Dry composition containing flap end nuclease

Abstract

To provide a dried composition comprising a flap endonuclease.SOLUTION: There is provided a composition of an aqueous solution that comprises, consists, or consists essentially of a flap endonuclease, a bulking agent, and an organic buffer. The aqueous solution has inorganic salt concentration of 5 mM or less. The composition is substantially free of glycerol. The present invention seeks to provide improvements in lyophilized compositions, especially when used in nucleic acid based assays, such as non-amplification-based assays or non-amplification-based assays combined with amplification based assays.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of each of the provisional patent applications 62 / 508,975, filed May 19, 2017; 62 / 508,990, filed May 19, 2017; and 62 / 540,478, filed Aug. 2, 2017, each of which is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] Commercially available kits for performing nucleic acid - based assays often contain reagents such as enzymes, nucleotides, surfactants, buffers, primers, probes, and inorganic salts including MnCl2, MgCl2, NaCl, and KCl (Innis et al., (1990) PCR Protocols: A Guide to Methods and Applications, Ch. 1, Optimizations of PCRs).

[0003] Nucleic acid-based assays can be "nucleic acid amplification-based assays," i.e., assays that use one or more steps to amplify a nucleic acid target sequence. Various amplification methods used in 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 depend on any step to amplify a nucleic acid target sequence. An exemplary non-nucleic acid amplification-based assay is a "cleavage-based assay," which is an assay that relies on the specific cleavage by a flap endonuclease of a linear double-strand cleavage structure formed by the specific hybridization of a duplicate oligonucleotide to the target nucleic acid. In these assays, an invader oligonucleotide (also called an "invader probe") is designed to stably anneal to the target nucleic acid at the assay reaction temperature. A signal probe oligonucleotide, containing the target hybridize sequence and a non-target hybridize flap region covering the invader oligonucleotide, is designed with a melting temperature that is approximately the melting temperature of the assay temperature. As a result, the signal probe is in a state to anneal to / dissociate from the target nucleic acid under the reaction conditions. When a signal probe is annealed to a target nucleic acid in the presence of a flap endonuclease, the non-target hybridizing flap region is cleaved overlap-dependently by the flap endonuclease, releasing the cleavage product. The cleaved flap region then anneals with a hairpin-configured probe (often referred to as a "FRET probe") containing a signaling and quenching portion, forming an overlap between a portion of the cleaved flap and a portion of the FRET probe that binds to either the signaling or quenching portion. In the presence of the flap endonuclease, the FRET probe is cleaved overlap-dependently, releasing either the signaling or quenching portion from the FRET probe, thereby generating a detectable signal. These assays often refer to signal amplification assays because they utilize an excess of signal and FRET probes compared to the invader probe.The principles of cleavage-based assays are well known in the art, and exemplary assays are described, for example, by Lyamichev et al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al. (Mol. Diagn. 4:135-144, 1999), Allawi et al. (J. Clin. Microbiol. 44:3443-3447, 2006), U.S. Patent Nos. 5,846,717 and 6,706,471 to Brow et al., and U.S. Patent No. 5,614,402 to Dahlberg et al. Cleavage-based assays include, for example, the commercially available Invader® assay (Hologic, Inc., Marlborough, MA).

[0004] Depending on the situation, it may be desirable to perform both an "amplification-based assay" (e.g., PCR) and a "non-amplification-based assay" (an assay using the Cleavase® enzyme) in the same reaction. The Cleavase® enzyme is a thermally stable, structure-specific endonuclease that cleaves at the junction of single-stranded and double-stranded deoxyribonuclei.

[0005] Magnesium ions have been reported to enhance the activity of polymerases and other enzymes. Potassium chloride has been reported to promote nucleic acid hybridization. Several inorganic salts have been reported to protect proteins under various stress conditions, including heat, exposure to chaotropic agents, and freeze-drying (Liu et al (2007) FEBS Letters. 581:1047; Kanaya et al (1996) J. Biol. Chem. 271:32729; Innis et al, (1990) PCR Protocols:A Guide to Methods and Applications,Ch.1,Optimizations of PCRs;Menendez et al(1998)J.Biol.Chem.273:167;Janeway et al(1993)Biochemistry.32:1601,Fox et al al(1971)J.Biol.Chem.246:5739,Chang et al(2002)J.Biol.Chem.277:277:4663,Rutter et al(1958)J.Biol.Chem.233:374,Huszar et al(1981)J.Virol.37:580-588,Wang(2000)Int.J.Pharmaceutics.203:1-60). Salts are also used in assays such as "amplification-based assays" and "non-amplification-based assays." For example, typical reaction conditions for performing the Invader® assay include potassium chloride in the enzyme stock and magnesium chloride in the reaction buffer.

[0006] However, the stability of freeze-dried materials is affected by the hygroscopicity of any salts present in the freeze-dried cake. The hygroscopicity of freeze-dried materials affects the time available for packaging, and thus affects the period and conditions under which freeze-dried materials can be stored and shipped. Unwanted rehydration of freeze-dried materials adversely affects the activity of the freeze-dried components. To minimize the adverse effects of unwanted rehydration of freeze-dried materials, long-term storage of such materials is usually carried out by refrigeration. The present invention aims to provide improvements to lyophilized compositions, particularly when used in nucleic acid-based assays, for example, in non-amplification assays or non-amplification assays combined with amplification assays. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Innis et al,(1990) PCR Protocols:A Guide to Methods and Applications,Ch.1,Optimizations of PCRs [Overview of the project] [Means for solving the problem]

[0008] A composition is disclosed comprising, or essentially comprising, a flap endonuclease, an extender, and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 6 mM or less, and the composition is substantially glycerol-free.

[0009] Preferably, the aqueous solution further comprises at least one oligonucleotide useful for performing molecular assays.

[0010] Preferably, the aqueous solution further comprises at least one oligonucleotide useful for performing nucleic acid-based assays.

[0011] Preferably, the flap end nuclease is a Cleavase® enzyme.

[0012] Preferably, the composition further comprises at least one polymerase.

[0013] Preferably, at least one polymerase is present in the aqueous solution at a concentration of about 0.10 U / μl to about 0.25 U / μl.

[0014] Preferably, at least one polymerase is present in the aqueous solution at a concentration selected from 0.11 U / ul, 0.12 U / ul, 0.14 U / ul, 0.146 U / ul, 0.1687 U / ul, 0.2 U / ul, and 0.022 U / ul.

[0015] Preferably, at least one polymerase is a hot-start polymerase.

[0016] Preferably, the hot-start polymerase is a recombinant Taq DNA polymerase conjugated with an antibody that specifically blocks the polymerase activity of the polymerase.

[0017] Preferably, the hot start polymerase is a chemically modified recombinant Taq DNA polymerase, and the chemical modification inhibits the polymerase activity of the polymerase.

[0018] Preferably, at least one polymerase preferably contains a reverse transcriptase present in an aqueous solution at a concentration of about 0.1 U / ul to about 4.0 U / ul.

[0019] Preferably, the reverse transcriptase is AMV reverse transcriptase.

[0020] Preferably, the reverse transcriptase is MMLV reverse transcriptase.

[0021] Preferably, at least one oligonucleotide contains an Invader probe.

[0022] Preferably, the sequence of the Invader probe is partially or completely complementary to the target nucleic acid sequence.

[0023] Preferably, at least one oligonucleotide contains a signal transduction probe.

[0024] Preferably, the sequence of the signal transduction probe is partially complementary to the target nucleic acid sequence.

[0025] Preferably, the sequence of the signal transduction probe contains a flap region.

[0026] Preferably, the flap region at least partially overlaps with the Invader probe.

[0027] Preferably, at least one oligonucleotide contains a FRET probe.

[0028] Preferably, the sequence of the FRET probe is partially complementary to the flap region of the signal transduction probe.

[0029] Preferably, the FRET probe contains a label covalently attached thereto.

[0030] Preferably, the label is a fluorescent molecule.

[0031] Preferably, the label is located at the 5' end of the FRET probe.

[0032] Preferably, the FRET probe includes a quenching molecule that is covalently bound to the probe within a proximity range that can quench the fluorescent molecule and that can at least partially quench the fluorescence from the fluorescent molecule.

[0033] Preferably, at least one oligonucleotide comprises a target capture probe.

[0034] Preferably, the target capture probe has a target hybridizing moiety that hybridizes specifically or nonspecifically to a target nucleic acid under stringent conditions.

[0035] Preferably, the target capture probe has a target hybridizing moiety that nonspecifically hybridizes to the target nucleic acid under stringent conditions.

[0036] Preferably, the nonspecific target hybridization portion of the target capture probe contains randomly arranged K nucleotides or randomly arranged R nucleotides (IUPAB-IUB ambiguity code).

[0037] Preferably, the aqueous solution contains two or more oligonucleotides for performing a multimolecular assay.

[0038] Preferably, the bulking agent is trehalose.

[0039] Preferably, the bulking agent is present at a concentration of about 0.2 M to about 0.5 M, preferably about 0.36 M.

[0040] Preferably, the aqueous solution contains an inorganic salt concentration of 6 mM or less. Preferably, the aqueous solution contains an inorganic salt concentration of about 6 mM to about 0.5 mM.

[0041] Preferably, the inorganic salt is present in a mass of approximately 0.373 μg / μl to approximately 0.029 μg / μl per microliter.

[0042] Preferably, the inorganic salt is sodium chloride, and preferably, sodium chloride is present in a mass per microliter of about 0.35 μg / µl to about 0.029 μg / µl, preferably about 0.32 μg / µl.

[0043] Preferably, the inorganic salt is potassium chloride, preferably in a mass of about 0.373 ug / ul to about 0.019 ug / ul, and preferably about 0.03 ug / ul per microliter.

[0044] Preferably, the aqueous solution contains about 0.135 ug / ul of sodium ions to about 0.006 μg / μl of sodium ions, preferably about 0.127 ug / ul.

[0045] Preferably, the aqueous solution contains about 0.196 ug / ul of potassium ions to about 0.010 ug / ul of potassium ions, preferably about 0.016 ug / ul of potassium ions.

[0046] Preferably, the aqueous solution contains approximately 0.355 ug / ul of chloride ions to approximately 0.009 ug / ul of chloride ions, preferably approximately 0.337 ug / ul.

[0047] Preferably, the aqueous solution contains an inorganic salt concentration of 4 mM or less.

[0048] Preferably, the aqueous solution contains an inorganic salt with a mass of about 0.298 μg / μl to about 0.234 μg / μl per microliter.

[0049] Preferably, the aqueous solution contains chloride ions in a mass per microliter of about 0.284 μg / μl to about 0.071 μg / μl.

[0050] Preferably, the aqueous solution contains an inorganic salt concentration of 3 mM or less.

[0051] Preferably, the aqueous solution contains an inorganic salt with a mass of about 0.224 μg / μl to about 0.175 μg / μl per microliter.

[0052] Preferably, the aqueous solution contains chloride ions in a mass of about 0.213 μg / μl to about 0.053 μg / μl per microliter.

[0053] Preferably, the aqueous solution contains an inorganic salt concentration of 2 mM or less.

[0054] Preferably, the aqueous solution contains an inorganic salt with a mass per microliter of about 0.149 μg / μl to about 0.117 μg / μl.

[0055] Preferably, the aqueous solution contains chloride ions in a mass per microliter of about 0.142 μg / μl to about 0.036 μg / μl.

[0056] Preferably, the aqueous solution contains an inorganic salt concentration of 1 mM or less.

[0057] Preferably, the aqueous solution contains an inorganic salt with a mass per microliter of about 0.075 μg / μl to about 0.058 μg / μl.

[0058] Preferably, the aqueous solution contains chloride ions in a mass per microliter of about 0.071 μg / μl to about 0.018 μg / μl.

[0059] Preferably, the aqueous solution contains an inorganic salt concentration of 500 μM or less.

[0060] Preferably, the aqueous solution contains an inorganic salt with a mass of about 0.037 μg / μl to about 0.029 μg / μl per microliter.

[0061] Preferably, the aqueous solution contains chloride ions in a mass of about 0.036 μg / μl to about 0.009 μg / μl per microliter.

[0062] Preferably, the inorganic salt concentration of the aqueous solution is sodium chloride with a concentration of less than 1 mM.

[0063] Preferably, the aqueous solution does not contain sodium chloride.

[0064] Preferably, the aqueous solution contains less than 1 mM of magnesium ions.

[0065] Preferably, the aqueous solution contains less than 0.1 mM magnesium ions.

[0066] Preferably, the aqueous solution further comprises deoxynucleotide triphosphate (dNTP).

[0067] Preferably, the aqueous solution contains dATP at a concentration of 0.1 mM to 0.5 mM, preferably about 0.28 mM to about 0.46 mM.

[0068] Preferably, the dATP concentration in the aqueous solution is 0.3 mM to 0.4 mM, for example, 0.375 mM.

[0069] Preferably, the dNTP contains dGTP at a concentration of 0.1 mM to 0.4 mM in an aqueous solution.

[0070] Preferably, the concentration of dGTP in the aqueous solution is 0.3 mM to 0.4 mM, preferably 0.29 to 0.46 mM, for example, 0.375 mM.

[0071] Preferably, the dNTP contains dCTP at a concentration of 0.1 mM to 0.4 mM, preferably 0.29 to 0.46 mM, in an aqueous solution.

[0072] Preferably, the concentration of dCTP in aqueous solution is 0.3 mM to 0.4 mM, for example, 0.375 mM.

[0073] Preferably, the dNTP is present in an aqueous solution at a concentration of 0.1 mM to 0.4 mM, preferably 0.2 to 0.37 mM, for example, 0.284 mM.

[0074] Preferably, the dNTPs include dUTP at a concentration of 0.1 mM to 0.4 mM, preferably 0.125 to 0.234 mM, for example, 0.182 mM, in an aqueous solution.

[0075] Preferably, flap endonuclease is present in the aqueous solution at a concentration of about 0.010 μg / μl to about 0.050 ug / ul, preferably about 0.12 ug / ul to 0.047 ug / ul.

[0076] Preferably, flap endonuclease is present in the aqueous solution at a concentration of about 0.030 μg / μl to about 0.04 μg / μl, and preferably, flap endonuclease is present in the aqueous solution at a concentration of about 0.030 μg / μl to about 0.035 μg / μl.

[0077] Preferably, the organic buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

[0078] Preferably, the MOPS buffer is present in an aqueous solution at a concentration of 10 to 20 mM, and more preferably at a concentration of 12.5 mM to 20 mM.

[0079] Preferably, the organic buffer is tris(hydroxymethyl)aminomethane(Tris) buffer.

[0080] Preferably, the Tris buffer solution is present in the aqueous solution at a concentration of 40 mM to 60 mM, and preferably at a concentration of 50 mM.

[0081] Preferably, the composition contains globular proteins.

[0082] Preferably, the globular protein is bovine serum albumin (BSA).

[0083] Preferably, the bovine serum albumin (BSA) is unacetylated BSA, and preferably, ultra-high purity unacetylated BSA.

[0084] Preferably, the globular protein is present in an amount of 0.40 to 0.60 μg / μl, preferably 0.50 μg / μl.

[0085] Preferably, the composition comprises, or is essentially composed of, Cleavase® enzyme, trehalose, MOPS buffer, dNTPs, and an inorganic salt concentration of 5 mM or less, and the composition is substantially glycerol-free.

[0086] Preferably, the composition comprises or essentially consists of Cleavase® enzyme present in aqueous solution at approximately 0.030 μg / μl, trehalose present at a concentration of approximately 0.3 M, MOPS buffer at a concentration of approximately 12.5 mM, dNTPs at concentrations of approximately 0.3 mM each (optionally, dATP, dGTP, and dCTP are approximately 0.375 mM, dTTP is approximately 0.284 mM, and dUTP is approximately 0.182 mM), and inorganic salts with a concentration of 5 mM or less, and the composition is substantially glycerol-free.

[0087] Preferably, the composition comprises, or is essentially composed of, Cleavase® enzyme, trehalose, Tris buffer, dNTPs, bovine serum albumin, and an inorganic salt concentration of 5 mM or less, and the composition is substantially glycerol-free.

[0088] Preferably, the composition comprises or essentially consists of Cleavase® enzyme present in aqueous solution at approximately 0.030 μg / μl, trehalose present at a concentration of approximately 0.3 M, Tris buffer at a concentration of approximately 50 mM, dNTPs at concentrations of approximately 0.3 mM each (optionally, dATP, dGTP, and dCTP are approximately 0.375 mM, dTTP is approximately 0.284 mM, and dUTP is approximately 0.182 mM), bovine serum albumin at approximately 0.5 μg / μl, and inorganic salt concentrations of 5 mM or less, and the composition is substantially glycerol-free.

[0089] Preferably, the composition comprises or is essentially composed of Cleavase®, trehalose, MOPS buffer, dNTPs, bovine serum albumin, and an inorganic salt concentration of 5 mM or less, and the composition is substantially glycerol-free.

[0090] Preferably, the composition comprises or essentially consists of Cleavase® present in aqueous solution at approximately 0.035 μg / μl, trehalose at a concentration of approximately 0.36 M, MOPS buffer at a concentration of approximately 15 mM, dNTPs at a concentration of approximately 0.38 mM each, bovine serum albumin at approximately 0.5 μg / μl, and an inorganic salt concentration of 5 mM or less, and the composition is substantially glycerol-free.

[0091] Preferably, the composition contains α-cyclodextrin at a concentration of, for example, 0.1 to 0.5 μg / ml.

[0092] The dried forms of the compositions according to this disclosure are also disclosed. For example, drying a 24 µl aqueous solution as described herein provides dried compositions having masses in the range of about 0.003 g to about 0.004 g, about 0.0032 g to about 0.0037 g to 0.0033 g, 0.0034 g, 0.0035 g, or 0.0036 g. It should be understood that if multiple dried compositions are prepared from aliquots of a single bulk aqueous solution, the mass of each dried composition will vary. For example, drying two or more 24 µl aliquots of a bulk aqueous solution separately provides dried compositions having masses in the range of about 0.003 g to about 0.004 g. Therefore, the average weight of each of the multiple dry pellets is within the range of approximately 0.003g to 0.004g, 0.0032g to 0.0037g, 0.0033g, 0.0034g, 0.0035g, or 0.0036g. Changing the concentration of components in aqueous solution changes the mass of the dry composition. For example, changing the concentration of an enzyme to optimize the enzyme activity used in a particular reaction may change the mass of the dry composition. Similarly, changing the type of component used can change the mass of the dry composition. For example, using different flap endonucleases may require changing the concentration to provide the desired level of enzyme activity. Changing the concentration and / or type of one or more of the various components is understood to be within the scope of this disclosure, and therefore, the resulting change in the mass of the dry composition is also understood to be within the scope of this disclosure.

[0093] Also disclosed are a dry composition comprising, or essentially comprising, a flap endonuclease, a bulking agent, and an organic buffer, wherein one or more inorganic salts are present in the dry composition in an amount of 0.350% or less of the total mass of the dry composition, and the dry composition is substantially glycerol-free.

[0094] The dried composition is useful for nucleic acid-based assays after reconstitution. Surprisingly, the dried composition yields robust results after prolonged exposure to a humid environment. The dried form of the composition is useful in nucleic acid-based assays after exposure to a humid environment, where the absolute humidity level of the humid environment is greater than 2.3 grams of water per cubic meter of air for a period of up to 3 hours, preferably 90 to 180 minutes, preferably about 90 minutes, preferably about 180 minutes. Surprisingly, the dried composition yields robust results after prolonged incubation of the pre-dried aqueous solution.

[0095] Preferably, one or more inorganic salts are present in the dry composition in an amount of about 0.311% to about 0.024% by mass of the total mass of the dry composition.

[0096] Preferably, one or more inorganic salts are selected from the group consisting of sodium chloride, potassium chloride, and both sodium chloride and potassium chloride.

[0097] Preferably, the dried composition further comprises at least one oligonucleotide useful for performing molecular assays.

[0098] Preferably, the dried composition further comprises at least one oligonucleotide useful for performing nucleic acid-based assays.

[0099] Preferably, the probe oligonucleotide comprises at least one probe oligonucleotide, and preferably at least two probe oligonucleotides.

[0100] Preferably, the probe oligonucleotide(s) are partially or completely complementary to the target nucleic acid sequence.

[0101] Preferably, the flap end nuclease is a Cleavase® enzyme.

[0102] Preferably, the dry composition further comprises at least two probe oligonucleotides that can anneal to a target nucleic acid to form a three-dimensional structure that can be recognized by a flap endonuclease.

[0103] Preferably, the dry composition further comprises at least one polymerase.

[0104] Preferably, at least one polymerase is present in the aqueous solution at a concentration of about 0.10 U / μl to about 0.2 U / μl.

[0105] Preferably, at least one polymerase is present in the aqueous solution at a concentration selected from about 0.1 U / ul to about 0.25 U / ul.

[0106] Preferably, at least one polymerase is a hot-start polymerase.

[0107] Preferably, the hot-start polymerase is a recombinant Taq DNA polymerase conjugated with an antibody that specifically blocks the polymerase activity of the polymerase.

[0108] Preferably, the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase, where the chemical modification inhibits the polymerase activity.

[0109] Preferably, at least one polymerase includes a reverse transcriptase present in the aqueous solution at a concentration of about 0.1 U / μl to about 0.6 U / μl.

[0110] Preferably, the reverse transcriptase is AMV reverse transcriptase.

[0111] Preferably, the reverse transcriptase is MMLV reverse transcriptase.

[0112] Preferably, at least one oligonucleotide comprises a detection probe.

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

[0114] Preferably, the detection probe sequence is partially or completely complementary to the target nucleic acid sequence.

[0115] Preferably, the detection probe includes a label covalently bonded to it.

[0116] Preferably, the label is a fluorescent molecule or a chemiluminescent molecule.

[0117] Preferably, the label is located at the 5' end of the detection probe and the internal quenching molecule.

[0118] Preferably, at least one oligonucleotide comprises an invader probe.

[0119] Preferably, the invader probe sequence is partially or completely complementary to the target nucleic acid sequence.

[0120] Preferably, at least one oligonucleotide comprises a signal transduction probe.

[0121] Preferably, the sequence of the signal transduction probe is partially complementary to the target nucleic acid sequence.

[0122] Preferably, the sequence of the signal transduction probe includes a flap region.

[0123] Preferably, the flap region overlaps with the invader probe at least partially.

[0124] Preferably, at least one oligonucleotide comprises a FRET probe.

[0125] Preferably, the sequence of the FRET probe is partially complementary to the flap region of the signal transduction probe.

[0126] Preferably, the FRET probe includes a label covalently bonded to it.

[0127] Preferably, the label is a fluorescent molecule.

[0128] Preferably, the label is located at the 5' end of the FRET probe. Preferably, the FRET probe includes a quenching molecule that is covalently bound to the probe within a proximity range that can quench a fluorescent molecule and that can at least partially quench the fluorescence from the fluorescent molecule.

[0129] Preferably, at least one oligonucleotide comprises a target capture probe.

[0130] Preferably, the target capture probe has a target hybridizing moiety that hybridizes specifically or nonspecifically to a target nucleic acid under stringent conditions.

[0131] Preferably, the target capture probe has a target hybridizing moiety that nonspecifically hybridizes to the target nucleic acid under stringent conditions.

[0132] Preferably, the nonspecific target hybridization portion of the target capture probe includes randomly arranged K nucleotides or randomly arranged R nucleotides.

[0133] Preferably, the composition contains oligonucleotides for performing a multimolecular assay.

[0134] Preferably, the bulking agent is trehalose.

[0135] Preferably, the dried composition further comprises deoxynucleotide triphosphates (dNTPs).

[0136] Preferably, the organic buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

[0137] Preferably, the organic buffer is tris(hydroxymethyl)aminomethane(Tris) buffer.

[0138] Preferably, the dry composition contains globular proteins.

[0139] Preferably, the globular protein is bovine serum albumin (BSA).

[0140] Preferably, the bovine serum albumin (BSA) is unacetylated BSA, and preferably, ultra-high purity unacetylated BSA.

[0141] A method for forming a mixture used in nucleic acid-based assays is also disclosed, the method comprising combining a reconstitution solution with a dry composition described herein, the reconstitution solution comprising at least one inorganic salt.

[0142] Preferably, the reconstituted solution contains an inorganic salt concentration of less than 1 mM.

[0143] Preferably, the reconstitution solution contains magnesium ions.

[0144] Preferably, the reconstituted solution contains MgCl2 at a concentration of about 5 mM to about 15 mM, preferably 9 mM to 10 mM or 9 to 12 mM, and optionally 11.25 mM.

[0145] Preferably, the reconstituted 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.

[0146] Preferably, the concentration of methylparaben in the reconstituted solution is 0.016% w / v.

[0147] Preferably, the concentration of propylparaben in the reconstituted solution is 0.008% w / v.

[0148] Preferably, the concentration of anhydrous ethanol in the reconstituted solution is approximately 0.26% v / v.

[0149] A method for preparing a dry composition to be used in nucleic acid-based assays is also disclosed, the method comprising (i) freezing an aqueous solution of the present disclosure to form a frozen form of the aqueous solution, and (ii) exposing the frozen form from step (i) to freeze-drying conditions to form a dry composition.

[0150] Preferably, the dry composition is exposed to a humid environment, where the absolute humidity level of the humid environment is greater than 2.3 grams of water per cubic meter of air at 30°C.

[0151] Preferably, the dry composition is exposed to a humid environment for up to 3 hours.

[0152] Preferably, this method includes the step of storing the dried composition in a sealed container.

[0153] A method for preparing a dried composition used in nucleic acid-based assays is also disclosed, the method comprising the steps of drying an aqueous solution described in this disclosure using a drying method selected from the group consisting of dehydration, drying, freeze-drying, and spray-drying, and thereby forming a dried composition.

[0154] Preferably, the drying method is freeze-drying, and the dried composition is a freeze-dried composition.

[0155] Preferably, this method further includes the step of storing the dried composition in a sealed container.

[0156] A kit used in performing nucleic acid-based assays is also disclosed, the kit comprising a first container containing the dry composition described herein, and a second container containing a reconstituted solution containing MgCl2 at a concentration of about 3.8 mM to about 14.4 mM, preferably about 9.4 mM or about 11.25 mM.

[0157] Preferably, the first container is a multiwell plate containing one or more wells.

[0158] Preferably, each of one or more wells contains a dry single-unit dose pellet containing a mass percentage of inorganic salt relative to the mass of the pellet, up to 0.311%.

[0159] Preferably, the first and second containers are incorporated into a device adapted for the automated transfer of the reconstituted solution from the second container to the first container.

[0160] Preferably, each of one or more wells contains a dry single-unit dose pellet weighing about 0.000125 g to about 0.000667 g per microliter of a dried aqueous solution so as to form a dry single-unit dose pellet in each well.

[0161] Preferably, each of one or more wells contains a dry single-unit dose pellet having a weight in the range of approximately 0.003g to approximately 0.004g, approximately 0.0032g to approximately 0.0037g, 0.0033g, 0.0034g, 0.0035g, or 0.0036g.

[0162] Dialysis compositions comprising, consisting of, or essentially comprising an aqueous solution containing an organic buffer, a bulking agent, chloride ions, and a chelating agent are also disclosed.

[0163] Preferably, the bulking agent is trehalose.

[0164] Preferably, the bulking agent is present at a concentration of approximately 100 mM to 300 mM, preferably 200 mM.

[0165] Preferably, the organic buffer is tris(hydroxymethyl)aminomethane(Tris) buffer.

[0166] Preferably, the Tris buffer solution is present in the aqueous solution at a concentration of 10 mM to 30 mM, preferably at a concentration of 20 mM. Preferably, the pH is approximately 8.0.

[0167] Preferably, the chloride ion is KCl.

[0168] Preferably, KCl is present at a concentration of approximately 40-60 mM, preferably 50 mM.

[0169] Preferably, the chelating agent is EDTA.

[0170] Preferably, EDTA is present in the aqueous solution at a concentration of 0.05 to 0.2 mM, preferably 0.1 mM.

[0171] Preferably, the composition comprises flap endonuclease, preferably Cleavase® enzyme.

[0172] A method for preparing a flap endonuclease composition substantially free of glycerol is also disclosed, the method comprising (i) providing an aqueous solution comprising, consisting of or essentially consisting of, a flap endonuclease and glycerol; (ii) dialyzing the aqueous solution to the dialysis composition described herein; and (iii) obtaining a flap endonuclease composition substantially free of glycerol.

[0173] Preferably, the aqueous solution of step (i) contains about 0.0 to about 0.5% (w / v) glycerol, preferably about 0.35% (w / v) glycerol, and more preferably less than 0.2% (w / v) glycerol.

[0174] The use of the dried compositions described herein for preparing dried flap end nuclease-containing compositions is also disclosed.

[0175] The use of the dry compositions described herein in combination with reconstitution solutions for performing nucleic acid-based assays is also disclosed.

[0176] The use of the dialysis compositions described herein for preparing a flap endonuclease composition that is substantially free of glycerol is also disclosed.

[0177] definition The term "approximately" indicates a very small variation in the amount of a component of the composition that does not have any significant effect on the activity or stability of the composition.

[0178] "Bulking agents" provide a matrix for the precipitation of proteins and other reagents 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 "cake" or other structure of the product, preventing protein loss from the vial during drying and increasing protein stability.

[0179] "Chelating agents" are Mg, which is necessary for enzyme activity. 2+ or Mn 2+ It is a drug that sequesters divalent ions such as those mentioned above.

[0180] The terms "freeze-dried," "freeze-dried," and "freeze-dried" refer to a process in which the material to be dried is first frozen, and then the ice or freezing solvent is removed by sublimation in a vacuum environment. "Freeze-dried material" refers to a substance that has been freeze-dried.

[0181] The term "stringent" in relation to nucleic acid hybridization (including "stringent hybridization conditions" or "stringent conditions") refers to conditions under which a particular oligonucleotide can hybridize with a target nucleic acid more effectively than other nucleic acids present in the test sample. It should be understood that these conditions can vary depending on factors including the GC content and length of the oligonucleotide, the hybridization temperature, the composition of the hybridization reagent or solution, and the desired degree of hybridization specificity. Appropriate hybridization conditions for probes, oligonucleotides, target capture nucleotides, blockers, and other nucleotides are well known in the art and can be predicted based on sequence composition or determined by routine testing methods (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, especially §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

[0182] The term "cleavage structure" refers to a structure formed by the interaction of several nucleic acids such that two nucleic acids form a structure containing nucleic acid bases configured to hybridize at a single nucleic acid base position on a third nucleic acid, with an overlap formed between the first two nucleic acids, and the resulting non-hybridized flap region (e.g., cleavage structure) is cleavable by flap endonucleases. Cleavage structures are substrates for specific cleavage by flap endonucleases (i.e., the formation of a double structure is not required), in contrast to nucleic acid molecules that are substrates for nonspecific cleavage by drugs such as phosphodiesterases, which cleave nucleic acid molecules regardless of their secondary structure. For further discussion of cleavage structures and other embodiments of flap endonuclease detection assays, see U.S. Patent No. 5,846,717.

[0183] As used herein, "flap endonucleases" refer to a class of nucleases that act as structure-specific 5' endonucleases for double-stranded nucleic acid structures in which one of the strands is substituted by another strand of nucleic acid and contains a single-stranded 5' overhang or flap (i.e., such that there are duplicate nucleotides that adjacent first and second probes hybridize to the target). Flap endonucleases may also be referred to as "5' endonucleases," or abbreviated as "FEN." FENs catalyze the hydrolytic cleavage of phosphodiester bonds at the junctions of single-stranded and double-stranded nucleic acids, releasing the overhang or flap. FENs have been studied by Ceska and Savers (Trends Biochem. Sci. 23:331-336, 1998) and Liu et al. (Annu. Rev. Biochem. 73:589-615, 2004).

[0184] The "overlap region" or "flap region" consists of one or more bases of a first probe oligonucleotide (e.g., a signal probe) that hybridizes to the target and is overlapped by a second probe oligonucleotide (e.g., an invader probe). The 3' terminal base of the second probe oligonucleotide determines the end of the overlap region and may or may not hybridize to the target.

[0185] In cleavage-based assays, the “first probe oligonucleotide” refers to an oligonucleotide (e.g., a signal probe) that interacts with the target nucleic acid and forms a cleavage structure in the presence of a “second probe oligonucleotide” that hybridizes with the upstream region of the first probe oligonucleotide (e.g., an invader probe). Upon annealing to the target nucleic acid, the first probe oligonucleotide and the target form a cleavage structure, and cleavage by a flap endonuclease may occur within the first probe oligonucleotide. In the presence of an overlapping second probe oligonucleotide upstream of the first probe oligonucleotide along the target nucleic acid, the cleavage site within the first probe oligonucleotide occurs after the last overlapping base (the cleavage depends on at least one overlapping base of the second probe having the target hybridizing base of the first probe). In addition to the target hybridizing region that hybridizes to the target sequence in the target nucleic acid, the first probe oligonucleotide includes a non-target hybridizing region (also referred to as the “flap region”) at its 5' end. When the first and second probe oligonucleotides are annealed to the target nucleic acid, site-specific cleavage occurs by flap endonucleases, generating a cleavage product that includes the flap region and the overlapping region of the first probe oligonucleotide.

[0186] In cleavage-based assays, a “second probe oligonucleotide” refers to an oligonucleotide whose 3' end, when annealed to the target nucleic acid, overlaps with the 5' end of the target hybridize sequence in a downstream first probe oligonucleotide. Typically, these regions compete for hybridization to the same segment along the complementary target nucleic acid. The second probe may, in some cases, be used not only as a primer but also as an invader probe. The 3' terminal nucleotide of the second probe oligonucleotide may or may not form a 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 hybridize sequence of the first probe oligonucleotide. The cleaved second probe oligonucleotide can participate in a secondary reaction and act as a probe to a fluorescence resonance energy transfer cassette (e.g., FRET probe or FRET cassette) resulting in the formation of a duplicate structure recognized by the Cleavase® enzyme. When the FRET cassette is cleaved, a fluorophore is released from the quencher of the FRET cassette, which produces a fluorescent signal.

[0187] As used herein, the term "FRET cassette" refers to a hairpin oligonucleotide comprising a fluorophore moiety and a nearby quencher moiety that quenches the fluorophore. Hybridization of the cleavage product with the FRET cassette generates a secondary substrate for the flap endonuclease. Once this substrate is formed, the 5' fluorophore-containing base is cleaved from the cassette, thereby generating a fluorescent signal.

[0188] An "amplifying oligomer" is a primer or promoter primer that can support template-dependent replication. An amplified oligomer pair is a pair of such oligomers that support template-dependent replication of the counter-strand of the template. Multiple amplification is amplification that occurs simultaneously with multiple amplified oligomer pairs.

[0189] A "detection probe" is an oligonucleotide that can hybridize to an amplified product or initial target nucleic acid to reveal the presence or amount of the amplified product. Such detection probes often incorporate molecules that produce fluorescence or other detectable signals, in which case they are referred to as detectably labeled probes.

[0190] A "primer-probe set" is a combination of primers and detection probes configured to generate amplification products from a template nucleic acid.

[0191] "Reconstitution time" is the time required to rehydrate the dry formulation with a solution to obtain a solution that is free of particles and turbidity visible to the naked eye.

[0192] Relative fluorescence units (RFU) are a measure of unquenched fluorophores. In nucleic acid-based amplification reactions, the presence of a product is determined by measuring RFU at several cycle times (Ct).

[0193] "Ct" refers to the number of cycles required to reach the logarithmic phase in real-time PCR. Ct is inversely correlated with the amount of analyte in the sample.

[0194] When referring to assay results, "positive" refers to the percentage of samples that exceeded the exponential threshold in a test involving multiple samples. For example, if there are 12 samples and it is determined that 6 samples exceeded the threshold, the positive result is "50%".

[0195] A "single unit dose" or "SUD" refers to the volume of a reaction mixture used to perform an assay on a single sample. A single unit dose can be in liquid or dry form. For example, a single unit dose may be a dry pellet containing a reagent useful for detecting a single sample in a single container.

[0196] "LOD" is the detection limit of the analyte. LOD+1 is the logarithm of LOD+1 detected by the user. In other words, LOD+1 is 10 times the number of analytes that meet the LOD.

[0197] A composition disclosed as containing dTTP and dUTP may contain dTTP or dUTP or both at the indicated concentrations. Similarly, if a composition is disclosed as containing dTTP or dUTP, the disclosure should be understood to alternatively include a composition containing both dTTP and dUTP at the indicated concentrations. In embodiments of the present invention, for example, the following items are provided. (Item 1) A composition comprising or essentially consisting of a flap endonuclease, a bulking agent, and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 5 mM or less, and the composition is substantially free of glycerol. (Item 2) The composition according to item 1, wherein the aqueous solution further comprises at least one oligonucleotide useful for performing a molecular assay. (Item 3) The composition according to item 1 or item 2, wherein the aqueous solution further comprises at least one oligonucleotide useful for performing nucleic acid-based assays. (Item 4) The composition according to item 3, wherein the at least one oligonucleotide comprises a probe oligonucleotide, preferably at least two probe oligonucleotides. (Item 5) The composition according to item 4, wherein the probe oligonucleotide(s) are partially or completely complementary to the target nucleic acid sequence. (Item 6) The composition according to any one of items 1 to 5, wherein the flap endonuclease is the Cleavase® enzyme. (Item 7) A composition according to any one of items 2 to 6, comprising at least two probe oligonucleotides that can anneal to a target nucleic acid to form a three-dimensional structure recognizable by the flap endonuclease. (Item 8) The compositions according to items 1 to 7, further comprising at least one polymerase. (Item 9) The composition according to item 8, wherein the at least one polymerase is a polymerase present in the aqueous solution at a concentration of about 0.10 U / μl to about 0.2 U / μl. (Item 10) The composition according to item 8 or 9, wherein the at least one polymerase comprises a polymerase present in the aqueous solution at a concentration selected from 0.14 U / μl, 0.146 U / μl, and 0.1686 U / μl. (Item 11) The composition according to any one of items 8 to 10, wherein the at least one polymerase is a hot-start polymerase. (Item 12) The composition according to item 11, wherein the hot-start polymerase is recombinant Taq DNA polymerase, and preferably the polymerase is conjugated by an antibody that specifically blocks the polymerase activity of the polymerase. (Item 13) The composition according to item 12, wherein the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase, and the chemical modification inhibits the polymerase activity of the polymerase. (Item 14) The composition according to any one of items 8 to 13, wherein the at least one polymerase preferably comprises a reverse transcriptase present in the aqueous solution at a concentration of about 0.1 U / μl to about 4.0 U / μl. (Item 15) The composition according to item 14, wherein the reverse transcriptase is AMV reverse transcriptase. (Item 16) The composition according to item 14, wherein the reverse transcriptase is MMLV reverse transcriptase. (Item 17) The composition according to any one of items 2 to 16, wherein the at least one oligonucleotide comprises an invader probe. (Item 18) The composition according to item 17, wherein the sequence of the invader probe is partially or completely complementary to the target nucleic acid sequence. (Item 19) The composition according to any one of items 2 to 18, wherein the at least one oligonucleotide comprises a signal transduction probe. (Item 20) The composition according to item 19, wherein the sequence of the signal transduction probe is partially complementary to the target nucleic acid sequence. (Item 21) The composition according to item 19 or item 20, wherein the sequence of the signal transduction probe includes a flap region. (Item 22) The composition according to item 21, wherein the flap region at least partially overlaps with the invader probe. (Item 23) The composition according to any one of items 2 to 22, wherein the at least one oligonucleotide comprises a FRET probe. (Item 24) The composition according to item 23, wherein the sequence of the FRET probe is partially complementary to the flap region of the signal transduction probe. (Item 25) The composition according to item 23 or item 24, wherein the FRET probe comprises a label covalently bonded thereto. (Item 26) The composition according to item 25, wherein the label is a fluorescent molecule. (Item 27) The composition according to item 26, wherein the label is located at the 5' end of the FRET probe. (Item 28) The composition according to any one of items 23 to 27, wherein the FRET probe contains a quenching molecule that is covalently bonded to the probe within a proximity range that quenches the fluorescent molecule and can at least partially quench the fluorescence from the fluorescent molecule. (Item 29) The composition according to any one of items 2 to 28, wherein the at least one oligonucleotide comprises a target capture probe. (Item 30) The composition according to item 29, wherein the target capture probe has a target hybridize moiety that hybridizes specifically or nonspecificly to a target nucleic acid under stringent conditions. (Item 31) The composition according to any one of items 2 to 30, wherein the aqueous solution contains two or more oligonucleotides for performing a multimolecule assay. (Item 32) The composition according to any one of items 1 to 31, wherein the volume extender is trehalose. (Item 33) The composition according to any one of items 1 to 32, wherein the bulking agent is present at a concentration of about 0.2 M to about 0.5 M, preferably about 0.36 M or about 0.47 M. (Item 34) The composition according to any one of items 1 to 33, wherein the inorganic salt is present in a mass of approximately 0.373 μg / μl to approximately 0.029 μg / μl per microliter. (Item 35) The composition according to any one of items 1 to 34, wherein the inorganic salt is sodium chloride, and preferably the sodium chloride is present in a mass per microliter of about 0.292 μg / μl to about 0.35 μg / μl, preferably about 0.32 ug / ul. (Item 36) The composition according to any one of items 1 to 34, wherein the inorganic salt is potassium chloride, preferably present in a mass of about 0.373 μg / μl to about 0.019 μg / μl of potassium chloride, and preferably about 0.03 ug / ul per microliter. (Item 37) The composition according to any one of items 1 to 36, wherein the aqueous solution contains approximately 0.135 μg / μl of sodium ions to approximately 0.006 μg / μl of sodium ions, preferably approximately 0.127 ug / ul. (Item 38) The composition according to any one of items 1 to 37, wherein the aqueous solution contains approximately 0.196 μg / μl of potassium ions to approximately 0.010 μg / μl of potassium ions, preferably approximately 0.016 ug / ul of potassium ions. (Item 39) The composition according to any one of items 1 to 38, wherein the aqueous solution contains approximately 0.355 μg / μl of chloride ions to approximately 0.009 μg / μl of chloride ions, preferably approximately 0.337 ug / ul. (Item 40) The composition according to any one of items 1 to 39, wherein the aqueous solution contains an inorganic salt concentration of 4 mM or less. (Item 41) The composition according to item 40, wherein the aqueous solution contains an inorganic salt in a mass per microliter of approximately 0.298 μg / μl to approximately 0.234 μg / μl. (Item 42) The composition according to item 40 or 41, wherein the aqueous solution contains chloride ions in a mass per microliter of about 0.284 μg / μl to about 0.071 μg / μl. (Item 43) The composition according to any one of items 1 to 42, wherein the aqueous solution contains an inorganic salt concentration of 3 mM or less. (Item 44) The composition according to item 43, wherein the aqueous solution contains an inorganic salt in a mass per microliter of approximately 0.224 μg / μl to approximately 0.175 μg / μl. (Item 45) The composition according to item 43 or item 44, wherein the aqueous solution contains chloride ions in a mass per microliter of about 0.213 μg / μl to about 0.053 μg / μl. (Item 46) The composition according to any one of items 1 to 45, wherein the aqueous solution contains an inorganic salt concentration of 2 mM or less. (Item 47) The composition according to item 46, wherein the aqueous solution contains an inorganic salt in a mass per microliter of approximately 0.149 μg / μl to approximately 0.117 μg / μl. (Item 48) The composition according to item 46 or 47, wherein the aqueous solution contains chloride ions in a mass per microliter of about 0.142 μg / μl to about 0.036 μg / μl. (Item 49) The composition according to any one of items 1 to 48, wherein the aqueous solution contains an inorganic salt concentration of 1 mM or less. (Item 50) The composition according to item 49, wherein the aqueous solution contains an inorganic salt in a mass per microliter of approximately 0.075 μg / μl to approximately 0.058 μg / μl. (Item 51) The composition according to item 49 or 50, wherein the aqueous solution contains chloride ions in a mass per microliter of about 0.071 μg / μl to about 0.018 μg / μl. (Item 52) The composition according to any one of items 1 to 51, wherein the aqueous solution contains an inorganic salt concentration of 500 μM or less. (Item 53) The composition according to item 52, wherein the aqueous solution contains an inorganic salt in a mass per microliter of approximately 0.037 μg / μl to approximately 0.029 μg / μl. (Item 54) The composition according to item 52 or 53, wherein the aqueous solution contains chloride ions in a mass per microliter of about 0.036 μg / μl to about 0.009 μg / μl. (Item 55) The composition according to any one of items 1 to 54, wherein the inorganic salt concentration of the aqueous solution is less than 1 mM sodium chloride. (Item 56) The composition according to any one of items 1 to 55, wherein the aqueous solution does not contain sodium chloride. (Item 57) The composition according to any one of items 1 to 56, wherein the aqueous solution contains less than 1 mM of magnesium ions. (Item 58) The composition according to any one of items 1 to 57, wherein the aqueous solution contains less than 0.1 mM of magnesium ions. (Item 59) The composition according to any one of items 1 to 58, wherein the aqueous solution further comprises deoxynucleotide triphosphate (dNTP). (Item 60) The composition according to item 59, wherein the dNTP contains dATP at a concentration of 0.1 mM to 0.4 mM, preferably 0.29 to 0.46 mM, in the aqueous solution. (Item 61) The composition according to item 59 or 60, wherein dATP is present in the aqueous solution at a concentration of 0.3 mM to 0.4 mM, for example, 0.375 mM. (Item 62) The composition according to any one of items 1 to 61, wherein the dNTP contains dGTP at a concentration of 0.1 mM to 0.4 mM, preferably 0.29 to 0.46 mM, in the aqueous solution. (Item 63) The composition according to item 62, wherein the dGTP is concentrated in the aqueous solution at a concentration of 0.3 mM to 0.4 mM, for example, 0.375 mM. (Item 64) The composition according to any one of items 1 to 63, wherein the dNTP contains dCTP at a concentration of 0.1 mM to 0.4 mM, preferably 0.29 to 0.46 mM, in the aqueous solution. (Item 65) The composition according to item 64, wherein the dCTP is concentrated in the aqueous solution at a concentration of 0.3 mM to 0.4 mM, for example, 0.375 mM. (Item 66) The composition according to any one of items 1 to 65, wherein the dNTP contains dTTP at a concentration of 0.1 mM to 0.4 mM, preferably 0.2 to 0.37 mM, for example, 0.284 mM, in the aqueous solution. (Item 67) The composition according to item 66, wherein the dTTP is concentrated at a concentration of 0.3 mM to 0.4 mM in the aqueous solution. (Item 68) The composition according to any one of items 1 to 67, wherein the dNTP contains dUTP at a concentration of 0.1 mM to 0.4 mM, preferably 0.125 to 0.234 mM, for example, 0.182 mM, in the aqueous solution. (Item 69) The composition according to item 68, wherein the dUTP is concentrated at a concentration of 0.3 mM to 0.4 mM in the aqueous solution. (Item 70) The composition according to any one of items 1 to 69, wherein the flap end nuclease is present in the aqueous solution at a concentration of approximately 0.020 μg / μl to approximately 0.040 μg / μl. (Item 71) The composition according to item 70, wherein the flap endonuclease is present in the aqueous solution at a concentration of approximately 0.030 μg / μl to approximately 0.04 μg / μl, and preferably at a concentration of approximately 0.030 μg / μl to approximately 0.035 μg / μl. (Item 72) The composition according to any one of items 1 to 71, wherein the organic buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. (Item 73) The composition according to item 72, wherein the MOPS buffer is present in the aqueous solution at a concentration of 10 to 20 mM, preferably at a concentration of 12.5 mM to 15 mM. (Item 74) The composition according to any one of items 1 to 71, wherein the organic buffer is tris(hydroxymethyl)aminomethane (Tris) buffer. (Item 75) The composition according to item 74, wherein the Tris buffer solution is preferably present in the aqueous solution at a concentration of 40 mM to 60 mM, and more preferably at a concentration of 50 mM. (Item 76) The composition according to any one of items 1 to 75, wherein the composition comprises a globular protein. (Item 77) The composition according to item 76, wherein the globular protein is bovine serum albumin (BSA). (Item 78) The composition according to item 77, wherein the bovine serum albumin (BSA) is non-acetylated BSA, preferably ultra-high purity non-acetylated BSA. (Item 79) The composition according to any one of items 76 to 78, wherein the globular protein is present in an amount of 0.40 to 0.60 μg / μl, preferably 0.50 μg / μl. (Item 80) A composition according to any one of items 1 to 79, comprising, or essentially consisting of, Cleavase® enzyme, trehalose, MOPS buffer, dNTP, and an inorganic salt concentration of 5 mM or less, wherein the composition is substantially free of glycerol. (Item 81) The composition according to item 80, comprising or essentially consisting of Cleavase® present in the aqueous solution at a concentration of approximately 0.030 μg / μl, trehalose present at a concentration of approximately 0.3 M, MOPS buffer at a concentration of approximately 12.5 mM, dNTPs at a concentration of approximately 0.3 mM each, and an inorganic salt concentration of 5 mM or less, wherein the composition is substantially free of glycerol. (Item 82) A composition according to any one of items 1 to 81, comprising, or essentially consisting of, Cleavase® enzyme, trehalose, Tris buffer, dNTP, bovine serum albumin, and an inorganic salt concentration of 5 mM or less, wherein the composition is substantially free of glycerol. (Item 83) The composition according to item 82, comprising, or essentially consisting of, the following: Cleavase® enzyme present in the aqueous solution at approximately 0.030 μg / μl; trehalose present at a concentration of approximately 0.3 M; Tris buffer at a concentration of approximately 50 mM; dNTPs at a concentration of approximately 0.3 mM each; bovine serum albumin at approximately 0.5 μg / μl; and an inorganic salt concentration of 5 mM or less, wherein the composition is substantially free of glycerol. (Item 84) A composition according to any one of items 1 to 83, comprising, or essentially consisting of, Cleavase® enzyme, trehalose, MOPS buffer, dNTP, bovine serum albumin, and an inorganic salt concentration of 5 mM or less, wherein the composition is substantially free of glycerol. (Item 85) The composition according to item 84, comprising, or essentially consisting of, the following: Cleavase® enzyme present in the aqueous solution at approximately 0.035 μg / μl; trehalose present at a concentration of approximately 0.36 M; MOPS buffer at a concentration of approximately 15 mM; dNTPs at a concentration of approximately 0.38 mM each; bovine serum albumin at approximately 0.5 μg / μl; and an inorganic salt concentration of 5 mM or less, wherein the composition is substantially free of glycerol. (Item 86) For example, a composition according to any one of items 1 to 85, further comprising 0.1 to 0.5 μg / ml of α-cyclodextrin. (Item 87) The dry form of any of the compositions described in items 1 to 85. (Item 88) A dry composition comprising or essentially comprising a flap endonuclease, a bulking agent, and an organic buffer, wherein one or more inorganic salts are present in the dry composition in an amount of 0.350% or less of the total mass of the dry composition, and the dry composition is substantially glycerol-free. (Item 89) The dry composition according to item 88, wherein one or more inorganic salts are present in the dry composition in an amount of about 0.311% to about 0.024% by mass of the total mass of the dry composition. (Item 90) The dried composition according to item 88 or 89, wherein the one or more inorganic salts are selected from the group consisting of sodium chloride, potassium chloride, and both sodium chloride and potassium chloride. (Item 91) A dried composition according to any one of items 88-90, further comprising at least one oligonucleotide useful for performing molecular assays. (Item 92) A dried composition according to any one of items 88 to 91, further comprising at least one oligonucleotide useful for performing nucleic acid-based assays. (Item 93) The dry composition according to item 91 or 92, wherein the at least one oligonucleotide comprises a probe oligonucleotide, preferably at least two probe oligonucleotides. (Item 94) The dried composition according to item 93, wherein the probe oligonucleotide(s) are partially or completely complementary to the target nucleic acid sequence. (Item 95) The dried composition according to any one of items 88 to 94, wherein the flap end nuclease is the Cleavase® enzyme. (Item 96) The dried composition according to item 95, comprising at least two probe oligonucleotides that can anneal to a target nucleic acid to form a three-dimensional structure that can be recognized by the flap endonuclease. (Item 97) A dry composition according to any one of items 88 to 96, further comprising at least one polymerase. (Item 98) The dry composition according to item 97, wherein the at least one polymerase is a polymerase present in the aqueous solution at a concentration of about 0.10 U / μl to about 0.2 U / μl. (Item 99) The dry composition according to item 97, wherein the at least one polymerase comprises a polymerase present in the aqueous solution at a concentration selected from 0.14 U / μl, 0.146 U / μl, and 0.1686 U / μl. (Item 100) A dry composition according to any one of items 97 to 99, wherein the at least one polymerase is a hot-start polymerase. (Item 101) The dry composition according to item 100, wherein the hot-start polymerase is recombinant Taq DNA polymerase, and preferably the polymerase is conjugated by an antibody that specifically blocks the polymerase activity of the polymerase. (Item 102) The dried composition according to item 101, wherein the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase, and the chemical modification inhibits the polymerase activity of the polymerase. (Item 103) The dry composition according to any one of items 97 to 102, wherein the at least one polymerase preferably comprises a reverse transcriptase present in the aqueous solution at a concentration of about 0.1 U / μl to about 0.6 U / μl. (Item 104) The dried composition according to item 103, wherein the reverse transcriptase is AMV reverse transcriptase. (Item 105) The dried composition according to item 103, wherein the reverse transcriptase is MMLV reverse transcriptase. (Item 106) The dried composition according to any one of items 92 to 105, wherein the at least one oligonucleotide comprises an invader probe. (Item 107) The dried composition according to item 106, wherein the sequence of the invader probe is partially or completely complementary to the target nucleic acid sequence. (Item 108) The dried composition according to any one of items 91 to 107, wherein the at least one oligonucleotide comprises a signal transduction probe. (Item 109) The dried composition according to item 108, wherein the sequence of the signal transduction probe is partially complementary to the target nucleic acid sequence. (Item 110) The dry composition according to item 108 or item 109, wherein the sequence of the signal transduction probe includes a flap region. (Item 111) The dry composition according to item 110, wherein the flap region at least partially overlaps with the invader probe. (Item 112) The dried composition according to any one of items 91 to 111, wherein the at least one oligonucleotide comprises a FRET probe. (Item 113) The dried composition according to item 112, wherein the sequence of the FRET probe is partially complementary to the flap region of the signal transduction probe. (Item 114) The dry composition according to item 112 or item 113, wherein the FRET probe comprises a label covalently bonded thereto. (Item 115) The dried composition according to item 114, wherein the label is a fluorescent molecule. (Item 116) The dry composition according to item 115, wherein the label is located at the 5' end of the FRET probe. (Item 117) A dry composition according to any one of items 112 to 116, comprising a quenching molecule that is covalently bonded to the FRET probe within a proximity range that quenches the fluorescent molecule, and that can at least partially quench the fluorescence from the fluorescent molecule. (Item 118) The dried composition according to any one of items 91 to 117, wherein the at least one oligonucleotide comprises a target capture probe. (Item 119) The dried composition according to item 118, wherein the target capture probe has a target hybridize moiety that hybridizes specifically or nonspecificly to a target nucleic acid under stringent conditions. (Item 120) The dried composition according to any one of items 91 to 119, wherein the aqueous solution contains oligonucleotides for performing a multimolecule assay. (Item 121) The dry composition according to any one of items 91 to 120, wherein the bulking agent is trehalose. (Item 122) The dry composition according to any one of items 91 to 121, wherein the composition further comprises deoxynucleotide triphosphate (dNTP). (Item 123) The dried composition according to any one of items 91 to 122, wherein the organic buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. (Item 124) The dry composition according to any one of items 91 to 122, wherein the organic buffer is tris(hydroxymethyl)aminomethane (Tris) buffer. (Item 125) The dried composition according to any one of items 91 to 124, wherein the dried composition comprises a globular protein. (Item 126) The dried composition according to item 125, wherein the globular protein is bovine serum albumin (BSA). (Item 127) The dried composition according to item 126, wherein the bovine serum albumin (BSA) is non-acetylated BSA, preferably ultra-high purity non-acetylated BSA. (Item 128) A dry composition according to any one of items 87 to 127, wherein the mass of the dry composition is about 0.003 g to about 0.004 g, or about 0.0032 g to about 0.0037 g, or 0.0033 g, or 0.0034 g, or 0.0035 g, or 0.0036 g, or about 0.000125 g to about 0.000667 g per microliter of aqueous solution dried to form the dry composition. (Item 129) A method for forming a mixture used in performing nucleic acid-based assays, comprising combining a reconstitution solution with a dried composition described in any one of items 87 to 128, wherein the reconstitution solution comprises at least one inorganic salt. (Item 130) The method according to item 129, wherein the reconstituted solution contains an inorganic salt concentration of less than 1 mM. (Item 131) The method according to item 129 or 130, wherein the reconstitution solution contains magnesium ions. (Item 132) The method according to item 131, wherein the reconstituted solution contains MgCl2 at a concentration of about 5 mM to about 15 mM, preferably 9 mM to 12 mM, for example, 11.25 mM. (Item 133) The method according to any one of items 129 to 132, wherein the reconstituted 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. (Item 134) The method according to item 133, wherein the concentration of methylparaben in the reconstituted solution is 0.016% w / v. (Item 135) The method according to item 133 or item 134, wherein the concentration of propylparaben in the reconstituted solution is 0.008% w / v. (Item 136) The method according to any one of items 133 to 135, wherein the concentration of the anhydrous ethanol is present in the reconstituted solution at approximately 0.26% v / v. (Item 137) A method for preparing a dry composition used in nucleic acid-based assays, comprising: (i) freezing an aqueous solution described in any one of items 1 to 78 to form a frozen form of the aqueous solution; and (ii) exposing the frozen form from step (i) to freeze-drying conditions to form a dry composition. (Item 138) The method according to item 137, wherein the dry composition is exposed to a humid environment, and the absolute humidity level of the humid environment is greater than 2.3 grams of water per cubic meter of air at 30°C. (Item 139) The method according to item 138, wherein the dried composition is exposed to the humid environment for up to 3 hours. (Item 140) The method according to any one of items 137 to 139, further comprising the step of storing the dried composition in a sealed container. (Item 141) The method according to any one of items 137 to 140, wherein the mass of the dried composition is from about 0.003 g to about 0.004 g, or from about 0.0032 g to about 0.0037 g, or 0.0033 g, or 0.0034 g, or 0.0035 g, or 0.0036 g, or about 0.000125 g to about 0.000667 g per microliter of the aqueous solution dried to form the dried composition. (Item 142) A method for preparing a dried composition used in performing a nucleic acid-based assay, comprising drying the aqueous solution according to any one of items 1 to 85 using a drying method selected from the group consisting of dehydration, drying, freeze-drying, and spray-drying, thereby forming a dried composition. (Item 143) The method according to item 142, wherein the drying method is freeze-drying and the dried composition is a freeze-dried composition. (Item 144) The method according to item 142 or 143, further comprising the step of storing the dried composition in a sealed container. (Item 145) A kit for performing a nucleic acid-based assay, comprising a first container containing the dried composition according to any one of items 86 to 127, and a second container containing a reconstitution solution containing MgCl2 at a concentration of about 3.8 mM to about 4.4 mM. (Item 146) The kit according to item 145, wherein the first container is a multi-well plate containing one or more wells. (Item 147) The kit according to item 145, wherein each of the one or more wells contains a dried single unit dose pellet containing an inorganic salt mass percentage of not more than 0.311% based on the mass of the pellet. (Item 148) The kit according to any one of items 145 to 147, wherein the first and second containers are incorporated into a device adapted for the automated transfer of the reconstituted solution from the second container to the first container. (Item 149) A dialysis composition comprising, or essentially comprising, an aqueous solution containing an organic buffer, a bulking agent, chloride ions, and a chelating agent. (Item 150) The dialysis composition according to item 149, wherein the volume expander is trehalose. (Item 151) The dialysis composition according to item 149 or item 150, wherein the volume extender is present at a concentration of approximately 100 mM to 300 mM, preferably 200 mM. (Item 152) The dialysis composition according to any one of items 149 to 151, wherein the organic buffer is tris(hydroxymethyl)aminomethane (tris) buffer. (Item 153) The dialysis composition according to item 152, wherein the Tris buffer solution is present in the aqueous solution at a concentration of 10 mM to 30 mM, preferably at a concentration of 50 mM. (Item 154) The dialysis composition according to any one of items 149 to 153, wherein the chloride ion is KCl. (Item 155) The dialysis composition according to item 154, wherein the KCl is present at a concentration of approximately 40 to 60 mM, preferably 50 mM. (Item 156) The dialysis composition according to any one of items 151 to 155, wherein the chelating agent is EDTA. (Item 157) The dialysis composition according to item 156, wherein the EDTA is present in the aqueous solution at a concentration of 0.05 to 0.2 mM, preferably 0.1 mM. (Item 158) A dialysis composition according to any one of items 149 to 157, further comprising a flap endonuclease, preferably the Cleavase® enzyme. (Item 159) A method for preparing a flap endonuclease composition substantially free of glycerol, comprising: (i) providing an aqueous solution comprising, consisting of, or essentially composed of, a flap endonuclease and glycerol; (ii) dialyzing the aqueous solution to a dialysis composition described in any of items 149 to 158; and (iii) obtaining a flap endonuclease composition substantially free of glycerol. (Item 160) The method according to item 159, wherein the aqueous solution in step (i) contains about 0.3 to about 0.5% (w / v) glycerol, preferably about 0.35% (w / v) glycerol. (Item 161) Use of any of the aqueous compositions described in items 1 to 86 for preparing a composition containing dried flap end nuclease. (Item 162) Use of any of the aqueous compositions described in items 1 to 86, in combination with a reconstitution solution for performing nucleic acid-based assays. (Item 163) Use of any of the dialysis compositions described in items 149 to 158 for preparing a flap endonuclease composition that is substantially free of glycerol. [Modes for carrying out the invention]

[0198] I. Overview This disclosure is at least partly based on the finding that the instability of conventional lyophilized kits for performing nucleic acid-based assays is due to the presence of inorganic salts. These salts can produce undesirable hybridization products or other by-products before, during, and after drying. These salts also make the dried composition hygroscopic, requiring limited exposure to moisture, storage by refrigeration or cryogenic freezing, and / or storage in the presence of a desiccant. The presence of water and salts can cause premature loss of activity of the enzymatic components in such kits and can also promote hybridization of nucleic acids to each other within such kits. This disclosure overcomes these problems by drying reagents for performing nucleic acid-based assays from bulk reagents that are essentially free of inorganic salts. Such salts are supplied during the reconstitution of the dried composition. Contrary to the expectation that inorganic salts are necessary for the stability of enzymes used in nucleic acid-based assays, it has been found that reaction mixtures dried to be essentially free of inorganic salts can be stored for extended periods at temperatures above freezing while retaining full or substantially the same activity during reconstitution.

[0199] II. Bulk Reagents and Dry Pellets The bulk reagents described herein (which may also be referred to as pre-lyophilized mixtures, solutions, aqueous solutions, or compositions) typically comprise a flap endonuclease, an extender, and an organic buffer. The bulk reagents may or may not comprise one or more nucleic acids and / or dNTPs. The bulk reagents may also comprise a chelating agent and an RNase inhibitor.

[0200] Such bulk reagents are essentially free of inorganic salts, meaning that the concentrations of inorganic salts, individually and collectively, are less than 5 mM, 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.

[0201] Such bulk reagents are substantially glycerol-free. As used herein, the term “substantially glycerol-free” means that glycerol is not present in amounts commonly used in enzyme preparations. Preferably, glycerol is present in amounts 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). Preferably, glycerol is present in amounts undetectable by conventional methods known in the art. For example, the free glycerol concentration can be measured using the Abcam (Cambridge, UK) Glycerol Assay Kit by enzymatically oxidizing glycerol and reacting it with a probe to produce a product that produces color and fluorescence. This kit can detect glycerol in a variety of samples from 50 pmol to 10 nmol. Preferably, glycerol is present at concentrations less than 50 pmol.

[0202] The nucleotides incorporated into the reaction mixture are typically provided as dNTPs. Exemplary concentrations of dNTPs are 0.1 - 0.4 mM dATP, preferably 0.3 - 0.4 mM dATP, preferably 0.29 - 0.46 mM dATP; 0.1 - 0.4 mM dGTP, preferably 0.3 - 0.4 mM dGTP, preferably 0.29 - 0.4 mM dGTP; 0.1 - 0.4 mM dCTP, preferably 0.3 - 0.4 mM dCTP, preferably 0.29 - 0.4 mM dCTP; 0.1 - 0.4 mM dTTP, preferably 0.2 - 0.3 mM dATP; and 0.1 - 0.4 mM dUTP, preferably preferably 0.125 - 0.234 mM dUTP. Exemplary concentrations in a 1.5× lyophilized mixture or final reaction (i.e., after reconstitution at 1.5× volume) are as follows.

[0203] [Chemical formula]

[0204] Such mixtures can be customized for any type of reaction using flap endonucleases. Such mixtures can be customized for any type of reaction using flap endonucleases and polymerases.

[0205] Flap endonucleases may be commercially available or prepared by the user. Flap endonucleases are not limited to enzymes having only 5' nuclease activity. For example, flap endonucleases can be natural DNA polymerases having 5' nuclease activity (e.g., Taq DNA polymerase, E. coli DNA polymerase I), or modified DNA polymerases having 5' nuclease activity by lacking synthetic activity (e.g., Cleavase® enzyme).

[0206] Such mixtures can be customized to suit different types of amplification, including PCR, RT-PCR, and transcription-mediated amplification, by selecting polymerase enzymes and other components.

[0207] DNA polymerase enzymes are commercially available or may be prepared by the user. One example of a polymerase enzyme is Taq polymerase (Germantown, MD, catalog number 201203) commercially available from Qiagen. Another example of Taq polymerase is GoTaq® G2 Flexi DNA Polymerase (Promega, Madison, WI, catalog number M7801) commercially available. Other commercially available DNA polymerases include, but are not limited to, Tth DNA polymerase (e.g., Sigma-Aldrich, St. Louis, MO, catalog number 11480022001) and chimeric DNA polymerases such as Phusion® High-Fidelity DNA Polymerase (NEB, Ipswich, MA, catalog number M0530S). Hot-start DNA polymerase enzymes are also commercially available. For example, Taq polymerase is commercially available as GoTaq® Hot Start Polymerase (Promega, catalog number M5001). GoTaq® Hot Start Polymerase is an antibody-mediated hot start enzyme in which Taq polymerase is conjugated to an antibody that blocks polymerase activity. The blocking antibody is denatured using high heat, so the antibody is denatured during the initial heating step of the PCR reaction, and polymerase activity is restored. Various antibodies can be used with the hot start method, for example, TAQSTART antibody (Clontech Laboratories, Mountain View, CA, catalog number R028A). Similarly, other hot start polymerase enzymes, including chemically mediated hot start polymerases, are available. Equivalent polymerases and antibodies are available from various commercial suppliers and may also be prepared by the user as alternatives.

[0208] Reverse transcriptase may be commercially available or prepared by the user. Examples of commercially available reverse transcriptases, but not limited to, MMLV (Moloni's mouse leukemia virus) reverse transcriptase and SuperScript® III Reverse Transcriptase (e.g., ThermoFisher 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® reverse transcriptase (Promega, catalog number A50003). GoScript reverse transcriptase comprises the reverse transcriptase and a set of reagents for the synthesis of single-stranded cDNA optimized for quantitative PCR amplification. Equivalent reverse transcriptases and reagents are available from various commercial suppliers and may, as an alternative, be prepared by the user.

[0209] Exemplary concentrations of flap endonuclease in a single unit dose are about 0.01 μg / μl to about 1.0 μg / μl, preferably about 0.01 μg / μl to about 0.8 μg / μl, or about 0.01 μg / μl to about 0.6 μg / μl, or about 0.01 μg / μl to about 0.4 μg / μl, or about 0.01 μg / μl to about 0.2 μg / μl, or about 0.01 μg / μl to about 0.1 μg / μl. More preferably, the concentration of flap endonuclease in a single unit dose is about 0.02 μg / μl to about 0.04 μg / μl. More preferably, the concentration of flap endonuclease in a single unit dose is about 0.030 μg / μl to about 0.04 μg / μl, and more preferably about 0.030 μg / μl to about 0.035 μg / μl. Preferably, flap endonuclease is present in the aqueous solution at a concentration of about 0.010 μg / μl to about 0.050 ug / ul, and preferably about 0.012 ug / ul to 0.047 ug / ul.

[0210] Exemplary concentrations of DNA polymerase enzyme in a single unit dose are 0.1–0.2 U / μl (including all integers and fractions within this range). For example, 0.14 U / μl, 0.146 U / μl, and 0.1687 U / μl.

[0211] 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 in 30 minutes at 74°C. An exemplary concentration of reverse transcriptase in a single unit dose is -1.0 U / μl (including all integers and fractions). 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-precipitating substance in 10 minutes at 37°C.

[0212] The preferred organic buffer is MOPS or Tris.

[0213] Preferably, the MOPS buffer is present in the aqueous solution at a concentration of about 10 mM to about 20 mM, and preferably at a concentration of about 12.5 mM to about 15 mM.

[0214] Preferably, the Tris buffer solution is present in an aqueous solution at a concentration of approximately 5 mM to approximately 60 mM, preferably at a concentration of approximately 40 mM to 60 mM, preferably at a concentration of approximately 50 mM, and preferably at a concentration of approximately 10 mM.

[0215] Alternative organic buffers that can be incorporated into the bulk reagent include Good's buffers, as well as combinations of buffers, such as phosphates, citrates, acetates, CHES, histidine, and HEPES, MES, tricine, and glycinamide. Other organic buffers include succinates, citrates, glucons, and phosphates. Preferred buffers are effective in a pH range of about 6.0 to about 10.0 or about 7.0 to about 9.0, preferably at a pH of about 7.5 or 8.0 or 8.5.

[0216] The preferred bulking agent is trehalose. Other bulking agents that may be considered include raffinose, sucrose, mannitol, trehalose and mannitol, sucrose and mannitol, sucrose and glycine, and hydroxyethyl starch. See Cleland et al (2001) J.Pharm.Sci.90:310; Meyer et al (2009) Eur.J.Pharm.Sci.38:29; Webb et al (2003) J.Pharm.Sci.92:715; Garzon Rodrigues et al (2004) J.Pharm.Sci.93:684; Qiu et al (2012) Int.J.Pharmaceuticals.437:51); Van Dijk-Wolthuis et al (1997) Polymer. 38:6235 6242. Hydroxyethyl starch is classified as hetastarch, hexastarch, pentastarch, and tetrastarch (see, for example, Chow's WO2014 / 099198). The bulking agent is preferably present at a concentration of about 0.2 M to about 0.5 M, preferably about 0.2 M to about 0.4 M, preferably about 0.3 M to about 0.4 M, preferably about 0.47 M, and preferably about 0.36 M.

[0217] The bulk reagent may contain one or more nucleic acids, such as probe oligomers, amplified oligomers, capture probes, positive control templates, and negative control templates. The bulk reagent may contain one or more nucleic acids for performing non-amplification assays that include one or more probe oligonucleotides. The bulk reagent may contain one or more nucleic acids for performing non-amplification assays and amplification-based assays that include one or more probe oligonucleotides and / or one or more amplified oligomers and / or one or more amplified oligomer pairs and / or multiple amplified oligomer pairs.

[0218] Optional additions to the bulk reagent include RNase inhibitors, surfactants, amphoteric surfactants, anionic surfactants, cationic surfactants, nonionic surfactants, surfactants, primers, probes, templates, polymers, biomacromolecules, oligosaccharides, polysaccharides, polyglucose, amylose, chelating agents, methylparaben, and propylparaben. Exemplary concentrations of methylparaben are 0.01–0.024% by weight, e.g., about 0.016%, or alternatively, about 0.010%, about 0.014%, about 0.016%, about 0.020%, about 0.024%, and any range with these values ​​as boundaries. The exemplary concentration range for propylparaben is 0.002–0.016% 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 with these values ​​as boundaries. One unit is equivalent to the amount of RNasin® Ribonuclease required to inhibit the activity of ribonuclease A by 50% at a dose of 5 ng. It is defined as the amount of inhibitor. Activity is measured by inhibition of hydrolysis of cytidine 2',3' cyclic monophosphate by ribonuclease A.

[0219] The chelating agent comprises one or more of the following: EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), EDDS (ethylenediamine-N,N'-disuccinic acid), MGDA (methylglycine diacetic acid), and DTPA (diethylenetriaminepentaacetic acid). An exemplary concentration of the chelating agent is 1.0 mM to 2.5 mM. In one embodiment, the use of EDTA is preferred. Preferably, EDTA is present at a concentration of about 0.05 to about 0.2 mM, preferably about 0.1 mM.

[0220] RNase inhibitor proteins are naturally occurring, and recombinants are 50 kDa proteins that inhibit the RNase A family and human placental RNase by non-covalently binding to RNase in a 1:1 ratio (Promega Corp., Madison, WI). Botella-Estrada et al (2001) Cancer Gene See Ther. 8:278; Polakowski et al (1992) EXS. 61:428. Typical concentrations of RNase inhibitors are approximately 0.04 U / μl to 0.4 U / μl.

[0221] Bulk reagents may contain low concentrations of surfactants. Surfactants include ionic (cationic or anionic), nonionic, and amphoteric surfactants, which are available from many commercial suppliers (e.g., Geno Technology, Inc., St. Louis, MO). Examples, but not limited to, lithium lauryl sulfate, amprolium hydrochloride, benzalkonium chloride, choline p-toluenesulfonate, dodecyltrimethylammonium chloride, 3-[(3-coramidopropyl)dimethylammonio]-1-propanesulfonic acid, ethylhexadecyldimethylammonium bromide, hexadecylpyridinium chloride, hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, hexadecyltrimethylammonium p-toluenesulfonate, luviquat (trademark), methylbenzethonium chloride, myristyltrimethylammonium bromide, N,N',N'-polyoxyethylene(10)-N-fat-1,3-diaminopropane solution, oxyphenonium bromide, tetraheptylammonium bromide, tetrakis(decyl)ammonium bromide, tricaprylylmethylammonium chloride, amidosulfobetaine-16, toridodecylmethylammonium chloride, trimethyloctadecylammonium bromide, Nonidet Examples include P-40 (registered trademark), Tween-20 (registered trademark), Tween-80 (registered trademark), Brij-35 (registered trademark), and TritonX-100 (registered trademark).

[0222] Exemplary volumes of the bulk reagent include approximately 1 μL, 5 μL, 10 μL, 20 μL, 24 μL, 50 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1,000 μL (1 mL), 2 mL, 5 mL, 10 mL, 20 mL, 50 mL, etc. The reconstituted composition can be formed in the same volume as the bulk reagent, a smaller volume, or a larger volume. A smaller volume may be approximately 90%, 80%, 60%, 40%, 20%, 10%, or 5% of the bulk reagent. Larger volumes can be approximately 120%, 140%, 160%, 180%, 200% (twice), 4 times, 6 times, 8 times, 10 times, or 20 times the volume of the bulk reagent.

[0223] The sample to be analyzed can be added to the bulk reagent before, during, or after reconstruction. In a preferred embodiment, the entire reconstituted dry composition is used to combine with the sample, where the relative volume of the reconstituted solution / sample may 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.

[0224] Unless otherwise specified, the concentration of the reagent in the bulk reagent may be 0.0% (no reagent), 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. Reagents are also provided that fall within a range of approximately the above concentrations, below the above concentrations, above the above concentrations, or including any two of the above concentrations.

[0225] Exemplary bulk reagent compositions are substantially glycerol-free, have an inorganic salt concentration of 5 mM or less, and contain 0.1 to 0.4 mM, preferably about 0.3 mM, of each of dATP, dGTP, dCTP, and dUTP or dTTP, as well as 0.02 μg / μl to 0.40 ug / μl of flap endonuclease, preferably about 0.03 μg / μl of flap endonuclease. Some compositions contain about 0.2 M to about 0.4 M, preferably about 0.3 M, of a volume extender, preferably trehalose. Some compositions preferably contain a buffer with a pH of about 7.5, preferably MOPS buffer.

[0226] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and contains 0.1–0.4 mM, preferably about 0.375 mM, each of dATP, dGTP, and dCTP, and about 0.182 mM dUTP or about 0.284 mM dTTP, 0.02 μg / μl–0.40 ug / μl flap endonuclease, preferably about 0.03 μg / μl flap endonuclease, and 0.1 U / μl–0.2 U / μl, preferably 0.146 U / μl polymerase. Some compositions contain about 0.2 M–0.4 M, preferably about 0.3 M, of a volume extender, preferably trehalose. Some compositions preferably contain a buffer at pH about 7.5, preferably MOPS buffer.

[0227] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and comprises 0.1 to 0.4 mM, preferably about 0.375 mM, each of dATP, dGTP, and dCTP, and about 0.182 mM of dUTP or about 0.284 mM of dTTP, 0.02 μg / μl to 0.40 ug / μl of flap endonuclease, preferably about 0.035 μg / μl of flap endonuclease, 0.1 U / μl to 0.2 U / μl, preferably 0.146 U / μl of polymerase, about 0.2 M to about 0.4 M, preferably about 0.3 M of extender, preferably trehalose, and preferably a buffer at pH about 7.5, preferably MOPS buffer.

[0228] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and contains 0.1–0.4 mM, preferably about 0.375 mM, each of dATP, dGTP, and dCTP, and about 0.182 mM dUTP or about 0.284 mM dTTP, as well as 0.02 μg / μl–0.40 μg / μl of flap endonuclease, preferably about 0.03 μg / μl of flap endonuclease. Some compositions contain about 0.2 M–0.4 M, preferably about 0.3 M, of a volume extender, preferably trehalose. Some compositions contain a globular protein, preferably bovine serum albumin (BSA), more preferably ultra-high purity unacetylated BSA. The globular protein may be present in an amount of 0.40–0.60 μg / μl, preferably 0.50 μg / μl. Some compositions preferably include a buffer with a pH of about 8.5, preferably Tris buffer.

[0229] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and contains 0.1–0.4 mM, preferably about 0.375 mM, each of dATP, dGTP, and dCTP, and about 0.182 mM dUTP or about 0.284 mM dTTP, as well as 0.02 μg / μl–0.40 ug / μl of flap endonuclease, preferably about 0.03 μg / μl of flap endonuclease, and 0.1 U / μl–0.2 U / μl, preferably 0.146 U / μl of polymerase. Some compositions contain about 0.2 M–0.4 M, preferably about 0.3 M, of a volume extender, preferably trehalose. Some compositions contain a globular protein, preferably bovine serum albumin (BSA), more preferably ultra-high purity unacetylated BSA. Spheroidal proteins may be present in amounts of 0.40–0.60 μg / μl, preferably 0.50 μg / μl. Some compositions preferably contain a buffer with a pH of approximately 8.5, preferably Tris buffer.

[0230] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and contains 0.1 to 0.4 mM, preferably 0.3 mM, of each of dATP, dGTP, dCTP, and dUTP or dTTP, as well as 0.02 μg / μl to 0.40 ug / μl of flap endonuclease, preferably about 0.03 μg / μl of flap endonuclease, and 0.1 U / μl to 0.2 U / μl, preferably The solution preferably contains 0.146 U / μl polymerase, a volume extender of about 0.2 M to about 0.4 M, preferably about 0.3 M, preferably trehalose, globular protein, preferably bovine serum albumin (BSA), more preferably ultra-high purity non-acetylated BSA (the globular protein is present in an amount of 0.40 to 0.60 μg / μl, preferably 0.50 μg / μl), and preferably a buffer solution with a pH of about 8.5, preferably Tris buffer solution.

[0231] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and contains 0.1–0.4 mM, preferably 0.375 mM, of each of dATP, dGTP, dCTP, and dUTP or dTTP, as well as 0.02 μg / μl–0.40 μg / μl of flap endonuclease, preferably about 0.035 μg / μl of flap endonuclease. In some compositions, the flap endonuclease is the Cleavase® enzyme. Some compositions contain about 0.2 M–0.4 M, preferably about 0.36 M, of a bulking agent, preferably trehalose. Some compositions contain a globular protein, preferably bovine serum albumin (BSA), more preferably ultra-high purity unacetylated BSA. The globular protein may be present in an amount of 0.40–0.60 μg / μl, preferably 0.50 μg / μl. Some compositions preferably include a buffer with a pH of approximately 7.5, and preferably a MOPS buffer.

[0232] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and contains 0.1–0.4 mM, preferably 0.375 mM, of each of dATP, dGTP, dCTP, and dUTP or dTTP, as well as 0.02 μg / μl–0.40 ug / μl of flap endonuclease, preferably about 0.035 μg / μl of flap endonuclease, and 0.1 U / μl–0.2 U / μl, preferably 0.167 U / μl of polymerase. Some compositions contain about 0.2 M–about 0.4 M, preferably about 0.36 M, of a volume extender, preferably trehalose. Some compositions contain a globular protein, preferably bovine serum albumin (BSA), more preferably ultra-high purity unacetylated BSA. Spheroidal proteins may be present in amounts of 0.40–0.60 μg / μl, preferably 0.50 μg / μl. Some compositions preferably contain a buffer with a pH of approximately 7.5, preferably MOPS buffer.

[0233] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and contains 0.1 to 0.4 mM, preferably 0.375 mM, of each of dATP, dGTP, dCTP, and dUTP or dTTP, as well as 0.02 μg / μl to 0.40 ug / μl of flap endonuclease, preferably about 0.035 μg / μl of flap endonuclease, and 0.1 U / μl to 0.2 U / μl, preferably The solution preferably contains 0.167 U / μl of polymerase, about 0.2 M to about 0.4 M, preferably about 0.36 M, of a volume extender, preferably trehalose, globular protein, preferably bovine serum albumin (BSA), more preferably ultra-high purity non-acetylated BSA (the globular protein is present in an amount of 0.40 to 0.60 μg / μl, preferably 0.50 μg / μl), and preferably a buffer solution with a pH of about 7.5, preferably MOPS buffer (e.g., 15 mM MOPS buffer).

[0234] Another exemplary bulk reagent composition is substantially glycerol-free, has an inorganic salt concentration of 5 mM or less, and contains 0.1 to 0.4 mM, preferably 0.375 mM, of each of dATP, dGTP, dCTP, and dUTP or dTTP, as well as 0.02 μg / μl to 0.40 ug / μl of flap endonuclease, preferably about 0.035 μg / μl of flap endonuclease, and 0.1 U / μl to 0.2 U / μl Preferably, the solution contains polymerase at a concentration of 0.169 U / μl, a bulking agent at a concentration of approximately 0.2 M to approximately 0.4 M, preferably approximately 0.36 M, trehalose, globular protein, preferably bovine serum albumin (BSA), more preferably ultra-high purity non-acetylated BSA (the globular protein is present in an amount of 0.40 to 0.60 μg / μl, preferably 0.51 μg / μl), preferably a buffer at a pH of approximately 7.5, preferably MOPS buffer (e.g., 15 mM MOPS buffer), and a chelating agent, preferably EDTA at a concentration of approximately 0.05 to 0.2 mM, preferably approximately 0.15 mM.

[0235] After the formation of the bulk reagent composition, the composition may be left at room temperature for a significant period before drying. This period may be up to 8 hours before the drying step is initiated, or alternatively, up to 1, 2, 4, 6, 10, 12, or 14 hours before the drying step is initiated. If the bulk reagent contains salts, undesirable hybridization products and other by-products may occur during this incubation period. Such undesirable hybridization and by-products can be reduced or eliminated by forming a bulk reagent composition that is essentially free of inorganic salts.

[0236] The presence of inorganic salts in bulk reagents can result in one or more of the following undesirable properties: Nucleic acids may hybridize together, and hybridization is stimulated by the presence of inorganic salts such as potassium, sodium, manganese, magnesium, and / or chlorides. Undesirable enzymatic activity may also occur in the presence of inorganic salts such as manganese and magnesium. Undesirable byproducts may begin to form as a result of nucleic acid hybridization and enzymatic activity in the presence of salts. Furthermore, inorganic salts are hygroscopic and draw moisture into the dry pellet. Rehydration of the dry pellet reduces storage stability and enzymatic stability and allows for the formation of additional false byproducts.

[0237] A dry pellet may contain reagents to provide one single unit dose (SUD), or optionally two or more SUDs. A single unit dose is a set of reagents necessary to perform a nucleic acid-based assay on only one sample. A single unit dose may refer to a liquid reagent or a dry pellet. As referred to herein, it should be noted that a single unit dose does not necessarily contain all the reagents necessary to perform a nucleic acid-based assay on a single sample. A single unit dose may lack the reagents necessary to perform a nucleic acid-based assay reaction. Similarly, a single unit dose may contain an insufficient amount of reagent to perform a nucleic acid-based assay reaction. As just one example, a dry single unit dose pellet may contain enough units of flap endonuclease to perform a nucleic acid-based assay, but may not contain magnesium. In such an example, magnesium can be added to the dry single unit dose pellet, for example, by a reconstitution solution. Also, as just one example, a dry single unit dose may contain an insufficient amount of dNTPs to perform a nucleic acid-based assay. In such an example, the remainder of the dNTPs can be added to the dry single unit dose pellet, for example, by a reconstitution solution. Since these examples are not limiting, a person skilled in the art with a good understanding of this disclosure will be able to easily produce SUD and dry pellet SUD of various compositions.

[0238] In preferred embodiments, the bulk reagent contains an inorganic salt content of 5 mM or less, more preferably 4 mM or less, more preferably 3 mM or less, more preferably 2 mM or less, more preferably 1 mM or less, or more preferably 500 μM or less. Therefore, the preferred concentration range of inorganic salts in the bulk reagent is an inorganic salt content of about 5 mM to 0 mM (including all integers and fractions within that range). Common inorganic salts for nucleic acid-based assay reaction mixtures include, to name a few, one or more of sodium, potassium, manganese, magnesium, and chloride.

[0239] In one embodiment, the bulk reagent contains an inorganic salt of 5 mM or less, and the inorganic salt is present in a mass per microliter of 0.373 μg / μl or less, or 0.332 μg / μl or less, or 0.292 μg / μl or less. In a further embodiment, the bulk reagent contains an inorganic salt of 5 mM or less, and sodium chloride is present in a mass per microliter of 0.292 μg / μl or less, 0.146 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 5 mM or less, and sodium is present in a mass per microliter of 0.115 μg / μl or less, 0.057 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 5 mM or less, and potassium chloride is present in a mass per microliter of 0.373 μg / μl or less, 0.186 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 5 mM or less, and potassium is present in a mass per microliter of 0.196 μg / μl or less, 0.098 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 5 mM or less, and chloride is present in 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 a further embodiment, the dry pellet is prepared by drying a liquid bulk reagent containing 5 mM or less of an inorganic salt, wherein the mass percentage of the inorganic salt relative to the mass of the pellet is 0.311% or less, 0.277% or less, or 0.244% or less. In a further embodiment, a container is provided containing a dry single-unit dose pellet in which the mass percentage of the inorganic salt relative to the mass of the pellet is 0.311% or less, 0.277% or less, or 0.244% or less. In a further embodiment, a multi-well plate is provided, each of the one or more wells, containing a dry single-unit dose pellet in which the mass percentage of the inorganic salt relative to the mass of the pellet is 0.311% or less, 0.277% or less, or 0.244% or less.

[0240] In one embodiment, the bulk reagent contains an inorganic salt of 4 mM or less, and the inorganic salt is present in a mass per microliter of 0.298 μg / μl or less, or 0.266 μg / μl or less, or 0.234 μg / μl or less. In a further embodiment, the bulk reagent contains an inorganic salt of 4 mM or less, and sodium chloride is present in a mass per microliter of 0.234 μg / μl or less, 0.117 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 4 mM or less, and sodium is present in a mass per microliter of 0.092 μg / μl or less, 0.046 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 4 mM or less, and potassium chloride is present in a mass per microliter of 0.298 μg / μl or less, 0.149 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 4 mM or less, and potassium is present in a mass per microliter of 0.156 μg / μl or less, 0.078 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 4 mM or less, and chloride is present in 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 a further embodiment, the dry pellet is prepared by drying a liquid bulk reagent containing 4 mM or less of an inorganic salt, wherein the mass percentage of the inorganic salt relative to the mass of the pellet is 0.249% or less, 0.222% or less, or 0.195% or less. In a further embodiment, a container is provided containing a dry single-unit dose pellet in which the mass percentage of the inorganic salt relative to the mass of the pellet is 0.249% or less, 0.222% or less, or 0.195% or less. In a further embodiment, a multi-well plate is provided having one or more wells, each of which contains a dry single-unit dose pellet containing a mass percentage of the inorganic salt relative to the mass of the pellet of 0.249% or less, 0.222% or less, or 0.195% or less.

[0241] In one embodiment, the bulk reagent contains an inorganic salt of 3 mM or less, and the inorganic salt is present in a mass per microliter of 0.224 μg / μl or less, or 0.199 μg / μl or less, or 0.175 μg / μl or less. In a further embodiment, the bulk reagent contains an inorganic salt of 3 mM or less, and sodium chloride is present in a mass per microliter of 0.175 μg / μl or less, 0.088 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 3 mM or less, and sodium is present in a mass per microliter of 0.069 μg / μl or less, 0.034 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 3 mM or less, and potassium chloride is present in a mass per microliter of 0.224 μg / μl or less, 0.112 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 3 mM or less, and potassium is present in a mass per microliter of 0.117 μg / μl or less, 0.059 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 3 mM or less, and chloride is present in 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 a further embodiment, the dry pellet is prepared by drying a liquid bulk reagent containing 3 mM or less of an inorganic salt, wherein the mass percentage of the inorganic salt relative to the mass of the pellet is 0.186% or less, 0.166% or less, or 0.146% or less. In a further embodiment, a container is provided containing a dry single-unit dose pellet in which the mass percentage of the inorganic salt relative to the mass of the pellet is 0.186% or less, 0.166% or less, or 0.146% or less. In a further embodiment, a multi-well plate is provided having one or more wells, each of which contains a dry single-unit dose pellet containing a mass percentage of the inorganic salt relative to the mass of the pellet of 0.186% or less, 0.166% or less, or 0.146% or less.

[0242] In one embodiment, the bulk reagent contains an inorganic salt of 2 mM or less, and the inorganic salt is present in a mass per microliter of 0.149 μg / μl or less, or 0.133 μg / μl or less, or 0.117 μg / μl or less. In a further embodiment, the bulk reagent contains an inorganic salt of 2 mM or less, and sodium chloride is present in a mass per microliter of 0.117 μg / μl or less, 0.058 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 2 mM or less, and sodium is present in a mass per microliter of 0.046 μg / μl or less, 0.023 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 2 mM or less, and potassium chloride is present in a mass per microliter of 0.149 μg / μl or less, 0.075 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 2 mM or less, and potassium is present in a mass per microliter of 0.078 μg / μl or less, 0.039 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 2 mM or less, and chloride is present in 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 a further embodiment, the dry pellet is prepared by drying a liquid bulk reagent containing 2 mM or less of an inorganic salt, wherein the mass percentage of the inorganic salt relative to the mass of the pellet is 0.124% or less, 0.111% or less, or 0.097% or less. In a further embodiment, a container is provided containing a dry single-unit dose pellet in which the mass percentage of the inorganic salt relative to the mass of the pellet is 0.124% or less, 0.111% or less, or 0.097% or less. In a further embodiment, a multi-well plate is provided having one or more wells, each of which contains a dry single-unit pellet containing a mass percentage of the inorganic salt relative to the mass of the pellet of 0.124% or less, 0.111% or less, or 0.097% or less.

[0243] In one embodiment, the bulk reagent contains an inorganic salt of 1 mM or less, and the inorganic salt is present in a mass per microliter of 0.075 μg / μl or less, or 0.066 μg / μl or less, or 0.058 μg / μl or less. In a further embodiment, the bulk reagent contains an inorganic salt of 1 mM or less, and sodium chloride is present in a mass per microliter of 0.058 μg / μl or less, 0.029 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 1 mM or less, and sodium is present in a mass per microliter of 0.023 μg / μl or less, 0.011 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 1 mM or less, and potassium chloride is present in a mass per microliter of 0.075 μg / μl or less, 0.037 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 1 mM or less, and potassium is present in a mass per microliter of 0.039 μg / μl or less, 0.020 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 1 mM or less, and chloride is present in 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 a further embodiment, the dry pellet is prepared by drying a liquid bulk reagent containing 1 mM or less of an inorganic salt, wherein the mass percentage of the inorganic salt relative to the mass of the pellet is 0.062% or less, 0.055% or less, or 0.049% or less. In a further embodiment, a container is provided containing a dry single-unit dose pellet in which the mass percentage of the inorganic salt relative to the mass of the pellet is 0.062% or less, 0.055% or less, or 0.049% or less. In a further embodiment, a multi-well plate is provided having one or more wells, each of which contains a dry single-unit dose pellet containing a mass percentage of the inorganic salt relative to the mass of the pellet of 0.062% or less, 0.055% or less, or 0.049% or less.

[0244] In one embodiment, the bulk reagent contains an inorganic salt of 500 μM or less, and the inorganic salt is present in a mass per microliter of 0.037 μg / μl or less, or 0.033 μg / μl or less, or 0.029 μg / μl or less. In a further embodiment, the bulk reagent contains an inorganic salt of 500 μM or less, and sodium chloride is present in a mass per microliter of 0.029 μg / μl or less, 0.015 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 500 μM or less, and sodium is present in a mass per microliter of 0.011 μg / μl or less, 0.006 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 500 μM or less, and potassium chloride is present in a mass per microliter of 0.037 μg / μl or less, 0.019 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 500 μM or less, and potassium is present in a mass per microliter of 0.020 μg / μl or less, 0.010 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the bulk reagent contains an inorganic salt of 500 μM or less, and chloride is present in a mass per microliter of 0.036 μg / μl or less, 0.018 μg / μl or less, 0.009 μg / μl or less, or 0.0 μg / μl. In a further embodiment, the dry pellet is prepared by drying a liquid bulk reagent containing an inorganic salt of 500 μM or less, wherein the mass percentage of the inorganic salt relative to the mass of the pellet is 0.031% or less, 0.028% or less, or 0.024% or less. In a further embodiment, a container is provided containing a dry single-unit dose pellet in which the mass percentage of the inorganic salt relative to the mass of the pellet is 0.031% or less, 0.028% or less, or 0.024% or less. In a further embodiment, a multi-well plate is provided having one or more wells, each of which contains a dry single-unit dose pellet containing a mass percentage of the inorganic salt relative to the mass of the pellet of 0.031% or less, 0.028% or less, or 0.024% or less.

[0245] In one embodiment, the bulk reagent contains approximately 5 mM to approximately 500 μM of inorganic salt, with the inorganic salt present in a mass of approximately 0.373 μg / μl to approximately 0.029 μg / μl per microliter. In a further embodiment, the bulk reagent contains approximately 5 mM to approximately 500 μM of inorganic salt, with sodium chloride present in a mass of approximately 0.292 μg / μl to approximately 0.029 μg / μl per microliter. In a further embodiment, the bulk reagent contains approximately 5 mM to approximately 500 μM of inorganic salt, with sodium present in a mass of approximately 0.115 μg / μl to approximately 0.006 μg / μl per microliter. In a further embodiment, the bulk reagent contains approximately 5 mM to approximately 500 μM of inorganic salt, with potassium chloride present in a mass of approximately 0.373 μg / μl to approximately 0.019 μg / μl per microliter. In a further embodiment, the bulk reagent contains approximately 5 mM to approximately 500 μM of inorganic salts, with potassium present at a mass of 0.196 μg / μl to approximately 0.010 μg / μl per microliter. In a further embodiment, the bulk reagent contains approximately 5 mM to approximately 500 μM of inorganic salts, with chloride present at a mass of 0.355 μg / μl to approximately 0.009 μg / μl per microliter. In a further embodiment, the bulk reagent contains approximately 5 mM to approximately 500 μM of inorganic salts, with inorganic salts present at a mass of approximately 0.373 μg / μl to approximately 0.029 μg / μl per microliter, and sodium chloride present at a mass of approximately 0 μg / μl per microliter. In a further embodiment, the bulk reagent contains approximately 5 mM to approximately 500 μM of inorganic salts, with the inorganic salts present at a mass of approximately 0.373 μg / μl to approximately 0.029 μg / μl per microliter, and potassium chloride present at a mass of approximately 0 μg / μl per microliter. In a further embodiment, the dried pellets are prepared by drying the liquid bulk reagent containing 5 mM to 500 μM of inorganic salts, with the mass percentage of inorganic salts relative to the mass of the pellets being approximately 0.311% to 0.024%.In a further embodiment, the dry pellet is prepared by drying a liquid bulk reagent containing 5 mM to 500 μM of inorganic salt, wherein the mass percentage of inorganic salt relative to the mass of the pellet is approximately 0.311% to 0.024%, and the mass percentage of sodium chloride relative to the mass of the pellet is approximately 0%, or the mass percentage of potassium chloride relative to the mass of the pellet is approximately 0%. In a further embodiment, a container is provided containing a dry single-unit dose pellet in which the mass percentage of inorganic salt relative to the mass of the pellet is approximately 0.311% to 0.024%, and the mass percentage of sodium chloride relative to the mass of the pellet is approximately 0%, or the mass percentage of potassium chloride relative to the mass of the pellet is approximately 0%. In a further embodiment, a multi-well plate is provided containing one or more wells, each of which contains a dry single-unit dose pellet containing approximately 0.311% to 0.024% of the mass of the pellet in which inorganic salt is relative to the mass of the pellet, wherein the mass percentage of sodium chloride relative to the mass of the pellet is approximately 0%, or the mass percentage of potassium chloride relative to the mass of the pellet is approximately 0%.

[0246] In one embodiment, a multiwell plate comprising one or more wells is provided. In one embodiment, one or more wells comprises walls constructed from a material comprising low water vapor permeability, thermal conductivity, light transmittance, low autofluorescence, or a combination thereof. In one embodiment, one or more wells comprises conical walls. In one embodiment, a well comprises walls configured to fit into a heat conduction tube receiving region of a device for performing nucleic acid-based assay reactions. In one embodiment, one or more wells of a multiwell plate comprises an opening for accessing the chamber of the well. In one embodiment, each of the one or more wells comprises a cap that seals the opening of the associated well. In one embodiment, each opening of one or more wells is sealed with a cap that is a low water vapor permeable foil. In one embodiment, each opening of one or more wells is sealed with a cap that is a low water vapor permeable elastomer material. In one embodiment, the multiwell plate comprises one or more wells as described herein, wherein the chamber of the well comprises a dry single-unit dose pellet comprising a flap endonuclease and an inorganic salt, wherein the mass percentage of the inorganic salt relative to the mass of the pellet is approximately 0.311% to 0.024%.

[0247] III. Drying equipment and drying methods Bulk reagents can be lyophilized using standard methods and equipment. Lyophilizers are available, for example, from GEA Process Engineering (Columbia, MD). Contract lyophilization services are provided, for example, by Biopharma Technology Ltd. (Winchester, Hampshire, Great Britain) and BioPharma Solutions Sterile Contract Manufacturing (Baxter Healthcare Corp, Deerfield, IL). Lyophilization guidance can be found, for example, in L. Rey, J.C. May (eds.) (2010) Freeze Drying / Lyophilization of Pharmaceuticals and Biological Products, 3. rdIt is available from ed. Informa Healthcare, NY, or Methods in Enzymology, Vol. 22, Pages 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).

[0248] Various containers are suitable for drying. If the container is sealed and stored under partial vacuum, it must be able to withstand external pressure. The container should be made of a material that allows for proper heat transfer from the outside to the inside. The size of the container is preferably such that the solution to be dried occupies no more than 20% of the total volume to avoid overflow.

[0249] Samples can be dried in separate containers or multi-sample containers. A multi-sample container means a continuous container that can contain at least two samples so that they can be stored and handled simultaneously but separately. Standard formats for multi-sample receptacles include 6, 24, 96, 384, or 1536 wells. In an example of a 96-well format, the volume of each well is approximately 300–400 microliters, and the working volume is approximately 75–200 microliters. The volume generally varies inversely to the number of wells, typically ranging from 1 nL to 10 mL per well, although other sizes are also contemplated. Exemplary wells may have flat bottoms, round bottoms, or V-shaped bottoms, in particular. As used herein, a container is also referred to as a well. As used herein, a multi-sample container is also referred to as a multi-well plate. In addition, wells may also be referred to as reaction wells. The term reaction well does not require that any reaction actually occur within the reaction well. Rather, this term is used to refer to a container or well that contains a reagent in which a reaction cannot occur, in which a partial reaction may occur, or in which a complete reaction may occur.

[0250] In some embodiments of this specification, a multiwell plate can undergo lyophilization to form a dry composition from an aqueous solution. Lyophilization may be carried out in a nested device (see concurrently pending U.S. Patent Application No. 62 / 200,370). The nest is a container for a cartridge with vents that can be closed by a mechanism operable from outside a sealed lyophilization chamber. The nest containing the multiwell plate is placed in the lyophilization chamber with one or more vents in the open position. The chamber is then sealed, and a lyophilization atmosphere is applied to the entire chamber, including the space within the nest. One or more vents are then closed, thereby sealing the nest. The seal of the lyophilization chamber is subsequently released, and the nest containing the multiwell plate is removed. The nest can then be repositioned, and the multiwell plate can be placed inside and stored until it is ready for the operator to use the lyophilized composition contained therein, or to reseal the multiwell plate containing the lyophilized sample for further storage or sale. The wells of the multiwell plate can then be sealed to substantially prevent moisture from entering from the ambient air. The intrusion of small amounts of moisture into sealed multiwell plates can be prevented by storing the sealed multiwell plates in pouches containing a desiccant. Similarly, freeze-drying can be performed in separate containers, and nested freeze-drying is also possible.

[0251] Other drying methods include spray drying, fluidized bed drying, dehumidifiers, and batch contact drying, which dries the filtered cake under vacuum and at low temperatures to produce a free-flowing dried product (NP Cheremisinoff (2000) Handbook of Chemical Processing Equipment, Butterworth Heinemann, Boston, MA). Dehumidifiers are available from Bry Air, Inc. (Sunbury, Ohio) and DST Seibu Giken (Wyomissing, PA). Rotary dryers, conical dryers, and shelf dryers are available (McGill AirPressure LLC, Columbus, Ohio). In one embodiment, a vacuum dryer removes moisture by exposing the material to reduced pressure, using just enough heat to compensate for the heat lost by vaporization. Desiccants include silica gel desiccants, molecular sieve desiccants such as aluminosilicates and synthetic zeolites, and bentonite desiccants.

[0252] It is preferable to dry the reaction mixture in the same container in which it will be reconstituted for use.

[0253] The freeze-dried or otherwise dried formulations have low water content, such as less than 5% by weight, less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1.0% by weight, less than 0.5% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.05% by weight, less than 0.02% by weight, and less than 0.01% by weight.

[0254] IV.Save The freeze-dried or otherwise dried composition is stored before use. The storage period may include the period during which the dried composition is exposed to ambient air and stored at room temperature. Such a period may be up to 3 hours, or alternatively, up to 1.0 hour, up to 1.5 hours, up to 2.0 hours, up to 2.5 hours, or any range of 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, etc. The absolute humidity during such storage is at least 2.3 g of water per cubic meter of air at 30°C, or alternatively, more than 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3.0 g of water per cubic meter of air at 30°C.

[0255] Storage may also include a longer period during which the dry composition is sealed and substantially prevented from coming into contact with ambient air outside the seal. This storage period may 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. A period of one month to two years is illustrative.

[0256] For long-term storage, or for long-term or short-term stability testing, the storage temperature range 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 temperatures below 0°C such as -4--2°C, -6--2°C, -8--2°C, -10--2°C, -20°C, -40°C, -60°C, and -80°C under liquid nitrogen. Preferably, storage is above the freezing point, in the range of about 4-8°C. Accelerated degradation testing can be conducted at about 25°C, about 30°C, about 35°C, and about 40°C for periods such as 1 hour, 2 hours, 4 hours, 24 hours, 2 days, 4 days, 8 days, and 1 month. For storage purposes, or as an alternative, conditions for stability testing may include temperature fluctuations, such as those ranging from temperatures above freezing to temperatures below freezing.

[0257] The inherent absence of inorganic salts reduces the loss of enzyme activity and the formation of by-products during storage of bulk reagents before drying, short-term storage of dried compositions before sealing, and long-term storage after sealing. Preferably, all post-storage enzyme activity is within a range of 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%, and at least 20% of the value immediately before storage began, and these percentages relative to the value before storage began, or alternatively, to the value of a comparative sample stored under optimal conditions.

[0258] V. Reconstruction A preferred reconstituted solution provides about 3.0 to about 12.0 mM MgCl2 and about 0 to about 80 mM KCl in water. The reconstituted 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% (v / v) anhydrous ethanol.

[0259] The reconstitution time may 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 60 seconds (1 minute) after contacting the dry composition with an aqueous solution suitable for the intended use of the dry composition, and the contact may be optionally facilitated by shaking, tapping, vortexing, agitation, aspiration and suction from a pipette tip, or folding or squeezing a flexible vial. An example reconstitution time is 2 to 10 seconds. The reconstitution time can be measured with a reconstitution solution at refrigerator temperature (approximately 4°C), ambient temperature solution at approximately 23°C, or warm solution at approximately 37°C. Typically, the dry composition is taken out of the refrigerator and is therefore cold before the addition of the reconstitution solution. For any of these procedures, the environment (room) is typically ambient temperature or approximately 23°C. The time at which a substance is judged to be reconstituted may be, for example, the time at which the substance is judged to be completely solubilized. Complete solubilization can be determined by visual inspection; for example, the absence of turbidity or the absence of a Schlieren pattern are indicators of complete solubilization. Alternatively, complete solubilization can be determined by optical instruments such as machines that measure light scattering.

[0260] VI. Stability of the composition The stability of a composition is typically assessed by determining its activity (i.e., detection rate or yield) or the formation of by-products in the composition after reconstitution of the dried product. Lack of stability can result from loss of activity or by-product formation during storage, either before or after drying. Activity or by-product formation can be absolute or relative measures. If relative, the baseline for comparison may be the bulk reagent mixture before drying and reconstitution, or a control reconstitution mixture different from the one tested by the defined method (Mg 2+ (or the presence of other salts). Activity can be assessed by the rate of cleavage or amplification in real time, or by the final yield of the cleavage or amplification product, or by the hit rate. By-products can be assayed by one or more of the following: gel electrophoresis, gel scanner, agarose gel, capillary electrophoresis, etc.

[0261] The activity of the reconstituted amplification mixture (corrected as necessary for any differences due to different volumes of the reconstituted material) is preferably within 75, 80, 85, 90, or 95% of the activity of the bulk reagent before drying, or indistinguishable from the activity of the bulk reagent before drying within the margin of error. By-products present in the reconstituted amplification mixture (corrected as necessary for any differences due to different volumes of the reconstituted material) are less than 20, 15, 10, 5, 4, 3, 2, or 1% by weight of the original compound present in the bulk reagent before drying, or their average number of moles. By-products may be below the detection limit.

[0262] VII. Kit The above-mentioned dry compositions may be provided in a kit. Such a kit may include the dry compositions in a container such as a test tube. In some embodiments, the kit includes a multiwell plate having one or more wells. Some kits include multiple dry compositions supplied in separate containers. Some kits include one or more multiwell plates containing multiple dry compositions in one or more sealed well members of a multiwall multiwell plate.

[0263] Some kits also include a reconstitution solution in a separate container from the dry composition. The reconstitution solution may be supplied in bulk for aliquoting into individual dry composition containers, or in the form of one or more unit doses for each to be combined with a single container containing the dry composition.

[0264] Optionally, the containers containing the dried composition and the containers containing the reconstituted solution can be separated by a brittle material. The brittle material may be aluminum foil, polypropylene, polyester, polyvinyl chloride (PVC), or polyethylene. The barrier may consist of one, two, three, or more layers, each having the same composition, or each layer having a different composition, such as a foil layer in contact with a PVC layer. The film can be sourced, for example, from Dow Chemical Co. (Midland, MI) or Arkema, Inc. (King of Prussia, PA). The reconstituted solution can be brought into contact with the freeze-dried composition by perforating the brittle material.

[0265] The kit can be designed to fit into a thermocycler or incubator, so that the enzymatic reaction takes place directly within the kit's compartment, avoiding the need to transfer the composition to a different reaction vessel or a container that holds such a vessel.

[0266] The kit can be adapted to allow the introduction of user-supplied reagents into containers within the kit, for example, by a syringe that punctures a port, hose, or septum (see US2014 / 0121515 and US2014 / 0276356), or alternatively, user-supplied reagents, such as nucleic acid templates, can be mixed with the reagents of this disclosure in user-supplied containers. One or more of the kit's compartments may be supplied empty and used as a mixing chamber.

[0267] VIII. Dialysis of flap endonucleases Some enzymes used in this disclosure are prepared in a glycerol-containing buffer. The glycerol content can hinder lyophilization; therefore, it is necessary to prepare a solution containing a glycerol-free enzyme solution and substantially free of inorganic salts by dialyzing the enzyme to a glycerol-free buffer. This dialyzing can be used to substantially remove glycerol and replace it with the buffer. The dialyzed enzyme can then be used to prepare a glycerol-free lyophilized formulation that is substantially free of inorganic salts. Accordingly, this specification also discloses dialyzing compositions comprising, consisting of, or essentially comprising an aqueous solution containing an organic buffer, an extender, a chloride ion, and a chelating agent. The extender may be trehalose. The extender may be present at a concentration of about 100 mM to 300 mM, preferably 200 mM. The organic buffer may be tris(hydroxymethyl)aminomethane (Tris) buffer. The Tris buffer may be present at a concentration of 10 mM to 30 mM, preferably 50 mM. The chloride ion is KCl. Chloride ions may be present at a concentration of approximately 40–60 mM, preferably 50 mM. The chelating agent may be EDTA. The chelating agent may be present in the aqueous solution at a concentration of 0.05–0.2 mM, preferably 0.1 mM. The dialysis composition may contain flap endonuclease.

[0268] An exemplary dialysis composition is an aqueous solution comprising, or essentially comprising, Tris buffer (pH 8.0), trehalose, KCl, and EDTA.

[0269] Another exemplary dialysis composition is an aqueous solution comprising, or essentially consisting of, approximately 20 mM Tris buffer (pH 8.0), approximately 200 mM trehalose, approximately 50 mM KCl, and approximately 0.1 mM EDTA.

[0270] Furthermore, a method for preparing a flap endonuclease composition that is substantially free of glycerol is disclosed, wherein the flap endonuclease and glycerol are dialyzed in order to obtain a flap endonuclease composition that is substantially free of glycerol. Preferably, glycerol is present in an amount of about 0 to about 0.35% (w / v), preferably about 0 to about 0.2% (w / v), preferably about 0.1% (w / v), and preferably about 0.01% (w / v). [Examples]

[0271] Example 1. Bulk Reagent

[0272] Examples 1-3 demonstrate the preparation of bulk reagents, the drying of a single unit dose (SUD) volume of bulk reagent to obtain a SUD dry pellet in a container, and the reconstitution of the dry pellet to obtain a SUD amplification and detection mixture. Tables 1 and 2 disclose the components of exemplary bulk reagents for drying. The two tables also disclose exemplary single unit dose concentrations. Master mix 1 was a 2X master mix containing 0.4 mM dATP, dGTP, dCTP, and dTTP, 0.8 mM dUTP, and BSA, and substantially free of inorganic salts. The Taq polymerases in Table 1 were present in a glycerol-free Tris buffer containing a cationic surfactant. 2X Master Mix 2 consists of 0.74 U / uL of GoTaq® MDx Hot Start Polymerase (glycerol-free) and 0.48 M trehalose in a proprietary buffer containing 0.4 mM dATP, dGTP, dCTP, 0.8 mM dUTP, Tris, and non-acetylated BSA. 50X GoScript RT Mix is ​​as follows: 20 U / μL GoScript® and 8 units / μL of RNasin® Plus RNase Inhibitor (Table 2), and 10 U / μL of GoScript® and 8 units / μL of RNasin™ Plus (Table 1).

[0273] GoScript® RT Custom is a concentrated solution of GoScript RT at 160 U / uL, glycerol-free, and RNase inhibitor-free. Possible stabilizers include: deamidation inhibitors, antioxidants, surfactants, and surfactants such as fatty acid esters of polyethoxylated sorbitan (e.g., polysorbate 20 or polysorbate 80), and poloxamer 188. [Table 1] [Table 2-1] [Table 2-2] [Table 3]

[0274] Each of the bulk amplification reagents in Tables 1-3 contained a set of primers and probes for amplification and detection reactions in the following examples. Those skilled in the art will understand how to prepare the primer / probe mix for amplification reactions (see, for example, Innis, Michael A. et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990)). In the following examples, the primers and probes were present in the bulk reagent at concentrations of approximately 8 μM to approximately 12 μM.

[0275] Example 2: Freeze-drying In this example, the bulk reaction mixture was dried using a freeze-drying oven. 24 microliters of the bulk amplification reagent outlined in Example 1 were added to a container and then loaded into the freeze-drying chamber. In the examples herein, 24 µl represents the amount of bulk reagent used to perform the amplification and detection reaction for a single sample (also referred to as a single unit dose or SUD). In this example, a multi-well plate (particularly a 12-well plate) was used for both the freeze-drying reaction and the storage of the freeze-dried pellets present in the wells of the multi-well plate. 24 µl aliquots of the bulk reaction mixture were placed in each of the 12 wells of the 12-well plate. The freeze-drying cycle was started (running for approximately 36 hours). After the freeze-drying cycle, the 12-well plate was recovered and moved to a location where the individual containers of the 12-well plate would be sealed. The containers were sealed with metal foil covering the container openings. The metal foil was a low water vapor permeability foil. The sealed 12-well plates were then placed in a bag with a desiccant.

[0276] Example 3 Reconstitution Solution This example describes one reconstitution solution. The purpose of the reconstitution solution is to rehydrate the dry pellet in preparation for performing a nucleic acid-based detection reaction on a sample using the reconstituted pellet. 24 µl of reconstitution solution was dispensed into each of the 12 wells of the 12-well plate from Example 2 by pre-perforating the foil cover on the wells and then dispensing the reconstitution solution into the wells. Table 4 discloses the reconstitution solutions used in these examples and provides both bulk reconstitution solution concentrations and final assay concentrations. After reconstitution of the dry pellet, target nucleic acids from the sample were added to the wells, and PCR amplification and detection reactions were performed. [Table 4]

[0277] Example 4: Adverse effects of inorganic salts on dried bulk reagents This example illustrates the adverse effects of the presence of inorganic salts in bulk reagents on dry pellets. Two bulk reagent mixtures were prepared in general accordance with Example 1 and Table 5. The difference between bulk reagent A and bulk reagent B in Table 5 was the presence or absence of MgCl2 in the reaction mixture. [Table 5]

[0278] Liquid bulk reagents A and B were each prepared on ice. Bulk reagents A and B were then aliquoted separately into the wells of several multi-well plates (specifically, 12-well plates were used in this case). These 12-well plates, each containing either 12 aliquots of bulk reagent A or 12 aliquots of bulk reagent B, were then divided under four different incubation conditions: (1) 90 minutes of incubation on ice, (2) 90 minutes of incubation at room temperature, (3) 180 minutes of incubation on ice, or (4) 180 minutes of incubation at room temperature. Thus, a portion of each bulk reagent mixture was incubated at room temperature for 180 minutes, and the other portion was incubated on ice for 180 minutes. Similarly, a portion of each bulk reagent mixture was incubated at room temperature for 90 minutes, and the other portion was incubated on ice for 90 minutes. In all cases, the addition of the nucleic acid component to the reaction mixture (added as the final component) marked the start of the incubation time.

[0279] After incubation time, the bulk reagents ali-coated in 12-well plates were freeze-dried until a substantially dry composition was obtained. Each of the dried compositions obtained in the wells of the 12-well plate represented a single dry unit dose of the triple amplification and detection reaction for identifying one or more of influenza A, influenza B, and respiratory syncytial virus B in the sample.

[0280] The dried composition was reconstituted with a reconstituted solution containing 65 mM KCl, 0.02% w / v and 0.01% w / v methyl and propylparaben, respectively, and 0.33% v / v anhydrous ethyl alcohol. The reconstituted solution of the dried composition prepared from bulk reagent A also contained 2.5 mM MgCl2.

[0281] All three samples—influenza A, influenza B, and respiratory syncytial virus B—were combined with the reconstituted reaction mixture at three times their LoD, so that all components of the reconstituted mixture were approximately 80% of the bulk reagent concentration. These positive samples were obtained by combining viruses extracted from negative plasma with a transport medium and serially diluting them to the desired concentrations (except that the serial dilutions of the RSVB samples differed by a 10-fold). As shown in Table 6, the primer and probe mixes were designed to specifically detect each of the three viral targets, namely influenza A, influenza B, and respiratory syncytial virus B, in separate fluorescence channels despite being single-molecule reactions.

[0282] Samples were assayed using a real-time PCR-compatible thermal cycler (ABI 7500FAST, Applied Biosystems, Carlsbad, CA). The results are shown in Table 6. The positive percentage values ​​in the table represent the number of samples with RFU values ​​exceeding the threshold, expressed as a percentage of the 12 samples tested. The viral load (viral particles per assay) was sufficient to yield at least 95% positivity compared to the positive control. [Table 6]

[0283] These results indicate that the bulk reagent (pre-lyophilized solution without MgCl2 and KCl) is stable at room temperature for at least 180 minutes. Dried SUD pellets from bulk reagent A, when reconstituted and combined with a sample, yielded more robust amplification and detection reactions than dried SUD pellets from bulk reagent B. When bulk reagent B was incubated for a short period of 90 minutes at room temperature or even on ice, then dried and reconstituted to produce an amplification reaction, it yielded an amplification reaction with a relatively low signal and numerous small byproducts compared to bulk reagent A under the same conditions. Bulk reagents containing little to no inorganic salts are useful for drying to produce dried compositions containing polymerase enzyme components, dNTPs, and components for amplification reactions, including nucleic acids.

[0284] Example 5: Stability of dry pellets with or without salt This example compares the stability of single-unit dose dry pellets containing salt with that of single-unit dose dry pellets without salt (inorganic salt less than 6 mM). Single-unit dose pellets were prepared by drying bulk reagents, as outlined in Table 5 above. Immediately after synthesis, bulk reagent A and bulk reagent B were ali-coated into separate multi-well reaction plates (12 wells) and dried using a freeze-drying oven. After freeze-drying, the multi-well plates containing the dry pellets were placed in a nitrogen gas environment with a relative humidity of approximately 5%, and the multi-well plates were sealed by covering the well openings with foil. The sealed plates were placed in an aluminum pouch containing a desiccant, and then the pouch was sealed. The sealed pouches containing the dry pellets in the multi-well plates were stored at 4°C for 8 days.

[0285] After 8 days, the pouched multiwell plates were transferred to one of the following three conditions: Condition 1. One subset of multiwell plates in a pouch containing dry pellets from bulk reagent A and another subset of multiwell plates in a pouch containing dry pellets from bulk reagent B were removed from the pouches and placed in an environment at 15°C with a relative humidity of 70%. Condition 2. One subset of multiwell plates in a pouch containing dry pellets from bulk reagent A and another subset of multiwell plates in a pouch containing dry pellets from bulk reagent B were removed from the pouches and placed in an environment at 45°C with a relative humidity of 15% (accelerated stability). Condition 3. One subset of multiwell plates in a pouch containing dry pellets from bulk reagent A and another subset of multiwell plates in a pouch containing dry pellets from bulk reagent B were placed at 4°C (the multiwell plates were in the pouch with the desiccant, and therefore the humidity was 0 percent). The plates were left under these conditions for another 30 days.

[0286] At the end of incubation, the dried pellets from each condition were reconstituted using a reconstitution solution containing 100 mM KCl and sufficient MgCl2 to a final concentration of 2.5 mM. The reconstituted reaction mixtures were tested for amplification and detection of influenza A targets using a real-time PCR thermal cycler (ABI PRISM 7000, Applied Biosystems, Carlsbad, CA). In short, influenza A targets were extracted at LOD 10^0 (+ / -1 log) in a negative pool along with an equivalent liquid control. The results are shown in Table 7. [Table 7]

[0287] These results indicate that single amplified reaction-dried pellets containing less than 2.5 mM of inorganic salt have higher RFU values ​​after storage under several different temperature and humidity conditions compared to single amplified reaction-dried pellets containing 2.5 mM or more of inorganic salt.

[0288] Example 6: Lyophilization of Cleavase® enzyme containing bulk reagent A bulk reagent containing the flap endonuclease Cleavase® was prepared to contain a final glycerol concentration of 0.35%. This bulk reagent was aliquoted and lyophilized as outlined above. Data showed that this level of glycerol content hindered the lyophilization of the SUD pellet by causing what is referred to as “meltback” (data not presented). Meltback generally refers to the breakdown of the lyophilized product. Meltback typically results from the presence of a substance during the primary drying stage that is detrimental to the formation of a robust lyophilized composition. Glycerol is one such substance. Therefore, a glycerol-free Cleavase®-containing solution substantially free of inorganic salts was prepared by dialyzing the Cleavase® enzyme (stored in 50% glycerol buffer) to a glycerol-free buffer using a 20 kDa MWCO Slyde-A-Lyzer cassette system (ThermoFisher P / N number 66005). The composition of the dialysis buffer is listed in Table 8. [Table 8]

[0289] Glycerol was removed from this buffer by dialyzing with Cleavase® enzyme and replaced with the buffer shown in Table 8. Then, a glycerol-free bulk reagent substantially free of inorganic salts was prepared using the dialyzed Cleavase® enzyme (Table 9). The bulk reagent was then alicoated into the wells of a multi-well plate as several SUDs and lyophilized. All SUD pellets lyophilized without showing any signs of meltback. [Table 9]

[0290] Dried compositions containing oligonucleotides for PCR amplification and assay detection based on cleavage of Staphylococcus aureus ("S. aureus") were rehydrated with a reconstitution solution (stock reconstitution solution containing 9.375 mM MgCl2, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, and 0.33% (v / v) was prepared to a total volume of 1 liter using water) to generate multiple reaction master mixes. S. aureus target DNA (positive) or water without nuclease (negative) was added to separate reconstituted master mixes and coated with silicone oil. Amplification and detection assays were performed using a Panther Fusion thermocycler. Non-freeze-dried bulk reagents were tested in parallel with freeze-dried formulations as controls.

[0291] The results in Table 10 below show that in this test, the amplification and detection system was able to successfully amplify and detect S. aureus-positive samples as positive in all three channels and negative samples as negative in all three channels, and that the results were comparable to those of the wet mixture control. [Table 10]

[0292] Example 7: Cleavase® Pre-Lyophilization Formulation Version 3 Modified versions of the pre-lyophilized S. aureus preparation were prepared, including ultra-high purity non-acetylated BSA and a change in buffer from MOPS buffer to Tris buffer (Table 11). [Table 11]

[0293] Promega X991X was formulated as a 2X master mix, and when diluted 1:1, it yielded a reaction mixture containing 0.24M trehalose, 0.2mM dNTP (0.4mM dUTP), and 9.25U GoTaq, as well as non-acetylated BSA and Tris, in a 25µl reaction mixture. A total of 25 cartridges were successfully prepared from this formulation and lyophilized.

[0294] Amplification and detection reaction products were prepared and carried out as outlined 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 12.5 mM MgCl2 in the reconstitution reaction mixture. As a control, several non-lyophilized reaction products were prepared and tested. The S. aureus target nucleic acids used in these reactions were S. aureus gDNA at 100 copies / mL and 1,000 copies / mL (approximately 3 times the assay LoD for this target). In the presence of 12.5 mM MgCl2, the Tris-buffered reaction product showed a delay in Ct, a decrease in RFU, and a decrease in T gradient value in S. aureus-positive samples. Furthermore, negative samples using the Tris-buffered formulation showed higher background generation compared to negative samples using the 10 mM MOPS-buffered formulation, regardless of the MgCl2 concentration in the reaction mixture. Therefore, based on these results, MOPS-buffered offers advantages for use in bulk formulations and can be used at concentrations up to approximately 15 mM in final cartridge formulations.

[0295] Further modifications of the formulations shown in Table 11 were tested. These modified formulations were prepared as a series of bulk reagents in which 11% trehalose was replaced with various trehalose / sugar / polymer combinations. These bulk reagent formulations contained the following reagent combinations and were lyophilized and tested as described above. a) 3% trehalose / 1% 10kDa polyvinyl propylene b) 3% trehalose / 2% 10kDa polyvinyl propylene c) 3% Trehalose / 3% 10kDa Polyvinyl Propylene d) 3% Trehalose / 1% 29kDa Polyvinyl Propylene e) 3% Trehalose / 2% 29kDa Polyvinyl Propylene f) 3% Trehalose / 3% 29kDa Polyvinyl Propylene g) 3% Trehalose / 1% 55kDa Polyvinyl Propylene h) 3% Trehalose / 2% 55kDa Polyvinyl Propylene i) 3% Trehalose / 3% 55kDa Polyvinyl Propylene j) 3% Trehalose / 1% Sucrose k) 3% Trehalose / 2% Sucrose l) 3% trehalose / 3% sucrose m) 11% trehalose (control)

[0296] Except for conditions i) and control condition m), all combinations were sufficiently freeze-dried and showed no initial decay. Furthermore, functional testing at t=0 showed no significant delay in Ct for any of the combinations a) to m). However, when placed under accelerated stability conditions at 37°C / 95%RH, all combinations except the control condition showed decay after 4 days. The control cartridge using 11% trehalose showed no decay at 37°C / 95%RH until day 14-16. Therefore, bulk reagent formulations containing 11% trehalose are suitable for long-term storage of freeze-dried pellets before use.

[0297] Example 8: Cleavase® Pre-Lyophilization Formulation Version 4 It is desirable to change the sample volume. Smaller sample volumes are useful for accommodating multiple tests on patient samples. Sample volumes from 210 µl to 420 µl were tested. To maintain sensitivity with smaller sample volumes, the amount of eluate used in the PCR reaction was increased from 5 µl to 10 µl. The excess 5 µl of eluate sample alters the concentration of reagents in the reaction mixture. The reagent concentrations in the bulk reagent were adjusted to accommodate different eluate volumes (see Table 13).

[0298] By comparing the conditions of 420µl / 5µl and 210µl / 10µl, it was found that the performance was equivalent under both conditions (Table 12). [Table 12-1] [Table 12-2]

[0299] To provide a total reaction mixture volume of 30 µl (e.g., 20 µl master mix + 10 µl eluate), the concentration of the SUD pellet was changed to "1.5X" by increasing the amount of eluate from 5 µl to 10 µl. Table 13 reflects the volume changes, with Tris buffer replaced by 10 mM MOPS buffer, pH 7.5. [Table 13]

[0300] This version 4 formulation contains a more concentrated reagent (5 μl → 10 μl) to accommodate the doubling of the sample elution volume in the PCR reaction. To ensure the robustness of the formulation as the concentration changes, a new retention time test was performed. On day 0, the liquid S. aureus reaction mixture was prepared according to Table 13 above and divided into three aliquots. The first aliquot was immediately lyophilized, the second aliquot of the mixture was stored at 2–8°C for 2 days and then lyophilized, and the final aliquot was stored at 2–8°C for 4 days and then lyophilized. The resulting cartridges were sealed and tested at baseline (data not shown) and simultaneously subjected to accelerated stability conditions of 4 weeks at 30°C / 95% relative humidity (RH). The tests were performed 10 replicates per condition using MRSA cell lines with a LoD (1,000 CFU / mL) 0.5 log above. The results showed that this formulation could detect MRSA at levels 0.5 log above the LoD, demonstrating robustness with retention times of 0–4 days before lyophilization and accelerated stability of up to 30 days at 30°C / 95%RH after lyophilization.

[0301] Example 9

[0302] To counteract the inhibitory effects of substances such as SDS that may be used to wash away captured nucleic acids, α-cyclodextrin can be added to the composition to be lyophilized. Some of the SDS remains in the nucleic acid solution and subsequently remains during the nucleic acid amplification and detection reactions. First, four versions of the formulation were prepared using S. aureus cells (GP1822) at 1,000 CFU / mL in simulated nasal fluid (SNF) similar to that in Table 13, with the following differences: (1) 0 ug / ul cyclodextrin, (2) 0.1 ug / ul cyclodextrin, (3) 0.25 ug / ul cyclodextrin, or (4) 0.5 ug / ul cyclodextrin. Samples were processed in a Panther Fusion Instrument (P182). 30% washing buffer was spiked into the elution buffer (i.e., normal WB carryover + 30%). The pellets were then used in test reactions for amplification and detection of MRSA target nucleic acids. No SDS was added to these amplification reactions. This experiment determines whether the addition of cyclodextrin to the mixture affected the nucleic acid reaction performed using the pellets. As seen in conditions 1-4 of Table 14, the ct values ​​were substantially the same under each test condition, suggesting that pellets containing at least 0.5 ug / ul of cyclodextrin did not adversely affect performance.

[0303] After the initial experiment, four versions of the formulation similar to those in Table 13 were prepared again, each containing one of the following: (1) 0 ug / ul cyclodextrin, (2) 0.1 ug / ul cyclodextrin, (3) 0.25 ug / ul cyclodextrin, or (4) 0.5 ug / ul cyclodextrin. The resulting pellets were then used in test reactions for amplification and detection of MRSA target nucleic acids. In this series of amplification reactions, SDS was added to the reaction mixture until a final concentration of 30% v / v was reached. Amplification and detection reactions were performed. As seen in conditions 5-8 in Table 14, SDS had an inhibitory effect on the nucleic acid reaction (condition 5). 0.1 ug / ul cyclodextrin almost completely neutralized the inhibition of SDS (condition 6), and 0.25 ug / ul and 0.5 ug / ul completely neutralized the inhibition of SDS (conditions 7 and 8).

[0304] [Table 14]

[0305] Further datasets were tested using S. aureus cells (GP1822) at 1,000 CFU / mL in simulated nasal fluid (SNF). Samples were processed using a Panther Fusion Instrument (P368). 30% wash buffer was spiked into elution buffer (i.e., normal WB carryover + 30%). [Table 15]

[0306] The table shows that higher concentrations of cyclodextrin, up to 5.0 ug / ml, are effective in neutralizing SDS.

[0307] Therefore, formulations like those shown in Table 13 can be supplemented with cyclodextrin, for example, 0.1 to 5 ug / ul of cyclodextrin, or 0.1 to 0.5 ug / ul of cyclodextrin.

[0308] From the foregoing, it should be understood that while certain embodiments are described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of the invention. Therefore, the invention is not limited by express disclosure. Unless otherwise evident from the context, any embodiment, feature, aspect, or step can be used in combination with any other. Unless otherwise evident from the context, any composition said to contain the enumerated components consists of or is essentially composed of those components. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for producing a dry composition comprising hot-start polymerase and reverse transcriptase, comprising the steps of: (a) forming an aqueous solution comprising a volume extender, the hot-start polymerase, the reverse transcriptase, and an organic buffer, wherein the aqueous solution has a potassium concentration of 0.01 to 0.196 μg / μL, a magnesiumone concentration of less than 1 mM, an inorganic salt concentration of 5 mM or less, and the aqueous solution contains 0 to 0.35% (w / v) glycerol; (b) The step of adding dNTPs to form a bulk reagent; (c) The step of freezing the bulk reagent; (d) The frozen bulk reagent from step (c) is subjected to freeze-drying conditions, thereby forming a dried formulation. Methods that include...

2. The method according to claim 1, wherein the bulking agent comprises trehalose.

3. The method according to claim 2, wherein the aqueous solution contains trehalose at a concentration of 0.2 to 0.5 M or 0.3 M.

4. The method according to any one of claims 1 to 3, wherein the hot-start polymerase comprises a hot-start Taq polymerase.

5. The method according to claim 4, wherein the aqueous solution contains the hot-start Taq polymerase at a concentration of 0.1 to 1.0 U / μl or 0.46 U / μl.

6. The method according to any one of claims 1 to 5, wherein the aqueous solution contains the reverse transcriptase at a concentration of 0.1 to 4.0 U / μl or 0.5 U / μl.

7. The method according to any one of claims 1 to 6, wherein the aqueous solution further comprises an RNase inhibitor.

8. The method according to claim 7, wherein the RNase inhibitor comprises RNasin.

9. The method according to claim 8, wherein the aqueous solution contains RNasin at a concentration of 0.2 U / μl.

10. The method according to any one of claims 1 to 9, wherein the dNTP includes 0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP, 0.25 mM dTTP, and 0.5 mM UTP.

11. The method according to any one of claims 1 to 10, further comprising the step of adding one or more oligonucleotides selected from the group consisting of at least one forward primer, at least one reverse primer, and at least one probe.

12. The method according to claim 11, wherein the aqueous solution comprises at least a forward primer, at least one reverse primer, and at least one probe.

13. The method according to claim 12, wherein the aqueous solution comprises two or more forward primers, two or more reverse primers, and two or more probes.

14. The method according to any one of claims 11 to 13, wherein each of the one or more oligonucleotides is present at a concentration of 400 nM.

15. The method according to any one of claims 11 to 13, wherein the total concentration of the one or more oligonucleotides is 7 μM.

16. The aforementioned aqueous solution contains 2.7 mM Na ＋ The method according to any one of claims 1 to 15, including the method described in any one of claims 1 to 15.

17. The aforementioned aqueous solution contains 0.035 mM K ＋ The method according to any one of claims 1 to 16, including the method described in any one of claims 1 to 16.

18. The method according to any one of claims 1 to 17, wherein the aqueous solution contains less than 0.1 mM of magnesium ions.

19. The method according to any one of claims 1 to 18, wherein the aqueous solution has an inorganic salt concentration of 4 mM or less, or 3 mM or less.

20. The method according to any one of claims 1 to 19, wherein the buffer solution comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer solution or tris(hydroxymethyl)aminomethane (Tris) buffer solution.

21. The aqueous solution contains: 0.3 M trehalose; 0.46 U / μl hot-start Taq polymerase; 0.5 U / μl reverse transcriptase; 0.2 U / μl RNasin; 0.25 mM dATP; 0.25 mM dCTP; 0.25 mM dGTP; 0.25 mM dTTP; and 0.5 mM UTP; at least one forward primer; at least one reverse primer; at least one probe; and 2.7 mM Na ＋ ; 0.035 mM K ＋ The method according to claim 1, further comprising less than 0.1 mM of magnesium ions.

22. An aqueous solution containing hot-start polymerase, reverse transcriptase, a bulking agent, an organic buffer, a potassium concentration of 0.01 to 0.196 μg / μL, a magnesiumone concentration of less than 1 mM, an inorganic salt concentration of 5 mM or less, and 0 to 0.35% (w / v) glycerol.

23. A bulk reagent comprising the aqueous solution and dNTPs described in claim 22.

24. (a) The aqueous solution according to claim 22; or (b) Dry formulation and reconstituted solution of the aqueous solution according to claim 22 A kit comprising, wherein the reconstitution solution rehydrates the dried formulation.

25. The reconstituted solution consists of 65 mM KCl; 0.02% w / v methylparaben; 0.01% w / v propylparaben; 0.33% v / v ethanol; and 2.5 mM MgCl ２ The kit according to claim 24, including the following:

26. An aqueous solution according to claim 22, or a bulk reagent according to claim 23, for use in performing nucleic acid-based assays.

27. Use of the method according to any one of claims 1 to 21 for performing a nucleic acid-based assay.

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