit dose may refer to a liquid reagent or a dried pellet. It is noteworthy that a single unit dose as mentioned herein need not contain all the reagents required for a nucleic acid-based assay for a single sample. A single unit dose may lack the reagents required for a nucleic acid-based assay reaction. Similarly, a single unit dose may contain insufficient amounts of reagents for performing a nucleic acid-based assay reaction. By way of example only, a dried single unit dose pellet may include sufficient units of a flap endonuclease for performing a nucleic acid-based assay, but may not contain magnesium. In such examples, magnesium may be added to a dried single unit dose pellet, such as by means of a recombinant solution. Also by way of example only, a dried single unit dose may include insufficient amounts of dNTPs for performing a nucleic acid-based assay. In such examples, the remainder of the dNTPs may be added to a dried single unit dose pellet, such as by means of a recombinant solution. Because these examples are non-limiting, one of ordinary skill in the art armed with the present disclosure will readily generate SUDs and dried pellet SUDs having different compositions.

[0242] In a preferred embodiment, the bulk reagent includes an inorganic salt content of 5mM or less, more preferably an inorganic salt content of 4mM or less, more preferably an inorganic salt content of 3mM or less, more preferably an inorganic salt content of 2mM or less, more preferably an inorganic salt content of 1mM or less, or more preferably an inorganic salt content of 500μM or less. Therefore, the preferred concentration range of the inorganic salt in the bulk reagent is an inorganic salt content of about 5mM to 0mM (including all total and partial values ​​therein). Common inorganic salts for nucleic acid-based assay reaction mixtures include one or more of sodium, potassium, manganese, magnesium, and chloride, etc.

[0243] On the one hand, the bulk reagent includes 5mM or less inorganic salts, and the inorganic salt is present in every microliter of mass of 0.373μg / μl or less, or 0.332μg / μl or less or 0.292μg / μl or less. On the other hand, the bulk reagent includes 5mM or less inorganic salts, and sodium chloride is present in every microliter of mass of 0.292μg / μl or less, 0.146μg / μl or less or 0.0μg / μl. On the other hand, the bulk reagent includes 5mM or less inorganic salts, and sodium is present in every microliter of mass of 0.115μg / μl or less, 0.057μg / μl or less or 0.0μg / μl. On the other hand, the bulk reagent includes 5mM or less inorganic salts, and potassium chloride is present in every microliter of mass of 0.373μg / μl or less, 0.186μg / μl or less or 0.0μg / μl. In another aspect, the bulk reagent includes 5 mM or less of an inorganic salt, and potassium is present at a mass per microliter of 0.196 μg / μl or less, 0.098 μg / μl or less, or 0.0 μg / μl. In another aspect, the bulk reagent includes 5 mM or less of an inorganic salt, and chloride is present at a mass per microliter of 0.355 μg / μl or less, 0.178 μg / μl or less, 0.089 μg / μl or less, or 0.0 μg / μl. In another aspect, the dry pellet is made by drying a liquid bulk reagent including 5 mM or less of an inorganic salt, and the mass of the inorganic salt to the mass of the pellet is 0.311% or less, 0.277% or less, or 0.244% or less. In another aspect, a vessel containing dried single unit dose granules is provided, wherein the mass of inorganic salt to the mass of granules is 0.311% or less, 0.277% or less, or 0.244% or less. In another aspect, a multiwell plate comprising one or more wells is provided, wherein each of the one or more wells contains dried single unit dose granules, wherein the mass of inorganic salt contained in the single unit dose granules is 0.311% or less, 0.277% or less, or 0.244% or less to the mass of granules.

[0244] On the one hand, the bulk reagent includes 4mM or less inorganic salts, and the inorganic salt is present in every microliter of mass of 0.298μg / μl or less, or 0.266μg / μl or less, or 0.234μg / μl or less. On the other hand, the bulk reagent includes 4mM or less inorganic salts, and sodium chloride is present in every microliter of mass of 0.234μg / μl or less, 0.117μg / μl or less, or 0.0μg / μl. On the other hand, the bulk reagent includes 4mM or less inorganic salts, and sodium is present in every microliter of mass of 0.092μg / μl or less, 0.046μg / μl or less, or 0.0μg / μl. On the other hand, the bulk reagent includes 4mM or less inorganic salts, and potassium chloride is present in every microliter of mass of 0.298μg / μl or less, 0.149μg / μl or less, or 0.0μg / μl. In another aspect, the bulk reagent includes 4 mM or less of an inorganic salt, and potassium is present at a mass per microliter of 0.156 μg / μl or less, 0.078 μg / μl or less, or 0.0 μg / μl. In another aspect, the bulk reagent includes 4 mM or less of an inorganic salt, and chloride is present at a mass per microliter of 0.284 μg / μl or less, 0.142 μg / μl or less, 0.071 μg / μl or less, or 0.0 μg / μl. In another aspect, the dry pellet is made by drying a liquid bulk reagent including 4 mM or less of an inorganic salt, and the mass of the inorganic salt to the mass of the pellet is 0.249% or less, 0.222% or less, or 0.195% or less. In another aspect, a vessel containing dried single unit dose granules is provided, wherein the mass of inorganic salt to the mass of granules is 0.249% or less, 0.222% or less, or 0.195% or less. In another aspect, a multiwell plate comprising one or more wells is provided, wherein each of the one or more wells contains dried single unit dose granules, wherein the mass of inorganic salt contained in the single unit dose granules is 0.249% or less, 0.222% or less, or 0.195% or less to the mass of granules.

[0245] On the one hand, the bulk reagent includes 3mM or less inorganic salts, and the inorganic salt is present in every microliter of mass of 0.224μg / μl or less, or 0.199μg / μl or less, or 0.175μg / μl or less. On the other hand, the bulk reagent includes 3mM or less inorganic salts, and sodium chloride is present in every microliter of mass of 0.175μg / μl or less, 0.088μg / μl or less, or 0.0μg / μl. On the other hand, the bulk reagent includes 3mM or less inorganic salts, and sodium is present in every microliter of mass of 0.069μg / μl or less, 0.034μg / μl or less, or 0.0μg / μl. On the other hand, the bulk reagent includes 3mM or less inorganic salts, and potassium chloride is present in every microliter of mass of 0.224μg / μl or less, 0.112μg / μl or less, or 0.0μg / μl. In another aspect, the bulk reagent includes 3 mM or less of an inorganic salt, and potassium is present at a mass per microliter of 0.117 μg / μl or less, 0.059 μg / μl or less, or 0.0 μg / μl. In another aspect, the bulk reagent includes 3 mM or less of an inorganic salt, and chloride is present at a mass per microliter of 0.213 μg / μl or less, 0.107 μg / μl or less, 0.053 μg / μl or less, or 0.0 μg / μl. In another aspect, the dry pellet is made by drying a liquid bulk reagent including 3 mM or less of an inorganic salt, and the mass of the inorganic salt to the mass of the pellet is 0.186% or less, 0.166% or less, or 0.146% or less. In another aspect, a vessel containing dried single unit dose granules is provided, wherein the mass of inorganic salt to the mass of granules is 0.186% or less, 0.166% or less, or 0.146% or less. In another aspect, a multiwell plate comprising one or more wells is provided, wherein each of the one or more wells contains dried single unit dose granules, wherein the mass of inorganic salt contained in the single unit dose granules is 0.186% or less, 0.166% or less, or 0.146% or less to the mass of granules.

[0246] On the one hand, the bulk reagent includes 2mM or less inorganic salts, and the inorganic salt is present in every microliter of mass of 0.149μg / μl or less, or 0.133μg / μl or less, or 0.117μg / μl or less. On the other hand, the bulk reagent includes 2mM or less inorganic salts, and sodium chloride is present in every microliter of mass of 0.117μg / μl or less, 0.058μg / μl or less, or 0.0μg / μl. On the other hand, the bulk reagent includes 2mM or less inorganic salts, and sodium is present in every microliter of mass of 0.046μg / μl or less, 0.023μg / μl or less, or 0.0μg / μl. On the other hand, the bulk reagent includes 2mM or less inorganic salts, and potassium chloride is present in every microliter of mass of 0.149μg / μl or less, 0.075μg / μl or less, or 0.0μg / μl. In another aspect, the bulk reagent includes 2 mM or less of an inorganic salt, and potassium is present at a mass per microliter of 0.078 μg / μl or less, 0.039 μg / μl or less, or 0.0 μg / μl. In another aspect, the bulk reagent includes 2 mM or less of an inorganic salt, and chloride is present at a mass per microliter of 0.142 μg / μl or less, 0.071 μg / μl or less, 0.036 μg / μl or less, or 0.0 μg / μl. In another aspect, the dry pellet is made by drying a liquid bulk reagent including 2 mM or less of an inorganic salt, and the mass of the inorganic salt to the mass of the pellet is 0.124% or less, 0.111% or less, or 0.097% or less. In another aspect, a vessel containing dried single unit dose granules is provided, wherein the mass of inorganic salt to the mass of granules is 0.124% or less, 0.111% or less, or 0.097% or less. In another aspect, a multiwell plate comprising one or more wells is provided, wherein each of the one or more wells contains dried single unit dose granules, wherein the mass of inorganic salt contained in the single unit dose granules is 0.124% or less, 0.111% or less, or 0.097% or less to the mass of granules.

[0247] On the one hand, the bulk reagent includes 1mM or less inorganic salt, and the inorganic salt is present in every microliter of mass of 0.075μg / μl or less, or 0.066μg / μl or less or 0.058μg / μl or less. On the other hand, the bulk reagent includes 1mM or less inorganic salt, and sodium chloride is present in every microliter of mass of 0.058μg / μl or less, 0.029μg / μl or less or 0.0μg / μl. On the other hand, the bulk reagent includes 1mM or less inorganic salt, and sodium is present in every microliter of mass of 0.023μg / μl or less, 0.011μg / μl or less or 0.0μg / μl. On the other hand, the bulk reagent includes 1mM or less inorganic salt, and potassium chloride is present in every microliter of mass of 0.075μg / μl or less, 0.037μg / μl or less or 0.0μg / μl. In another aspect, the bulk reagent includes 1 mM or less of an inorganic salt, and potassium is present at a mass per microliter of 0.039 μg / μl or less, 0.020 μg / μl or less, or 0.0 μg / μl. In another aspect, the bulk reagent includes 1 mM or less of an inorganic salt, and chloride is present at a mass per microliter of 0.071 μg / μl or less, 0.036 μg / μl or less, 0.018 μg / μl or less, or 0.0 μg / μl. In another aspect, the dry pellet is made by drying a liquid bulk reagent including 1 mM or less of an inorganic salt, and the mass of the inorganic salt to the mass of the pellet is 0.062% or less, 0.055% or less, or 0.049% or less. In another aspect, a vessel containing dried single unit dose granules is provided, wherein the mass of inorganic salt to the mass of granules is 0.062% or less, 0.055% or less, or 0.049% or less. In another aspect, a multiwell plate comprising one or more wells is provided, wherein each of the one or more wells contains dried single unit dose granules, wherein the mass of inorganic salt contained in the single unit dose granules is 0.062% or less, 0.055% or less, or 0.049% or less to the mass of granules.

[0248] On the one hand, the bulk reagent includes 500 μM or less of inorganic salts, and the inorganic salt is present at 0.037 μg / μl or less, or 0.033 μg / μl or less, or 0.029 μg / μl or less per microliter mass. On the other hand, the bulk reagent includes 500 μM or less of inorganic salts, and sodium chloride is present at 0.029 μg / μl or less, 0.015 μg / μl or less, or 0.0 μg / μl per microliter mass. On the other hand, the bulk reagent includes 500 μM or less of inorganic salts, and sodium is present at 0.011 μg / μl or less, 0.006 μg / μl or less, or 0.0 μg / μl per microliter mass. In another aspect, the bulk reagent includes 500 μM or less of an inorganic salt, and potassium chloride is present at 0.037 μg / μl or less, 0.019 μg / μl or less, or 0.0 μg / μl per microliter. In another aspect, the bulk reagent includes 500 μM or less of an inorganic salt, and potassium is present at 0.020 μg / μl or less, 0.010 μg / μl or less, or 0.0 μg / μl per microliter. In another aspect, the bulk reagent includes 500 μM or less of an inorganic salt, and chloride is present at 0.036 μg / μl or less, 0.018 μg / μl or less, 0.009 μg / μl or less, or 0.0 μg / μl per microliter. On the other hand, the dry granules are made by drying a liquid bulk reagent including 500 μM or less of an inorganic salt, and the mass of the inorganic salt to the mass of the granules is 0.031% or less, 0.028% or less, or 0.024% or less. On the other hand, a vessel containing dry single unit dose granules is provided, wherein the mass of the inorganic salt to the mass of the granules is 0.031% or less, 0.028% or less, or 0.024% or less. On the other hand, a multiwell plate including one or more wells is provided, wherein each of the one or more wells contains dry single unit dose granules, and the mass of the inorganic salt contained in the single unit dose granules is 0.031% or less, 0.028% or less, or 0.024% or less, to the mass of the granules.

[0249] On the one hand, the bulk reagent includes an inorganic salt of about 5mM to about 500μM, and the inorganic salt is present in a mass per microliter of about 0.373μg / μl to about 0.029μg / μl. On the other hand, the bulk reagent includes an inorganic salt of about 5mM to about 500μM, and sodium chloride is present in a mass per microliter of 0.292μg / μl to about 0.029μg / μl. On the other hand, the bulk reagent includes an inorganic salt of about 5mM to about 500μM, and sodium is present in a mass per microliter of 0.115μg / μl to about 0.006μg / μl. On the other hand, the bulk reagent includes an inorganic salt of about 5mM to about 500μM, and potassium chloride is present in a mass per microliter of about 0.373μg / μl to about 0.019μg / μl. In another aspect, the bulk reagent includes about 5 mM to about 500 μM of an inorganic salt, and potassium is present at 0.196 μg / μl to about 0.010 μg / μl per microliter. In another aspect, the bulk reagent includes about 5 mM to about 500 μM of an inorganic salt, and chloride is present at 0.355 μg / μl to about 0.009 μg / μl per microliter. In another aspect, the bulk reagent includes about 5 mM to about 500 μM of an inorganic salt, the inorganic salt is present at about 0.373 μg / μl to about 0.029 μg / μl per microliter, and sodium chloride is present at about 0 μg / μl per microliter. In another aspect, the bulk reagent includes about 5 mM to about 500 μM of an inorganic salt, the inorganic salt being present at about 0.373 μg / μl to about 0.029 μg / μl per microliter of mass, and potassium chloride being present at about 0 μg / μl per microliter of mass. In another aspect, the dry granules are made by drying a liquid bulk reagent including 5 mM to 500 μM of an inorganic salt, and the mass of the inorganic salt to the mass of the granules is about 0.311% to 0.024%. In another aspect, the dry granules are made by drying a liquid bulk reagent including 5 mM to 500 μM of an inorganic salt, and the mass of the inorganic salt to the mass of the granules is about 0.311% to 0.024%, and the mass of sodium chloride to the mass of the granules is about 0%, or the mass of potassium chloride to the mass of the granules is about 0%. In another aspect, a container containing dry single unit dose granules is provided, wherein the mass percentage of inorganic salt to the mass percentage of granules is about 0.311% to 0.024%, and the mass percentage of sodium chloride to the mass percentage of granules is about 0%, or the mass percentage of potassium chloride to the mass percentage of granules is about 0%.On the other hand, a porous plate comprising one or more wells is provided, wherein each of the one or more wells contains a dry single unit dose granule, wherein the mass percentage of the inorganic salt contained in the single unit dose granule is about 0.311% to 0.024% by mass of the granule, and the mass percentage of the sodium chloride contained in the granule is about 0%, or the mass percentage of the potassium chloride contained in the granule is about 0%.

[0250] In one embodiment, a porous plate comprising one or more holes is provided. On the one hand, one or more holes include walls, which are constructed of materials including low moisture vapor transmission rate, thermal conductivity, optical transparency, low autofluorescence or a combination thereof. On the one hand, one or more holes include conical walls. On the one hand, the hole includes a wall, which is configured to be assembled into a heat-conducting tube receiving area of ​​a device for performing a nucleic acid-based assay reaction. On the one hand, one or more holes of the porous plate include an opening for entering the chamber of the hole. On the one hand, one or more holes each include a lid that seals the opening of the relevant hole. On the one hand, the opening of each hole in the one or more holes is sealed with a lid, which is a foil with low moisture vapor transmission rate. On the one hand, the opening of each hole in the one or more holes is sealed with a lid, which is an elastomeric material with low moisture vapor transmission rate. On the one hand, the porous plate includes one or more holes as described herein, and the chamber of the hole contains a dry single unit dose granule including a flap endonuclease and an inorganic salt, wherein the mass of the inorganic salt to the mass of the granule is about 0.311% to 0.024%.

[0251] III. Drying Equipment and Methods

[0252] Bulk reagents can be lyophilized using standard methods and equipment. Freeze dryers are available, for example, from GEA Process Engineering, Columbia, Maryland. Contract freeze drying services are provided by companies such as Biopharma Technology Ltd., Winchester, Hampshire, England, and Baxter Healthcare Corp, Deerfield, Illinois, for BioPharma Solutions Sterile Contract Manufacturing. Freeze drying guidance can be obtained from, for example, L. Rey, JC May (eds.) (2010) Freeze Drying / Lyophilization of Pharmaceuticals and Biological Products, 3rd ed., Informa Healthcare, New York; or Methods in Enzymology, Vol. 22, pp. 33-39, Academic Press, New York (1971); or Freeze-Drying, EW Flosdorf, Rheinhold, New York (1949). Optionally, the oxygen content can be reduced during freeze-drying (Phillips et al. (2001) Biologics. 19:219).

[0253] A wide variety of containers are suitable for drying. The container should be able to withstand external pressure when it is sealed and stored under partial vacuum. The container should be made of a material that allows reasonable transfer of heat from the outside to the inside. The size of the container is preferably such that the solution to be dried does not occupy more than 20% of the total volume to avoid overflow.

[0254] The sample can be dried in a separate vessel or a multi-sample vessel. A multi-sample vessel means a continuous vessel that can contain at least two samples, so that the at least two samples can be stored and manipulated in parallel but individually. The standard format of the multi-sample utensil comprises 6, 24, 96, 384 or 1536 holes. In the example of the 96-hole format, the volume of each hole is about 300-400 microliters, and the working volume is about 75-200 microliters. The volume usually changes inversely with the number of holes, although other sizes are also envisioned, but typically in the range between 1nL and 10mL. Exemplary holes can have flat bottoms, round bottoms or V-shaped bottoms, etc. As used herein, vessels are also referred to as holes. As used herein, multi-sample vessels are also referred to as porous plates. In addition, holes are sometimes further referred to as reaction holes. The term reaction hole does not require any actual reaction to occur in the reaction hole. On the contrary, the term is used to refer to vessels or holes containing reagents and may not react therein, partially react therein or react completely therein.

[0255] In some embodiments herein, the porous plate can undergo freeze drying to form a dry composition from an aqueous solution. Freeze drying can occur in a nest device (see co-pending application U.S. Provisional Application No. 62 / 200,370). A nest is a container for a box with a vent that can be closed by a mechanism operable from the outside of a sealed freeze-drying chamber. A nest containing a porous plate is placed in a freeze-drying chamber having one or more vents in an open position. The chamber is then sealed, and a freeze-drying atmosphere is applied throughout the chamber containing the space in the nest. One or more vents are then closed, thereby sealing the nest. The seal on the freeze-drying chamber is subsequently released, and the nest containing the porous plate is removed. The nest can then be repositioned and stored with the porous plate located therein until the operator is ready to use the freeze-dried composition located therein or reseal the porous plate containing the freeze-dried sample for further storage or sale. The holes of the porous plate can then be sealed, which substantially inhibits moisture from entering from ambient air. Small amounts of moisture can be prevented from entering the sealed multiwell plates by storing the sealed multiwell plates in a pouch containing desiccant. Similarly, individual vessels can be lyophilized and can be lyophilized in nests.

[0256] Other drying methods include spray drying, fluidized bed drying, dehumidifiers, and batch contact drying, where the filter cake is dried under vacuum at low temperatures to a free-flowing dry product (NP Cheremisinoff (2000) Handbook of Chemical Processing Equipment, Butterworth Heinemann, Boston, Massachusetts). Dehumidifiers are available from Bry Air, Inc., Sunbury, Ohio, and DST SeibuGiken, Wyomiss, Pennsylvania. Rotary dryers, conical dryers, and rack dryers are available (McGill Air Pressure LLC, Columbus, Ohio). In one embodiment, a vacuum dryer removes moisture by exposing the material to reduced pressure, where just enough heat is used to replace the heat lost by evaporation. Desiccants include silica gel desiccants, molecular sieve desiccants such as aluminosilicates and synthetic zeolites, and natural active desiccants.

[0257] The reaction mixture is preferably dried in the same vessel in which it will be reconstituted.

[0258] Lyophilized or otherwise dried formulations have a low moisture content, e.g., less than 5% water by weight, less than 4%, less than 3%, less than 2%, less than 1.0%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.02%, less than 0.01%, etc. by weight.

[0259] IV. Storage

[0260] Freeze-dried compositions or otherwise dry compositions are subjected to storage before use. The shelf life can comprise the time period that the dried composition is exposed to ambient air storage at room temperature. This time period can be up to 3 hours or alternatively up to 1.0 hours, up to 1.5 hours, up to 2.0 hours, up to the scope of any time in 2.5 hours or the time, such as 1 minute to 180 minutes or 1 minute to 100 minutes or 1 minute to 60 minutes or 1 minute to 30 minutes or 1 minute to 20 minutes or 1 minute to 10 minutes. The absolute humidity during this storage period can be at least 2.3g water per cubic meter of air at 30 ℃ or alternatively at 30 ℃ per cubic meter of air greater than 1.8 grams, 2.0 grams, 2.2 grams, 2.4 grams, 2.6 grams, 2.8 grams, 3.0 grams of water.

[0261] Storage can also include longer time periods during which the dry composition is sealed, which substantially prevents contact with ambient air outside the seal. This storage period can be long, for example, at least one week, at least one month, at least six months, at least one year, or at least two years. Time periods from one month to two years are exemplary.

[0262] The range of storage temperatures for long-term storage or for long-term or short-term stability studies includes, for example, 0-2°C, 0-4°C, 2-4°C, 2-6°C, 20°C, 25°C, 30°C, 40°C, 50°C, 60°C and sub-zero temperatures under liquid nitrogen, such as -4°C to -2°C, -6°C to -2°C, -8°C to -2°C, -10°C to -2°C, -20°C, -40°C, -60°C, -80°C. Preferably, storage is above freezing and in the range of about 4-8°C. Accelerated degradation studies can be conducted at about 25°C, about 30°C, about 35°C, about 40°C for a period of time, for example, one hour, two hours, four hours, 24 hours, two days, four days, eight days, one month, etc. Storage conditions or alternatively stability conditions that can be tested can be conditions of temperature fluctuations, such as those that fluctuate from above freezing to below freezing.

[0263] During storage of the bulk reagent before drying, during short-term storage of the dried composition before sealing, and during long-term storage after sealing, the substantial lack of inorganic salts reduces the loss of enzyme activity and the formation of by-products. Preferably, the enzyme activity after all storage is at least 99%, at least 98%, at least 95%, at least 90%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20% of the value immediately before storage, and the range of these values ​​before storage or alternatively to the value of the comparator sample stored under optimal conditions defined by these percentages.

[0264] V. Reorganization

[0265] The preferred reconstitution solution provides about 3.0 mM to about 12.0 mM MgCl2 and about 0 mM to about 80 mM KCl in water. The reconstitution solution may also contain, among other components, about 0.012 to about 0.020% (w / v) methylparaben, about 0.006 to about 0.010% (w / v) propylparaben, and / or about 0.25 to about 0.35% (w / v) anhydrous ethanol.

[0266] The reconstitution time can be less than 1 second, less than 2 seconds, less than 5 seconds, less than 10 seconds, less than 15 seconds, less than 20 seconds, less than 50 seconds or less than 60 seconds (1 minute), after the aqueous solution suitable for the intended use of the dry composition is contacted with the dry composition, optionally by shaking, tapping vortex, shaking, pipette tip aspiration and aspiration, or folding or squeezing the ductile vial to promote contact. Exemplary reconstitution times are 2-10 seconds. The reconstitution time can be measured with a reconstitution solution at any refrigerator temperature (about 4°C), an ambient temperature solution of about 23°C, or a warm solution of about 37°C. Typically, the dry composition has been removed from the refrigerator and is ice cold before adding the reconstitution solution. The environment (room temperature) of any of these procedures is typically ambient temperature or about 23°C. Determining the time when the substance is reconstituted can be, for example, determining the time when the substance is completely dissolved. Complete dissolution can be determined by visual inspection, for example, where the absence of turbidity or the absence of a Schlieren pattern is a measure of complete dissolution. Alternatively, complete dissolution can be determined by optical instruments, such as a machine that measures light scattering.

[0267] VI. Stability of the composition

[0268] Typically, the stability of the composition is assessed after reconstitution of the dried product by determining the activity (i.e., the rate or yield of detection) or the formation of by-products produced in the composition. Lack of stability may be caused by loss of activity or by-product formation during storage before or after drying. Activity or by-product formation may be an absolute or relative measure. If relative, the baseline for comparison may be the bulk reagent mixture before drying and reconstitution or the bulk reagent mixture in a defined manner (e.g., the presence of Mg). 2+ or other salts) and the mixture under test. Activity can be assessed by the rate of real-time cleavage or amplification or the final yield or hit rate of the cleavage or amplification product. Byproducts can be measured by one or more of gel electrophoresis, gel scanner, agarose gel, capillary electrophoresis, etc.

[0269] The activity of the recombined amplification mixture (correcting for any differences due to different recombinant volumes, if necessary) is preferably indistinguishable from the experimental error of the bulk reagent before drying within 75%, 80%, 85%, 90% or 95% or within experimental error. The byproducts present in the recombined amplification mixture (correcting for any differences due to different recombinant volumes, if necessary) are preferably less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% by weight or the average number of moles of the original compound present in the bulk reagent before drying. Sometimes the byproducts are below the detection limit.

[0270] VII.Medicine Box

[0271] The dry composition described above can be provided in a test kit. Such a test kit can contain the dry composition in a vessel such as a tube. In certain embodiments, the test kit contains a porous plate comprising one or more holes. Some test kits contain a variety of dry compositions supplied in a separate vessel. Some test kits contain one or more porous plates, and the one or more porous plates contain a variety of dry compositions in one or more sealed hole members of a multi-walled porous plate.

[0272] Some kits also include a reconstitution solution in a separate vessel from the dry composition. The reconstitution solution may be provided in bulk for aliquoting into separate dry composition vessels, or may be provided in one or more unit doses, each unit dose being intended for combination with a single vessel containing the dry composition.

[0273] Optionally, the vessel containing the dry composition and the vessel containing the reconstitution solution can be separated by a frangible material. The frangible material can be aluminum foil, polypropylene, polyester, polyvinyl chloride (PVC), polyethylene. The partition can contain one, two, three or more layers, each layer having the same composition, or each layer having a different composition, such as a foil layer in contact with a PVC layer. The membrane can be obtained from, for example, Dow Chemical Co., Midland, Michigan, or Arkema, Inc., King of Prussia, Pennsylvania. Penetrating the frangible material allows the reconstitution solution to contact the lyophilized composition.

[0274] The kit may be designed to fit into a thermal cycler or incubator so that the enzymatic reaction occurs directly within the compartments of the kit to avoid the need to transfer the composition to different reaction vessels or containers containing such vessels.

[0275] The test kit can be adapted to introduce user-supplied reagents into vessels within the test kit, for example, through a port, a hose, a syringe piercing a septum (see US2014 / 0121515 and US 2014 / 0276356), or alternatively, user-supplied reagents such as nucleic acid templates can be mixed with reagents of the present disclosure in a user-supplied container. One or more of the test kit compartments can be supplied in an empty state and used as a mixing chamber.

[0276] VIII. Dialysis of flap endonucleases

[0277] Some enzymes used in the present disclosure are manufactured in buffers containing glycerol. Glycerol content may be detrimental to lyophilization. Therefore, it is necessary to prepare a glycerol-free enzyme solution and a solution substantially free of inorganic salts by dialyzing the enzyme into a glycerol-free buffer. This dialysis can be used to substantially remove the glycerol and replace it with a buffer. Then, the dialyzed enzyme can be used to prepare a glycerol-free lyophilized formulation substantially free of inorganic salts. Therefore, a dialysis composition is also disclosed herein, comprising, consisting of, or substantially consisting of an aqueous solution containing an organic buffer, a swelling agent, chloride ions, and a chelating agent. The swelling agent can be trehalose. The swelling agent can be present in a concentration of about 100 mM to about 300 mM, suitably 200 mM. The organic buffer can be a tris (hydroxymethyl) aminomethane (Tris) buffer. The Tris buffer can be present in a concentration of 10 mM to 30 mM, suitably 50 mM. The chloride ion can be KCl. The chloride ions may be present in a concentration of about 40mM to 60mM, suitably 50mM. The chelating agent may be EDTA. The chelating agent may be present in the aqueous solution in a concentration of 0.05mM to 0.2mM, suitably 0.1mM. The dialysis composition may include a flap endonuclease.

[0278] An exemplary dialysis composition is an aqueous solution comprising, consisting of, or consisting essentially of Tris buffer (pH 8.0), trehalose, KCl, and EDTA.

[0279] Another exemplary dialysis composition is an aqueous solution comprising, consisting of, or consisting essentially of about 20 mM Tris buffer (pH 8.0), about 200 mM trehalose, about 50 mM KCl, and about 0.1 mM EDTA.

[0280] Also disclosed is a method for preparing a flap endonuclease composition substantially free of glycerol, wherein the flap endonuclease and glycerol are dialyzed to obtain a flap endonuclease composition substantially free of glycerol. Suitably, the glycerol is present in an amount of about 0 to about 0.35% (w / v) glycerol, suitably about 0 to about 0.2% (w / v) glycerol, suitably about 0.1% (w / v) glycerol, suitably about 0.01% (w / v) glycerol.

[0281] Examples

[0282] Example 1 Bulk Reagent

[0283] Examples 1 to 3 demonstrate the preparation of bulk reagents, drying of single unit dose (SUD) volumes of bulk reagents to obtain SUD dry pellets in a vessel, and reconstitution of dry pellets to obtain SUD amplification and detection mixtures. Tables 1 and 2 disclose components of exemplary bulk reagents for drying. These two tables also disclose exemplary single unit dose concentrations. Master mix 1 is a 2X master mix, the 2X master mix including 0.4 mM each of dATP, dGTP, dCTP, and dTTP; 0.8 mM dUTP; BSA and essentially no inorganic salts. The Taq polymerase in Table 1 is in a glycerol-free Tris buffer containing a cationic detergent. 2X Master Mix 2 is: 0.4 mM dATP, dGTP, dCTP, 0.8 mM dUTP, 0.74 U / μl MDx hot start polymerase, proprietary buffer containing Tris and non-acetylated BSA without glycerol, 0.48 M trehalose. 50X GoScript RT mix as follows: 20 U / μL of 8 units / μL in Table 2 Plus RNase inhibitor; 10U / μL 8 units / μL of RNasin™ Plus from Table 1.

[0284] RT Custom is a 160U / μl, glycerol-free, RNase inhibitor-free concentrate of GoScript RT. Stabilizers that may be included are deamidation inhibitors, anti-oxidants, detergents and surfactants, such as surfactants: fatty acid esters of sorbitan polyethoxylates (e.g., polysorbate 20 or polysorbate 80) and poloxamer 188.

[0285] Table 1: Exemplary bulk reagents for drying

[0286]

[0287] Table 2: Exemplary bulk reagents for drying

[0288]

[0289]

[0290] Table 3: Exemplary bulk reagents for drying

[0291]

[0292] The 10X oligonucleotide mixture in each of the bulk amplification reagents of Tables 1 to 3 contains a collection of primers and probes for performing amplification reactions and detection reactions in the examples below. One of ordinary skill will understand how to prepare primer and probe mixtures for amplification reactions (see, e.g., Innis, Michael A et al., PCR Protocol: Methods and Application Guide, Academic Press (1990)). For the examples below, primers and probes are present in the bulk reagents at a concentration of about 8 μM to about 12 μM.

[0293] Example 2. Freeze-drying

[0294] In this example, a freeze dryer is used to dry the bulk reaction mixture. The 24 microliters of bulk amplification reagents generally described in Example 1 are added to the vessel and then loaded into the freeze drying chamber. For the example herein, 24 μl represents the amount of the bulk reagent for amplification reaction and detection reaction for a single sample (also referred to as single unit dose or SUD). In this example, a multi-well plate (particularly a 12-well plate) is used for freeze drying reaction and storage of the freeze-dried (drying) pellets present in the holes of the multi-well plate. Each hole in the 12 holes of the 12-well plate accommodates the bulk reaction mixture of 24 μl aliquots. The freeze drying cycle is opened (running for about 36 hours). After the freeze drying cycle, the 12-well plate is retrieved and transferred to the sealed position of the separate vessel of the 12-well plate. The vessel is sealed with the metal foil on the vessel opening. The metal foil is a low moisture vapor transmission rate foil. The sealed 12-well plate is then bagged with a desiccant.

[0295] Example 3. Reconstitution of solution

[0296] This example describes a reconstitution solution. The purpose of the reconstitution solution is to rehydrate the prepared dry pellets to perform a reaction based on the detection nucleic acid on the sample using the reconstitution pellets. For each of the 12 wells of the 12-well plate from Example 2, 24 μl of reconstitution buffer is dispensed by pre-piercing the foil cover on the well and then dispensing the reconstitution solution into the well. Table 4 discloses the reconstitution solutions used in these examples, thereby providing both the bulk reconstitution solution concentration and the final assay concentration. After the reconstitution dry pellets, the target nucleic acid from the sample is added to the well, and PCR amplification and detection reactions are performed.

[0297] Table 4: General reconstitution solution

[0298]

[0299] Example 4. Adverse effects of inorganic salts on drying bulk reagents

[0300] This example describes the adverse effect of the presence of inorganic salts in the bulk reagent on the drying of the pellets. Two bulk reagent mixtures were prepared generally according to Example 1 and Table 5. The difference between bulk reagent A and bulk reagent B in Table 5 is the presence or absence of MgCl2 in the reaction mixture.

[0301] Table 5: Bulk reagents with and without inorganic salts

[0302]

[0303] § The nucleic acid is a multiplex primer and probe mixture consisting of the following primer probe sets: for amplification and detection of influenza A, there are 2 forward primers, 3 reverse primers, and 3 probes (two different influenza A target regions); for influenza B, there is 1 forward primer, 1 reverse primer, and 1 probe; for RSV, there is 1 forward primer, 1 reverse primer, and 1 probe for RSVA, and there is 1 forward primer, 1 reverse primer, and 1 probe for RSVB; and for the internal control, there is 1 forward primer, 1 reverse primer, and 1 probe. The value of 7 micromolar is the sum of all primer concentrations, with each primer at approximately 400 nM.

[0304] Each bulk reagent in liquid bulk reagent A and liquid bulk reagent B is prepared on ice. Then, bulk reagent A and bulk reagent B are each aliquoted into the holes of many porous plates (specifically using 12 orifice plates here). Then, these 12 orifice plates containing 12 aliquots of bulk reagent A or 12 aliquots of bulk reagent B are divided into four different cultivation conditions: (1) cultivate 90 minutes on ice; (2) cultivate 90 minutes at room temperature; (3) cultivate 180 minutes on ice; or (4) cultivate 180 minutes at room temperature. Therefore, a part of each bulk reagent mixture is cultivated at room temperature for 180 minutes, and another part is cultivated on ice for 180 minutes. Similarly, a part of each bulk reagent mixture is cultivated at room temperature for 90 minutes, and another part is cultivated on ice for 90 minutes. In all cases, nucleic acid component is added to reaction mixture (added as final component), which indicates that cultivation time begins.

[0305] After the incubation time, the bulk reagents aliquoted in the 12-well plate are then lyophilized until a substantially dry composition is obtained. Each of the resulting dried compositions in the wells of the 12-well plate represents a dried single unit dose for a triplex amplification and detection reaction to identify one or more of influenza A, influenza B, and respiratory syncytial virus B in a sample.

[0306] The dry composition was reconstituted with a reconstitution solution of 65 mM KCl containing 0.02% w / v methylparaben and 0.01% w / v propylparaben respectively and 0.33% v / v absolute ethanol. The reconstitution solution of the dry composition made from bulk reagent A also contained 2.5 mM MgCl2.

[0307] All three types of influenza A, influenza B and respiratory syncytial virus B positive samples are combined into the reaction mixture of reorganization with 3 times of their LoD, so that all components of the recombinant mixture are about 80% of its bulk reagent concentration. These positive samples are viruses extracted in the negative plasma combined with the transport medium and serially diluted to the desired concentration (except that the RSVB sample serial dilution deviation is 10 times). As indicated in Table 6, primers and probe mixtures are designed to specifically detect each of the three virus targets in the independent fluorescence channel (although in a single molecule reaction), i.e. influenza A, influenza B or respiratory syncytial virus B type.

[0308] The samples were assayed using a real-time PCR compatible thermal cycler (ABI 7500FAST, Applied Biosystems, Carlsbad, California). The results are presented in Table 6. The percent positive values ​​in the table represent the number of samples with RFU values ​​exceeding the threshold as a percentage of the 12 samples tested. The amount of virus (viral particles per assay) was sufficient to give at least 95% positive numbers compared to the positive control.

[0309] Table 6: Results

[0310]

[0311] These results indicate that the bulk reagent (pre-lyophilized solution without MgCl and KCl) is stable at room temperature for at least 180 minutes. Once reconstituted and combined with the sample, the dried SUD pellets from the bulk reagent A provide amplification and detection reactions that are more robust than those provided by the dried SUD pellets from the bulk reagent B. When incubated at room temperature or even on ice for only 90 minutes, then dried and reconstituted to generate an amplification reaction mixture, the amplification reaction provided by the bulk reagent B has relatively less signal and a large number of small side products compared to the bulk reagent A under the same conditions. Bulk reagents containing little to almost no inorganic salts can be used for drying to generate a dry composition containing components for an amplification reaction, the composition comprising a polymerase component, dNTPs and nucleic acids.

[0312] Example 5. Stability of dried granules with or without salt

[0313] This example compares the stability of a single unit dose of dried granules containing salt with the stability of a single unit dose of dried granules containing no salt (less than 6mM inorganic salt). The granules of a single unit dose are usually made by drying the bulk reagents as described in Table 5 above. Immediately after synthesis, bulk reagent A and bulk reagent B are each aliquoted into a separate porous reaction plate (12 holes) and dried using a freeze dryer. After freeze drying, the porous plate containing the dried granules is placed in a nitrogen environment with a relative humidity of about 5%, and the porous plate is sealed by covering the hole opening with foil. The sealed plate is placed in an aluminum bag containing a desiccant, and then the pouch is sealed. The sealed pouch containing the dried granules in the porous plate is stored at 4°C for eight days.

[0314] After eight days, the bagged multiwell plates were transferred to one of the following three conditions: Condition #1—1 subset of bagged multiwell plates containing dried pellets from bulk reagent A and 1 subset of bagged multiwell plates containing dried pellets from bulk reagent B were removed from the pouches and placed in an environment with 70% relative humidity at 15°C; Condition #2—1 subset of bagged multiwell plates containing dried pellets from bulk reagent A and 1 subset of bagged multiwell plates containing dried pellets from bulk reagent B were removed from the pouches and placed in an environment with 15% relative humidity at 45°C (accelerated stability); Condition #3—1 subset of bagged multiwell plates containing dried pellets from bulk reagent A and 1 subset of bagged multiwell plates containing dried pellets from bulk reagent B were placed at 4°C (multiwell plates were in pouches with desiccant, so humidity was 0%). The plates were left in these conditions for an additional thirty days.

[0315] At the end of the cultivation, the dry pellets from each condition in the conditions were reconstituted using a reconstitution solution containing 100 mM KCl and sufficient MgCl2 to obtain a final concentration of 2.5 mM. The reconstitution reaction mixture was tested using a real-time PCR thermal cycler (ABI PRISM 7000, Applied Biosystems, Carlsbad, California) to amplify and detect the influenza A target. In brief, the influenza A target was extracted in a negative pool together with a comparable liquid control at LOD 10^0 (+ / -1 log). The results are shown in Table 7.

[0316] Table 7

[0317]

[0318] These results show that individual amplification reaction dried pellets containing less than 2.5 mM inorganic salts had higher RFU values ​​after storage under many different temperature and humidity conditions compared to individual amplification reaction dried pellets containing 2.5 mM or more inorganic salts.

[0319] Example 6. Lyophilization of bulk reagents Enzymes

[0320] Flapper endonuclease The bulk reagent was prepared to contain a final glycerol concentration of 0.35%. This bulk reagent was typically aliquoted and lyophilized as described above. Data showed that this level of glycerol content impaired the lyophilization of the SUD pellets by causing what is known as "meltback" (data not presented). Meltback generally refers to the collapse of the lyophilized product. Meltback is generally caused by the presence of substances in the primary drying stage that are detrimental to the formation of a robust lyophilized composition. Glycerol is one such substance. Therefore, the bulk reagent was aliquoted and lyophilized using a 20 kDa MWCO Slyde-A-Lyzer cartridge system (Thermo Fisher Scientific P / N #66005). The enzyme (which was stored in 50% glycerol buffer) was dialyzed into a buffer without glycerol to prepare a glycerol-free Table 8 lists the dialysis buffer compositions.

[0321] Table 8: Dialysis buffer 1 composition

[0322] Reagents concentration Tris 20mM Trehalose 200mM KCl 50mM EDTA 0.1mM pH 8.0

[0323] Dialyze against this buffer The glycerol was removed by enzyme and replaced with the buffer shown in Table 8. The dialyzed The enzyme was used to prepare a glycerol-free bulk reagent that was substantially free of inorganic salts (Table 9). The bulk reagent was then aliquoted into wells of a multiwell plate as a number of SUDs and lyophilized. All SUD pellets were lyophilized without showing any signs of remelting.

[0324] Table 9: 1.25x pre-lyophilized formulation (version 2 formulation)

[0325]

[0326] The oligonucleotide mixture includes primers for PCR reaction and first probe / second probe / FRET box for cleavage-based assay reaction. The FRET boxes are each labeled with one of the fluorophores FAM, HEX or ROX.

[0327] Dried compositions containing oligonucleotides for PCR amplification and cleavage-based assay detection of Staphylococcus aureus ("S. aureus") were rehydrated with reconstitution solution (stock reconstitution solution included 9.375 mM MgCl2, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, 0.33% (v / v) and the stock reconstitution solution was brought to a total volume of 1 liter with water) to generate multiple reaction master mixes. S. aureus target DNA (positive) or nuclease-free water (negative) was added to separate the reconstituted master mix and silicone oil was placed on top. Amplification and detection assays were performed using a Panther Fusion thermal cycler. Non-lyophilized bulk reagents were tested in parallel with lyophilized formulations as controls.

[0328] The results in Table 10 below show that in this test, the amplification and detection system was able to successfully amplify and detect positive samples of S. aureus that were positive in all three channels and negative samples that were negative in all three channels with comparable values ​​compared to the wet mixture control.

[0329] Table 10: Testing of Version 2 Body Reagents—Lyophilized and Non-Lyophilized Controls

[0330]

[0331] Example 7. Version 3 Pre-lyophilized formulations

[0332] A revised version of the S. aureus pre-lyophilized formulation was prepared containing ultrapure non-acetylated BSA and a buffer change from MOPS buffer to Tris buffer (Table 11).

[0333] Table 11: 1.25x pre-lyophilized formulation using 2X MMA GoTaq source (version 3 formulation)

[0334]

[0335] * The formulations in Table 11 are supplemented with trehalose, Tris buffer, dNTPs, and non-acetylated BSA. These reagents are added to supplement the materials already included in the Promega 2X Master Mix reagent.

[0336] Promega X991X was formulated into a 2X master mix that, when diluted 1: 1, would produce a reaction mixture with 0.24M trehalose, 0.2mM dNTPs (0.4mM dUTP) and 9.25U GoTaq and non-acetylated BSA and Tris in a 25 μl reaction mixture. A total of 25 cassettes from this formulation were prepared and successfully lyophilized.

[0337] Amplification reactions and detection reactions were prepared and performed as generally described in Example 6 above. Lyophilized pellets were reconstituted using a reconstitution solution similar to that described in Example 6, except that the concentration of MgCl2 was increased to provide 12.5 mM MgCl2 in the reconstituted reaction mixture. As a control, a number of non-lyophilized reaction mixtures were prepared and tested. The S. aureus target nucleic acid used in these reactions was 100 copies / mL and 1,000 copies / mL of S. aureus gDNA (approximately 3 times the assay LoD for this target). In the presence of 12.5 mM MgCl2, the Tris buffered reactions showed a delayed Ct, a reduced RFU, and a reduced T slope value for S. aureus positive samples. Further, negative samples using Tris buffer formulations showed an increase in background generation compared to negative samples using 10 mM MOPS buffer formulations—regardless of the MgCl2 concentration in the reaction mixture. Therefore, based on these results, MOPS buffer offers advantages for use in bulk formulations and can be used in final cassette formulations at concentrations up to about 15 mM.

[0338] The formulations shown in Table 11 were further varied and tested. Formulation variations were prepared as a series of bulk agents in which 11% trehalose was replaced with various trehalose / sugar / polymer combinations. These bulk agent formulations contained the following agent combinations and were lyophilized and tested as described above:

[0339] a) 3% trehalose / 1% 10 kDa polyethylene propylene.

[0340] b) 3% trehalose / 2% 10 kDa polyethylene propylene.

[0341] c) 3% trehalose / 3% 10 kDa polyethylene propylene.

[0342] d) 3% trehalose / 1% 29 kDa polyethylene propylene.

[0343] e) 3% trehalose / 2% 29 kDa polyethylene propylene.

[0344] f) 3% trehalose / 3% 29 kDa polyethylene propylene.

[0345] g) 3% trehalose / 1% 55 kDa polyethylene propylene.

[0346] h) 3% trehalose / 2% 55 kDa polyethylene propylene.

[0347] i) 3% trehalose / 3% 55 kDa polyethylene propylene.

[0348] j) 3% trehalose / 1% sucrose.

[0349] k) 3% trehalose / 2% sucrose.

[0350] 1) 3% trehalose / 3% sucrose.

[0351] m) 11 of trehalose (control).

[0352] Except for condition i) and control condition m), all combinations lyophilized well and did not show any initial collapse. Functional testing at t=0 also showed no significant Ct delay in any of the combinations a) to m). However, when accelerated stabilization was performed at 37°C / 95% RH, all combinations except the control condition showed collapse after 4 days. The control box with 11% trehalose did not show collapse at 37°C / 95% RH until the 14th-16th day. Therefore, the bulk agent formulation containing 11% is suitable for long-term storage of the lyophilized pellets before use.

[0353] Example 8. Version 4 Pre-lyophilized formulations

[0354] It is desirable to vary the sample input volume. Lower sample volumes can be used to accommodate multiple tests on patient samples. Sample input volumes between 210 μl and 420 μl were tested. In order to maintain sensitivity at lower sample draw volumes, the eluent volume used in the PCR reaction was increased from 5 μl to 10 μl. The additional 5 μl eluent sample changed the concentration of the reagents in the reaction mixture. The reagent concentrations of the bulk reagents were adjusted to account for the different eluent volumes (see Table 13).

[0355] Comparison of the 420 μl / 5 μl and 210 μl / 10 μl conditions showed equivalent performance under both conditions (Table 12):

[0356] Table 12: 420 / 5μl vs. 210 / 10μl

[0357]

[0358] The increase in eluent volume from 5 μl to 10 μl resulted in a "1.5x" SUD pellet concentration to provide a total reaction mixture volume of 30 μl (e.g., 20 μl master mix + 10 μl eluent). Table 13 reflects the volume change and also replaces the Tris buffer with 10 mM MOPS buffer at pH 7.5.

[0359] Table 13: Version 4 1.5x Staphylococcus aureus pre-lyophilized formulation with MOPS buffer

[0360]

[0361] * These reagents are added to supplement the materials already contained in the 2X Master Mix A reagents.

[0362] This version 4 formulation has more concentrated reagents to accommodate the doubling of the sample eluate volume for the PCR reaction (5μl→10μl). As the formulation concentration changes, new retention time studies were performed to ensure the robustness of the formulation. On day 0, a liquid S. aureus reaction mixture was prepared according to Table 13 above and divided into 3 equal parts. The first aliquot was lyophilized immediately, the second aliquot mixture was lyophilized after 2 days of storage at 2-8°C, and the last aliquot was lyophilized after 4 days of storage at 2-8°C. The resulting box was sealed and tested at baseline (data not shown) and also subjected to 4 weeks of accelerated stability at 30°C / 95% relative humidity (RH). The MRSA cell strain was tested under conditions of 0.5 logs above the LoD (1,000 CFU / mL), and each condition was repeated 10 times. The results showed that this formulation was able to detect MRSA at 0.5 logs above the LoD and exhibited robustness from 0-4 days of holding time before lyophilization and accelerated stability after lyophilization at 30°C / 95% RH for up to 30 days.

[0363] Example 9

[0364] Alpha-cyclodextrin can be added to the composition to be lyophilized to resist the inhibitory effects of substances such as SDS, which can be used to wash the captured nucleic acids. Some of the SDS will remain in the nucleic acid solution and subsequently remain in the nucleic acid amplification and detection reactions. We first used 1,000 CFU / mL of Staphylococcus aureus cells (GP1822) similar to the cells in Table 13 in simulated nasal fluid (SNF) to prepare four versions of formulations, the cells having the following differences: (1) 0 μg / μl cyclodextrin; (2) 0.1 μg / μl cyclodextrin; (3) 0.25 μg / μl cyclodextrin; or (4) 0.5 μg / μl cyclodextrin. The samples were processed on a PantherFusion instrument (P182). 30% of the wash buffer was incorporated into the elution buffer (i.e., normal WB residual amount + 30%). The pellets were then used in the test reactions to amplify and detect MRSA target nucleic acids. No SDS was added to these amplification reactions. This experiment determined whether the addition of cyclodextrin to the mixture would have an effect on the nucleic acid reactions performed with the pellets. As can be seen from conditions 1-4 in Table 14, the ct values ​​for each of the conditions tested were essentially the same, indicating that there was no adverse effect on the performance of the pellets containing at least up to 0.5 μg / μl cyclodextrin.

[0365] After the first experiment, four versions of formulations similar to those in Table 13 were prepared again, and the formulations contained one of the following: (1) 0 μg / μl cyclodextrin; (2) 0.1 μg / μl cyclodextrin; (3) 0.25 μg / μl cyclodextrin; or (4) 0.5 μg / μl cyclodextrin. The resulting pellets were then used in a test reaction to amplify and detect MRSA target nucleic acids. For this set of amplification reactions, SDS was added to bring the final concentration of the reaction mixture to 30% v / v. Amplification and detection reactions were performed. As can be seen in conditions 5-8 of Table 14, SDS has an inhibitory effect on nucleic acid reactions (condition 5). 0.1 μg / μl cyclodextrin almost completely neutralized the inhibition of SDS (condition 6), and 0.25 μg / μl and 0.5 μg / μl fully neutralized the inhibition of SDS (conditions 7 and 8).

[0366] Table 14

[0367]

[0368] An additional data set was also tested using 1,000 CFU / mL of S. aureus cells (GP1822) in simulated nasal fluid (SNF). Samples were processed on a Panther Fusion instrument (P368). 30% of wash buffer was spiked into the elution buffer (ie, normal WB carryover + 30%).

[0369] Table 15

[0370]

[0371] The table shows that higher concentrations of cyclodextrin, up to 5.0 μg / ml, can effectively neutralize SDS.

[0372] Thus, formulations such as those in Table 13 may be supplemented with cyclodextrin, for example 0.1 μg / μl to 5 μg / μl or 0.1 μg / μl to 0.5 μg / μl of cyclodextrin.

[0373] According to the foregoing, it should be understood that, although specific embodiments have been described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of this clear disclosure. Therefore, the present invention is not limited by the clear disclosure. Unless it is obvious from the context, any embodiment, feature, aspect or step can be used in combination with any other embodiment, feature, aspect or step. Unless it is obvious from the context, it is said that any composition including the enumerated components can also be composed of or substantially composed of those components. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. An aqueous solution composition comprising a flap endonuclease, a swelling agent and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 5 mM or less, a potassium concentration of 0.196 μg / μL or less, and wherein the composition contains 0 to 0.2% w / v glycerol, wherein the flap endonuclease comprises a nuclease that acts as a structure-specific 5' endonuclease, and wherein the organic buffer is a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

2. The composition of claim 1, wherein the aqueous solution further comprises at least one oligonucleotide useful for performing a molecular assay.

3. The composition of claim 1, wherein the aqueous solution further comprises at least one oligonucleotide useful for performing a nucleic acid-based assay.

4. The composition of claim 3, wherein the at least one oligonucleotide comprises a probe oligonucleotide.

5. The composition of claim 4, wherein the at least one oligonucleotide comprises at least two probe oligonucleotides.

6. The composition according to claim 4 or 5, wherein the one or more probe oligonucleotides are partially or completely complementary to the target nucleic acid sequence.

7. The composition of claim 1, wherein the flap endonuclease is Enzyme.

8. The composition according to claim 6, comprising at least two probe oligonucleotides capable of annealing to a target nucleic acid to form a three-dimensional structure that can be recognized by the flap endonuclease.

9. The composition of claim 8, further comprising at least one polymerase.

10. The composition of claim 9, wherein the at least one polymerase in the aqueous solution comprises a polymerase present in the aqueous solution at a concentration of 0.10 U / μl to 0.2 U / μl.

11. The composition of claim 10, wherein the at least one polymerase comprises a polymerase present in the aqueous solution at a concentration selected from the group consisting of: 0.14 U / μl, 0.146 U / μl, and 0.1686 U / μl.

12. The composition of claim 9, wherein the at least one polymerase comprises a polymerase that is a hot start polymerase.

13. The composition of claim 12, wherein the hot-start polymerase is a recombinant Taq DNA polymerase.

14. The composition of claim 13, wherein the polymerase is bound by an antibody that specifically blocks the polymerase activity of the polymerase.

15. The composition of claim 13, wherein the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase, wherein the chemical modification inhibits the polymerase activity of the polymerase.

16. The composition of claim 9, wherein the at least one polymerase comprises a reverse transcriptase present in the aqueous solution. 17 . The composition according to claim 16 , wherein the reverse transcriptase is present in the aqueous solution at a concentration of 0.1 U / μl to 4.0 U / μl.

18. The composition of claim 16 or 17, wherein the reverse transcriptase is AMV reverse transcriptase.

19. The composition of claim 16 or 17, wherein the reverse transcriptase is MMLV reverse transcriptase.

20. The composition of claim 4, wherein the at least one oligonucleotide comprises an invader probe.

21. The composition of claim 20, wherein the sequence of the invader probe is partially or completely complementary to the target nucleic acid sequence.

22. The composition of claim 4, wherein the at least one oligonucleotide comprises a signaling probe.

23. The composition of claim 22, wherein the sequence of the signaling probe is partially complementary to the target nucleic acid sequence.

24. The composition of claim 23, wherein the sequence of the signaling probe comprises a flap region.

25. The composition of claim 24, wherein the petal at least partially overlaps with the invader probe.

26. The composition of claim 4, wherein the at least one oligonucleotide comprises a FRET probe.

27. The composition of claim 26, wherein the sequence of the FRET probe is partially complementary to the flap region of the signaling probe.

28. The composition of claim 27, wherein the FRET probe comprises a label covalently attached thereto.

29. The composition of claim 28, wherein the label is a fluorescent molecule.

30. The composition of claim 29, wherein the label is located at the 5' end of the FRET probe.

31. The composition of claim 30, wherein the FRET probe comprises a quencher molecule covalently attached to the FRET probe, within the quenching proximity of the fluorescent molecule, and capable of at least partially quenching fluorescence from the fluorescent molecule.

32. The composition of claim 3, wherein the at least one oligonucleotide comprises a target capture probe.

33. The composition of claim 32, wherein the target capture probe has a target hybridizing portion that specifically or non-specifically hybridizes to a target nucleic acid under stringent conditions.

34. The composition of claim 3, wherein the aqueous solution comprises two or more oligonucleotides for performing a multiplex molecular assay.

35. The composition of claim 1, wherein the bulking agent is trehalose.

36. The composition of claim 35, wherein the bulking agent is present in a concentration of 0.2M to 0.5M.

37. The composition of claim 36, wherein the bulking agent is present at a concentration of 0.36M or 0.47M.

38. The composition of claim 1, wherein the inorganic salt is present in a mass per microliter of 0.373 μg / μl to 0.029 μg / μl.

39. The composition of claim 1, wherein the aqueous solution contains sodium chloride at a mass per microliter of 0.292 μg / μl to 0.35 μg / μl.

40. The composition of claim 39, wherein the aqueous solution contains sodium chloride at a mass per microliter of 0.32 μg / μl.

41. The composition of claim 1, wherein the aqueous solution contains potassium chloride at a mass per microliter of 0.098 μg / μl or less.

42. The composition of claim 41, wherein the potassium chloride is present at 0.0 μg / μl.

43. The composition of claim 1, wherein the aqueous solution contains 0.135 μg / μl sodium ions to 0.006 μg / μl sodium ions.

44. The composition of claim 43, wherein the aqueous solution contains 0.127 μg / μl of sodium ions.

45. The composition of claim 1, wherein the aqueous solution contains 0.196 μg / μl potassium ions to 0.010 μg / μl potassium ions.

46. ​​The composition of claim 45, wherein the aqueous solution contains 0.016 μg / μl potassium ions.

47. The composition of claim 1, wherein the aqueous solution contains 0.355 μg / μl chloride ions to 0.009 μg / μl chloride ions.

48. The composition of claim 47, wherein the aqueous solution contains 0.337 μg / μl of chloride ions.

49. The composition of claim 1, wherein the aqueous solution comprises an inorganic salt concentration of 4 mM or less.

50. The composition of claim 49, wherein the aqueous solution comprises an inorganic salt in an amount of 0.298 μg / μl to 0.234 μg / μl per microliter.

51. The composition of claim 50, wherein the aqueous solution comprises chloride ions at a mass per microliter of 0.284 μg / μl to 0.071 μg / μl.

52. The composition of claim 1, wherein the aqueous solution comprises an inorganic salt concentration of 3 mM or less.

53. The composition of claim 52, wherein the aqueous solution comprises an inorganic salt in an amount of 0.224 μg / μl to 0.175 μg / μl per microliter.

54. The composition of claim 53, wherein the aqueous solution comprises chloride ions at a mass per microliter of 0.213 μg / μl to 0.053 μg / μl.

55. The composition of claim 1, wherein the aqueous solution comprises an inorganic salt concentration of 2 mM or less.

56. The composition of claim 55, wherein the aqueous solution comprises an inorganic salt in an amount of 0.149 μg / μl to 0.117 μg / μl per microliter.

57. The composition of claim 56, wherein the aqueous solution comprises chloride ions at a mass per microliter of 0.142 μg / μl to 0.036 μg / μl.

58. The composition of claim 1, wherein the aqueous solution comprises an inorganic salt concentration of 1 mM or less.

59. The composition of claim 58, wherein the aqueous solution comprises an inorganic salt in an amount of 0.075 μg / μl to 0.058 μg / μl per microliter.

60. The composition of claim 59, wherein the aqueous solution comprises chloride ions at a mass per microliter of 0.071 μg / μl to 0.018 μg / μl.

61. The composition of claim 1, wherein the aqueous solution comprises an inorganic salt concentration of 500 μM or less.

62. The composition of claim 61, wherein the aqueous solution comprises an inorganic salt in an amount of 0.037 μg / μl to 0.029 μg / μl per microliter.

63. The composition of claim 62, wherein the aqueous solution comprises chloride ions at a mass per microliter of 0.036 μg / μl to 0.009 μg / μl.

64. The composition of claim 1, wherein the inorganic salt concentration of the aqueous solution is less than 1 mM sodium chloride.

65. The composition of claim 64, wherein the aqueous solution is free of sodium chloride.

66. The composition of claim 1, wherein the aqueous solution contains less than 1 mM magnesium ions.

67. The composition of claim 66, wherein the aqueous solution contains less than 0.1 mM magnesium ions.

68. The composition of claim 1, wherein the aqueous solution further comprises deoxynucleotide triphosphates (dNTPs).

69. The composition of claim 68, wherein the dNTPs comprise dATP at a concentration of 0.1 mM to 0.4 mM in the aqueous solution.

70. The composition of claim 68, wherein the dNTPs comprise dATP at a concentration of 0.29 mM to 0.46 mM in the aqueous solution.

71. The composition of claim 69 or 70, wherein the concentration of dATP in the aqueous solution is 0.3 mM to 0.4 mM.

72. The composition of claim 71, wherein the concentration of dATP in the aqueous solution is 0.375 mM.

73. The composition of claim 68, wherein the dNTP comprises dGTP at a concentration of 0.1 mM to 0.4 mM in the aqueous solution.

74. The composition of claim 68, wherein the dNTPs comprise dGTP at a concentration of 0.29 mM to 0.46 mM in the aqueous solution.

75. The composition of claim 73 or 74, wherein the concentration of dGTP in the aqueous solution is 0.3 mM to 0.4 mM.

76. The composition of claim 75, wherein the concentration of dGTP in the aqueous solution is 0.375 mM.

77. The composition of claim 68, wherein the dNTPs comprise dCTP at a concentration of 0.1 mM to 0.4 mM in the aqueous solution.

78. The composition of claim 68, wherein the dNTPs comprise dCTP at a concentration of 0.29 mM to 0.46 mM in the aqueous solution.

79. The composition of claim 77 or 78, wherein the concentration of the dCTP in the aqueous solution is 0.3 mM to 0.4 mM.

80. The composition of claim 79, wherein the concentration of the dCTP in the aqueous solution is 0.375 mM.

81. The composition of claim 68, wherein the dNTPs comprise dTTP at a concentration of 0.1 mM to 0.4 mM in the aqueous solution.

82. The composition of claim 81, wherein the dNTPs comprise dTTP at a concentration of 0.2 mM to 0.37 mM in the aqueous solution.

83. The composition of claim 82, wherein the dNTPs comprise dTTP at a concentration of 0.284 mM in the aqueous solution.

84. The composition of claim 81, wherein the concentration of the dTTP in the aqueous solution is 0.3 mM to 0.4 mM.

85. The composition of claim 68, wherein the dNTPs comprise dUTP at a concentration of 0.1 mM to 0.4 mM in the aqueous solution.

86. The composition of claim 85, wherein the dNTPs comprise dUTP at a concentration of 0.125 mM to 0.234 mM in the aqueous solution.

87. The composition of claim 86, wherein the dNTPs comprise dUTP at a concentration of 0.182 mM in the aqueous solution.

88. The composition of claim 85, wherein the concentration of the dUTP in the aqueous solution is 0.3 mM to 0.4 mM.

89. The composition of claim 1, wherein the flap endonuclease is present in the aqueous solution at 0.020 μg / μl to 0.040 μg / μl.

90. The composition of claim 89, wherein the flap endonuclease is present in the aqueous solution at 0.030 μg / μl to 0.04 μg / μl.

91. The composition of claim 90, wherein the flap endonuclease is present in the aqueous solution at 0.030 μg / μl to 0.035 μg / μl.

92. The composition of claim 1, wherein the MOPS buffer is present in the aqueous solution at a concentration of 10 mM to 20 mM.

93. The composition of claim 92, wherein the MOPS buffer is present in the aqueous solution at a concentration of 12.5 mM to 15 mM.

94. The composition of claim 1, wherein the composition comprises a globular protein.

95. The composition of claim 94, wherein the globular protein is bovine serum albumin (BSA).

96. The composition of claim 95, wherein the BSA is non-acetylated BSA.

97. The composition of claim 96, wherein the non-acetylated BSA is ultrapure non-acetylated BSA.

98. The composition of claim 94, wherein the globular protein is present in an amount of 0.40 μg / μl to 0.60 μg / μl.

99. The composition of claim 98, wherein the globular protein is present in an amount of 0.50 μg / μl.

100. The composition of claim 1, comprising the following: enzyme, trehalose, MOPS buffer, dNTPs, inorganic salts at a concentration of 5 mM or less, and wherein the composition contains 0 to 0.2% w / v glycerol.

101. The composition according to claim 100, comprising: Trehalose is present at a concentration of 0.3 M, MOPS buffer is at a concentration of 12.5 mM, dNTPs are each at a concentration of 0.3 mM, inorganic salts are at a concentration of 5 mM or less, and wherein the composition contains 0 to 0.2% w / v glycerol.

102. The composition of claim 1, comprising the following: enzyme, trehalose, MOPS buffer, dNTP, bovine serum albumin, inorganic salts at a concentration of 5 mM or less, and wherein the composition contains 0 to 0.2% w / v glycerol.

103. The composition of claim 102, comprising: enzyme, trehalose at a concentration of 0.36 M, MOPS buffer at a concentration of 15 mM, dNTPs at a concentration of 0.38 mM each, 0.5 μg / μl bovine serum albumin, inorganic salts at a concentration of 5 mM or less, and wherein the composition contains 0 to 0.2% w / v glycerol.

104. The composition of claim 1, comprising: trehalose, hot start Taq DNA polymerase, reverse transcriptase, dNTPs, UTP, 0 mM KCl and 0 mM MgCl2.

105. The composition according to claim 1, comprising: 0.30 M trehalose, 0.46 units / μL hot start Taq DNA polymerase, 0.5 units / μL reverse transcriptase, 0.2 units / μL Rnasin, 0.25 mM NTPs, 0.5 mM UTP, 0 mM MgCl2, and 2.7 mM Na + and 0.035 mM K+ buffer.

106. The composition of claim 1, further comprising 0.1 μg / ml to 0.5 μg / ml of alpha cyclodextrin.

107. A dried form of a composition according to any one of claims 1 to 106.

108. A method for preparing a dried composition for performing a nucleic acid-based assay, the method comprising the steps of: (i) freezing the aqueous solution according to any one of claims 1 to 106, thereby forming a frozen form of the aqueous solution; and (ii) exposing the frozen form from step (i) to lyophilization conditions, thereby forming a dry composition.

109. The method of claim 108, wherein the dry composition is exposed to a humid environment, wherein the humid environment has an absolute humidity level of greater than 2.3 grams of water per cubic meter of air at 30°C.

110. The method of claim 109, wherein the dry composition is exposed to the moist environment for up to 3 hours.

111. The method of claim 108, further comprising the step of storing the dry composition in a sealed container.

112. The method of claim 108, wherein the mass of the dry composition is 0.003 g to 0.004 g per microliter of the aqueous solution, and the aqueous solution is dried to form the dry composition.

113. The method of claim 112, wherein the mass of the dry composition per microliter of the aqueous solution is 0.0032 g to 0.0037 g, and the aqueous solution is dried to form the dry composition.

114. The method of claim 113, wherein the mass of the dry composition per microliter of the aqueous solution is 0.0033g, or 0.0034g, or 0.0035g or 0.0036g, and the aqueous solution is dried to form the dry composition.

115. The method of claim 108, wherein the mass of the dry composition per microliter of the aqueous solution is 0.000125 g to 0.000667 g, and the aqueous solution is dried to form the dry composition.

116. A method for preparing a dried composition for performing a nucleic acid-based assay, the method comprising the steps of: The aqueous solution according to any one of claims 1 to 106 is dried using a drying method selected from the group consisting of dehydration, dehumidification, freeze drying and spray drying, thereby forming a dry composition.

117. The method of claim 116, wherein the drying method is freeze-drying and the dried composition is a freeze-dried composition.

118. The method of claim 116, further comprising the step of storing the dry composition in a sealed container.

119. Use of an aqueous solution composition according to any one of claims 1 to 106 for preparing a dried flap endonuclease-containing composition.

120. Use of an aqueous solution composition according to any one of claims 1 to 106 in combination with a reconstitution solution for performing a nucleic acid based assay.

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