Dried amplification compositions

Abstract

To provide dried amplification compositions.SOLUTION: The disclosure provides a dried composition to provide reagents for nucleic acid amplification, which is essentially free of inorganic salts. Lack of inorganic salts increases the stability of the composition and decreases formation of byproducts. Salts required for use of enzyme in the composition are provided on reconstitution. The invention also provides a dried composition comprising an enzyme selected from the group consisting of a polymerase and a reverse transcriptase, a bulking agent, an organic buffer solution, and a detergent. The dried composition may contain one or more inorganic salts at a concentration of 0.350% or less of the total mass of the dried composition.SELECTED DRAWING: Figure 7

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 291,770, filed February 5, 2016, which is incorporated herein by reference.

[0002] background Commercially available kits for performing nucleic acid amplification and / or detection reactions often contain reagents such as enzymes, including one or more polymerases, such as DNA-dependent DNA polymerases or RNA-dependent DNA polymerases (e.g., reverse transcriptase), nucleotides, detergents, buffers, primers, probes, and inorganic salts, including MnCl, MgCl, NaCl, and KCl (Innis et al., (1990) PCR Protocols: A Guide to Methods and Applications, Ch. 1, Optimizations of PCRs). Inorganic salts are useful for stabilizing components of nucleic acid reaction mixtures and for performing certain steps of nucleic acid-based reactions. However, these same salts adversely affect the stability and desiccation of lyophilized compositions, making them undesirable components of formulations that are lyophilized and / or stored prior to use.

[0003] Magnesium ions have been reported to increase the activity of polymerases and other enzymes. Potassium chloride has been reported to promote nucleic acid hybridization. Many inorganic salts, including those containing magnesium, have also been reported to protect proteins under a variety of conditions of stress, including heat, exposure to chaotropic agents, and lyophilization (see, e.g., 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(1971)J.Biol.Chem.246:5739, Chang et al 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). The presence of salt in the reaction mixture is believed to be necessary to avoid enzyme denaturation. However, the stability of the freeze-dried material is affected by any salt present in the freeze-dried cake. The hygroscopicity of the freeze-dried material, in turn, affects the time available for packaging the freeze-dried material and influences the duration and conditions under which the freeze-dried material can be stored and transported. Unwanted rehydration of the freeze-dried material adversely affects the activity of the freeze-dried components. To minimize the adverse effects from unwanted rehydration of the freeze-dried material, long-term storage of such materials is usually performed under refrigeration. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Innis et al,(1990) PCR Protocols:A Guide to Methods and Applications,Ch.1,Optimizations of PCRs [Non-patent document 2] Liu et al(2007)FEBS Letters.581:1047 [Non-patent document 3] Kanaya et al(1996)J.Biol.Chem.271:32729 [Non-patent document 4] Menendez et al (1998) J. Biol. Chem. 273:167 [Non-Patent Document 5] Janeway et al(1993)Biochemistry.32:1601 [Non-patent document 6] Fox et al (1971) J. Biol. Chem. 246:5739 [Non-Patent Document 7] Chang et al(2002)J.Biol.Chem.277:277:4663 [Non-patent document 8] Rutter et al (1958) J. Biol. Chem. 233:374 [Non-Patent Document 9] Huszar et al (1981) J. Virol. 37:580-588 [Non-Patent Document 10] Wang(2000)Int.J.Pharmaceutics.203:1-60 Summary of the Invention [Means for solving the problem]

[0005] Disclosed herein are compositions comprising an aqueous solution containing a polymerase and / or a reverse transcriptase, a filler, a detergent, and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 7 mM or less.

[0006] In some embodiments of the aqueous solution, the aqueous solution further comprises at least one oligonucleotide useful for conducting a molecular assay. In some embodiments, the aqueous solution comprises oligonucleotides for conducting a multiplex molecular assay. In some embodiments, at least one oligonucleotide comprises an amplification oligomer. In some embodiments, at least one oligonucleotide comprises a detection probe. In some embodiments, the detection probe comprises a label covalently attached to the oligonucleotide. In some embodiments, the label is a fluorescent or chemiluminescent molecule. In some embodiments, the detection probe is a TaqMan detection probe. In some embodiments, the detection probe oligonucleotide is configured to form a hairpin. In some embodiments, at least one oligonucleotide comprises an adapter oligonucleotide. In some embodiments, at least one oligonucleotide comprises an adapter configured to form a hairpin. In some embodiments, at least one oligonucleotide comprises a target capture probe. In some embodiments, the target capture probe has a target-hybridizing portion that specifically hybridizes to a target nucleic acid under stringent conditions. In some embodiments, the molecular assay comprises a nucleic acid amplification assay. In some embodiments, the molecular assay comprises a nucleic acid detection assay. In some embodiments, the molecular assay comprises a nucleic acid sequencing assay. In some embodiments, the molecular assay comprises a nucleic acid hybridization assay.

[0007] In some embodiments of the aqueous solution, the bulking agent is trehalose, raffinose, or a combination thereof. In some embodiments, the bulking agent is present at a concentration of about 0.16 M to about 0.32 M.

[0008] In some embodiments of the aqueous solution, the inorganic salt is present at a mass per microliter of about 0.029 μg / ul to about 0.373 μg / ul. In some embodiments, the aqueous solution contains about 0.029 μg / ul to about 0.292 μg / ul sodium chloride. In some embodiments, the aqueous solution contains about 0.019 μg / ul to about 0.373 μg / ul potassium chloride. In some embodiments, the aqueous solution contains about 0.006 μg / ul to about 0.115 μg / ul sodium ion. In some embodiments, the aqueous solution contains about 0.010 μg / ul to about 0.196 μg / ul potassium ion. In some embodiments, the aqueous solution contains about 0.009 μg / ul to about 0.355 μg / ul chloride ion.

[0009] In some embodiments of the aqueous solution, the aqueous solution comprises an inorganic salt concentration of 4 mM or less. In some embodiments, the aqueous solution comprises an inorganic salt mass of about 0.234 μg to about 0.298 μg per microliter. In some embodiments, the aqueous solution comprises a chloride ion mass of about 0.071 μg to about 0.284 μg per microliter. In some embodiments, the aqueous solution comprises an inorganic salt concentration of 3 mM or less. In some embodiments, the aqueous solution comprises an inorganic salt mass of about 0.175 μg to about 0.224 μg per microliter. In some embodiments, the aqueous solution comprises a chloride ion mass of about 0.053 μg to about 0.213 μg per microliter. In some embodiments, the aqueous solution comprises an inorganic salt concentration of 2 mM or less. In some embodiments, the aqueous solution comprises an inorganic salt mass of about 0.117 μg to about 0.149 μg per microliter. In some embodiments, the aqueous solution comprises about 0.036 μg to about 0.142 μg of chloride ion by mass per microliter. In some embodiments, the aqueous solution comprises an inorganic salt concentration of 1 mM or less. In some embodiments, the aqueous solution comprises about 0.058 μg to about 0.075 μg of inorganic salt by mass per microliter. In some embodiments, the aqueous solution comprises about 0.018 μg to about 0.071 μg of chloride ion by mass per microliter. In some embodiments, the aqueous solution comprises an inorganic salt concentration of 500 μM or less. In some embodiments, the aqueous solution comprises about 0.029 μg to about 0.037 μg of inorganic salt by mass per microliter. In some embodiments, the aqueous solution comprises about 0.009 μg to about 0.036 μg of chloride ion by mass per microliter.

[0010] In some embodiments of the aqueous solution, the inorganic salt concentration of the aqueous solution is less than 1 mM sodium chloride. In some embodiments, the aqueous solution does not contain sodium chloride. In some embodiments, the aqueous solution contains less than 1 mM magnesium ions. In some embodiments, the aqueous solution contains less than 0.1 mM magnesium ions, and suitably does not contain magnesium ions.

[0011] In some embodiments of the aqueous solution, the aqueous solution further comprises deoxynucleotide triphosphates (dNTPs). In some embodiments, the dNTPs comprise dATP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution. In some embodiments, the dATP is at a concentration of 0.2 mM in the aqueous solution. In some embodiments, the dNTPs comprise dGTP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution. In some embodiments, the dGTP is at a concentration of 0.2 mM in the aqueous solution. In some embodiments, the dNTPs comprise dCTP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution. In some embodiments, the dNTPs comprise dCTP at a concentration of 0.2 mM to 0.6 mM in the aqueous solution. In some embodiments, the dNTPs comprise dUTP at a concentration of 0.2 mM to 0.6 mM in the aqueous solution. In some embodiments, the dNTPs comprise labeled dNTPs.

[0012] In some embodiments of the aqueous solution, the polymerase is at a concentration of about 0.20 U / ul to about 0.72 U / ul in the aqueous solution. In some embodiments, the polymerase is at a concentration selected from 0.25 U / ul, 0.30 U / ul, 0.32 U / ul, 0.4 U / ul, 0.5 U / ul, 0.45 U / ul, and 0.72 U / ul in the aqueous solution. In some embodiments, the polymerase is a hot-start polymerase. In some embodiments, the polymerase is a recombinant Taq DNA polymerase bound by an antibody that specifically inhibits the polymerase activity of the polymerase. In some embodiments, the polymerase is a chemically modified recombinant Taq DNA polymerase, wherein the chemical modification inhibits the polymerase activity of the polymerase. In some embodiments, the polymerase is modified for incorporation of labeled dNTPs into nucleic acid extension reaction products.

[0013] In some embodiments of the aqueous solution, the aqueous solution comprises a reverse transcriptase at a concentration of about 0.1 U / ul to about 0.6 U / ul. In some embodiments, the reverse transcriptase is AMV reverse transcriptase. In some embodiments, the reverse transcriptase is MMLV reverse transcriptase.

[0014] In some embodiments of the aqueous solution, the aqueous solution further comprises an RNase inhibitor, hi some embodiments, the RNase inhibitor is present in the aqueous solution at a concentration of about 0.12 U / ul to about 0.20 U / ul.

[0015] In some embodiments of the aqueous solution, the aqueous solution further comprises a chelating agent. In some embodiments, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine acetate (MGDA), diethylenetriaminepentaacetic acid (DTPA), and ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In some embodiments, the chelating agent is EDTA and is present in the aqueous solution at a concentration of 1.5 mM to 2.0 mM.

[0016] Disclosed herein are dried forms of the aqueous solutions described above.

[0017] Disclosed herein is a dry composition comprising an enzyme selected from the group consisting of a polymerase and a reverse transcriptase, a bulking agent, an organic buffer, and a detergent. The dry composition also comprises one or more inorganic salts, wherein the one or more inorganic salts are present in the dry composition in an amount by weight that is 0.350% or less of the total weight of the dry composition.

[0018] In some embodiments of the dry composition, the one or more inorganic salts are present in the dry composition in an amount by weight that is about 0.311% to about 0.024% of the total weight of the dry composition, hi some embodiments, the one or more inorganic salts are selected from the group consisting of sodium chloride, potassium chloride, and both sodium chloride and potassium chloride.

[0019] In some embodiments of the dry composition, the dry composition further comprises at least one oligonucleotide useful for conducting a molecular assay. In some embodiments, the dry composition comprises oligonucleotides for conducting a multiplex molecular assay. In some embodiments, at least one oligonucleotide comprises an amplification oligomer. In some embodiments, at least one oligonucleotide comprises a detection probe. In some embodiments, the detection probe comprises a label covalently attached to the oligonucleotide. In some embodiments, the label is a fluorescent or chemiluminescent molecule. In some embodiments, the detection probe is a TaqMan detection probe. In some embodiments, the detection probe oligonucleotide is configured to form a hairpin. In some embodiments, at least one oligonucleotide comprises an adapter oligonucleotide. In some embodiments, at least one oligonucleotide comprises an adapter configured to form a hairpin. In some embodiments, at least one oligonucleotide comprises a target capture probe. In some embodiments, the target capture probe has a target-hybridizing portion that specifically hybridizes to a target nucleic acid under stringent conditions. In some embodiments, the molecular assay comprises a nucleic acid amplification assay. In some embodiments, the molecular assay comprises a nucleic acid detection assay. In some embodiments, the molecular assay comprises a nucleic acid sequencing assay. In some embodiments, the molecular assay comprises a nucleic acid hybridization assay.

[0020] In some embodiments of the dry composition, the bulking agent is trehalose, raffinose, or a combination thereof.

[0021] In some embodiments of the dry composition, the dry composition further comprises deoxynucleotide triphosphates (dNTPs).

[0022] In some embodiments of the dry composition, the polymerase is a hot-start polymerase. In some embodiments, the polymerase is a recombinant Taq DNA polymerase bound to an antibody that specifically inhibits polymerase activity. In some embodiments, the polymerase is a chemically modified recombinant Taq DNA polymerase. In some embodiments, the polymerase is modified for incorporation of labeled dNTPs into nucleic acid extension reaction products.

[0023] In some embodiments of the dry composition, the reverse transcriptase is AMV reverse transcriptase or the reverse transcriptase is MMLV reverse transcriptase.

[0024] In some embodiments of the dry composition, the dry composition further comprises an RNase inhibitor.

[0025] In some embodiments of the dry composition, the dry composition further comprises a chelating agent, hi some embodiments, the chelating agent is selected from the group consisting of EDTA, EGTA, EDDS, DTPA, and MGDA.

[0026] Disclosed herein is a method of forming a mixture for use in performing a nucleic acid-based amplification reaction, the method comprising combining a reconstitution solution and the dry composition described above, wherein the reconstitution solution comprises at least one inorganic salt.

[0027] In some embodiments of the method, the reconstitution solution comprises an inorganic salt concentration of less than 1 mM. In some embodiments, the reconstitution solution comprises an inorganic salt selected from the group consisting of sodium ions, potassium ions, magnesium ions, manganese ions, chloride ions, and combinations thereof. In some embodiments, the reconstitution solution comprises MgCl2 at a concentration of about 3.8 mM to about 4.4 mM, or KCl at a concentration of about 50 mM to about 80 mM, or both. In some embodiments, the reconstitution solution comprises a MgCl2 concentration that exceeds the MgCl2 concentration required in the reconstituted dry composition (the amount of MgCl2 required in the reconstituted dry composition is determined based on numerous factors, such as enzyme requirements, molecular assay requirements, and molecular assay optimization results). These reconstitution solutions containing excess McCl2 are referred to as universal reconstitution solutions. Universal reconstitution solutions are useful for reconstituting dry compositions containing various components for performing different molecular assays. By way of example only, the universal reconstitution solution may comprise MgCl2 at a concentration X. Dry Composition #1 has a requirement for a 0.8X concentration of MgCl, and Dry Composition #2 has a requirement for a 0.95X concentration of MgCl. Both Dry Composition #1 and Dry Composition #2 are reconstituted with the same universal reconstitution solution, and the MgCl concentration in the reconstituted dry compositions is reduced to the desired level by the use of a chelating agent. Preferably, Dry Compositions #1 and #2 are each formulated (e.g., in the bulk reagents prior to drying) to include an amount of chelating agent that will sequester excess MgCl from the subsequently used universal reconstitution solution. After reconstitution, the chelating agent will sequester a portion of the MgCl, thereby leaving only the desired concentration of MgCl free in solution (e.g., 0.8X and 0.95X, respectively, for this illustrative description).

[0028] In some embodiments of the method, the reconstituted solution comprises methylparaben at a mass concentration of about 0.012% w / v to about 0.020% w / v, or propylparaben at a mass concentration of 0.006% w / v to about 0.010% w / v, or absolute ethanol at a volume concentration of about 0.20% v / v to about 0.30% v / v, or a combination thereof. In some embodiments, the concentration of methylparaben in the reconstituted solution is 0.016% w / v. In some embodiments, the concentration of propylparaben in the reconstituted solution is 0.008% w / v. In some embodiments, absolute ethanol is present in the reconstituted solution at about 0.26% v / v.

[0029] Disclosed herein are methods for preparing dry compositions used in conducting molecular assays, such as nucleic acid-based amplification reactions, nucleic acid-based detection reactions, nucleic acid-based sequencing reactions, nucleic acid-based hybridization reactions, and combinations thereof. In some embodiments, the methods include a drying step selected from the group of methods consisting of dehydration, drying, lyophilization, and spray drying. In some embodiments, the methods include (i) freezing an aqueous solution composition as described herein, thereby forming a frozen form of the composition, and (ii) exposing the frozen form of the composition to lyophilization conditions, thereby forming a dried form of the composition. In some embodiments, the aqueous composition is dried in a lyophilizer to produce a lyophilized composition.

[0030] The dried composition is useful for nucleic acid-based reactions (e.g., amplification and / or detection reactions) after reconstitution. Dried compositions that have been exposed to a humid environment for extended periods surprisingly provide robust amplification and / or detection results when reconstituted and used in nucleic acid amplification and / or detection assays. The dried form of the composition exposed to a humid environment for periods of up to 3 hours, preferably 90 to 180 minutes, preferably about 90 minutes, or preferably about 180 minutes, where the absolute humidity level of the humid environment is greater than 2.3 grams of water per cubic meter of air, is then reconstituted and useful in nucleic acid amplification and / or detection assays. In certain embodiments, the reconstituted form of the dried composition is useful in amplification and / or detection reactions even when the dried composition is exposed to a humid environment where the relative humidity level of the humid environment is 10% or less for periods of up to 8 hours. In certain embodiments, the reconstituted form of the dry composition is useful in amplification and / or detection reactions even when the dry composition is exposed to a humid environment, where the humid environment has an absolute humidity level of 2.3 grams of water per cubic meter of air at or below 25° C. for a period of up to 8 hours. In certain embodiments, the aqueous bulk reagents are incubated for an extended period of time, and then all or a portion of the aqueous bulk reagents are dried to form a dry composition, which, after reconstitution of the dry composition, surprisingly provides robust nucleic acid amplification and / or detection results when used in nucleic acid amplification and / or detection assays.

[0031] In some embodiments, the dry composition is stored in a sealed container. In some embodiments, the dry composition is exposed to a humid environment prior to storing the dry composition in a sealed container, the humid environment having an absolute humidity level greater than 2.3 grams of water per cubic meter of air. In some embodiments, the aqueous solution prior to drying is stored at room temperature for up to 8 hours before initiating the drying step. In some embodiments, the aqueous solution prior to drying is stored at room temperature for a period of about 45 minutes to about 8 hours before initiating the drying step. In some embodiments, the dry composition is exposed to a humid environment prior to storing the dry composition in a sealed container, the humid environment having a relative humidity level of less than 10%. In some embodiments, the dry composition is exposed to a humid environment prior to storing the dry composition in a sealed container, the humid environment having an absolute humidity level of 2.3 grams of water per cubic meter of air at 25° C. or below. In some embodiments, the dry aqueous solution is stored at room temperature for up to 8 hours before storing the dry composition in a sealed container.

[0032] Disclosed herein are kits for use in performing molecular assays. In some embodiments, the kits are for performing nucleic acid-based amplification reactions. In some embodiments, the kits are for performing nucleic acid-based detection reactions. In some embodiments, the kits are for performing nucleic acid-based sequencing reactions. In some embodiments, the kits are for performing nucleic acid-based hybridization reactions. In some embodiments, the kits are for performing combined molecular assays, such as nucleic acid-based amplification and detection reactions. In some embodiments, the kits include a dry composition as described herein in a container. In some embodiments, the kits include a solution for reconstituting the dry composition used in a molecular assay, such as an amplification reaction and / or a detection reaction, in the container. In some embodiments, the kits include a first container containing a dry composition as described herein and a second container containing a reconstitution solution comprising MgCl2 at a concentration of about 3.8 mM to about 4.4 mM. In some embodiments, the first container is a multiwell plate containing one or more wells. In some embodiments, at least one of the one or more wells contains the dry composition. In some embodiments, each of the one or more wells contains the dry composition. In some embodiments, two or more of the one or more wells contain dry compositions for performing different molecular assays. In one aspect of this embodiment, each dry pellet in the two or more wells has a different MgCl concentration requirement. Furthermore, in some aspects of this embodiment, each dry pellet with a different MgCl concentration requirement is composed of a universal reconstitution having an MgCl concentration equal to or greater than the additional MgCl required for each molecular assay. In some embodiments, at least one of the one or more wells contains dry single-unit-dose pellets containing an inorganic salt mass percentage relative to the pellet mass of 0.311% or less. In some embodiments, each of the one or more wells contains dry single-unit-dose pellets containing an inorganic salt mass percentage relative to the pellet mass of 0.311% or less.In some embodiments, the first container is made of a material that is thermally conductive, optically transparent, produces low autofluorescence, or a combination thereof, and has low moisture permeability. In one embodiment, the first container includes a cap that seals the opening of the container. In some embodiments, the cap is a foil, a plug, or an elastomeric material. In some embodiments, the cap has low moisture permeability. In some embodiments, the first and second containers are incorporated into a device suitable for automatic transfer of the reconstituted solution from the second container to the first container. In certain embodiments, for example, the following are provided: (Item 1) 1. A composition comprising an aqueous solution containing at least one polymerase, a filler, a detergent, and an organic buffer, wherein the aqueous solution has an inorganic salt concentration of 7 mM or less. (Item 2) 2. The composition of claim 1, wherein the aqueous solution further comprises at least one oligonucleotide useful for performing a molecular assay. (Item 3) 3. The composition of claim 2, wherein the at least one oligonucleotide is selected from the group consisting of an amplification oligonucleotide, a detection probe oligonucleotide, a target capture probe oligonucleotide, an adapter oligonucleotide, and combinations thereof. (Item 4) 4. The composition of claim 2 or 3, wherein the at least one oligonucleotide comprises a detection probe oligonucleotide, and the detection probe oligonucleotide further comprises at least one label. (Item 5) 5. The composition of claim 4, wherein the label is selected from the group consisting of a fluorescent molecule, a quencher molecule, a luminescent molecule, and combinations thereof. (Item 6) 6. The composition of claim 5, wherein the label is a fluorescent or luminescent molecule. (Item 7) 7. The composition of claim 5 or 6, wherein the detection probe is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. (Item 8) 8. The composition of any one of items 2 to 7, wherein the at least one oligonucleotide comprises a target capture probe oligonucleotide. (Item 9) 9. The composition of claim 8, wherein the target capture probe oligonucleotide has a target-hybridizing portion that specifically hybridizes to a target nucleic acid under stringent conditions. (Item 10) 10. The composition according to any one of items 2 to 9, wherein the aqueous solution comprises oligonucleotides for performing a multiplex molecular assay. (Item 11) 11. The composition of any one of items 2 to 10, wherein the at least one oligonucleotide comprises an adaptor oligonucleotide configured to form a hairpin. (Item 12) 12. The composition according to any one of items 2 to 11, wherein the aqueous solution comprises oligonucleotides for performing a nucleic acid sequencing assay. (Item 13) 13. The composition according to any one of items 1 to 12, wherein the filler is trehalose, raffinose, or a combination thereof. (Item 14) Item 14. The composition of item 13, wherein the filler is trehalose. (Item 15) 15. The composition of any one of items 1, 13, and 14, wherein the filler is present at a concentration of about 0.16M to about 0.32M. (Item 16) 16. The aqueous solution according to any one of items 1 to 15, wherein the aqueous solution has an inorganic salt concentration of 5 mM or less. The composition described. (Item 17) 17. The composition of any one of items 1 to 16, wherein the inorganic salt is present in a mass per microliter of from about 0.373 ug / ul to about 0.029 ug / ul. (Item 18) 18. The composition of any one of items 1 to 17, wherein the aqueous solution contains from about 0.292 ug / ul sodium chloride to about 0.029 ug / ul sodium chloride. (Item 19) 18. The composition of any one of items 1 to 17, wherein the aqueous solution contains from about 0.373 ug / ul potassium chloride to about 0.019 ug / ul potassium chloride. (Item 20) 18. The composition according to any one of items 1 to 17, wherein the aqueous solution contains from about 0.115 ug / ul of sodium ions to about 0.006 ug / ul of sodium ions. (Item 21) 21. The composition of any one of items 1-4 or 20, wherein the aqueous solution contains from about 0.196 ug / ul of potassium ions to about 0.010 ug / ul of potassium ions. (Item 22) 22. The composition of any one of items 1-17, 20, or 21, wherein the aqueous solution contains from about 0.355 ug / ul chloride ion to about 0.009 ug / ul chloride ion. (Item 23) 16. The composition according to any one of items 1 to 15, wherein the aqueous solution comprises an inorganic salt concentration of 4 mM or less. (Item 24) 24. The composition of claim 23, wherein the aqueous solution comprises about 0.298 ug / ul to about 0.234 ug / ul of inorganic salt by mass per microliter. (Item 25) 25. The composition of claim 23 or 24, wherein the aqueous solution comprises chloride ion at a mass per microliter of from about 0.284 ug / ul to about 0.071 ug / ul. (Item 26) 16. The composition according to any one of items 1 to 15, wherein the aqueous solution comprises an inorganic salt concentration of 3 mM or less. (Item 27) 27. The composition of claim 26, wherein the aqueous solution comprises about 0.224 ug / ul to about 0.175 ug / ul of inorganic salt by mass per microliter. (Item 28) 28. The composition of claim 26 or 27, wherein the aqueous solution comprises chloride ion at a mass per microliter of from about 0.213 ug / ul to about 0.053 ug / ul. (Item 29) 16. The composition according to any one of items 1 to 15, wherein the aqueous solution comprises an inorganic salt concentration of 2 mM or less. (Item 30) 30. The composition of claim 29, wherein the aqueous solution comprises from about 0.149 ug / ul to about 0.117 ug / ul of inorganic salt by mass per microliter. (Item 31) 31. The composition of claim 29 or 30, wherein the aqueous solution comprises chloride ions at a mass per microliter of from about 0.142 ug / ul to about 0.036 ug / ul. (Item 32) 16. The composition according to any one of items 1 to 15, wherein the aqueous solution comprises an inorganic salt concentration of 1 mM or less. (Item 33) 33. The composition of claim 32, wherein the aqueous solution comprises an inorganic salt mass per microliter of about 0.075 ug / ul to about 0.058 ug / ul. (Item 34) 34. The composition of claim 32 or 33, wherein the aqueous solution comprises chloride ions at a mass per microliter of from about 0.071 ug / ul to about 0.018 ug / ul. (Item 35) 16. The composition according to any one of items 1 to 15, wherein the aqueous solution comprises an inorganic salt concentration of 500 uM or less. (Item 36) 36. The composition of claim 35, wherein the aqueous solution comprises about 0.037 ug / ul to about 0.029 ug / ul of inorganic salt by mass per microliter. (Item 37) 37. The composition of claim 35 or 36, wherein the aqueous solution comprises chloride ions by mass per microliter of about 0.036 ug / ul to about 0.009 ug / ul. (Item 38) 16. The composition according to any one of items 1 to 15, wherein the inorganic salt concentration of the aqueous solution is less than 1 mM sodium chloride. (Item 39) 14. The composition according to any one of items 1 to 13, wherein the aqueous solution does not contain sodium chloride. (Item 40) 16. The composition according to any one of items 1 to 15, wherein the aqueous solution contains less than 1 mM magnesium ions. (Item 41) 41. The composition of any one of items 1 to 40, wherein the aqueous solution contains less than 0.1 mM magnesium ions, suitably no magnesium ions. (Item 42) 42. The composition according to any one of items 1 to 41, wherein the aqueous solution further comprises deoxynucleotide triphosphates (dNTPs). (Item 43) Item 43. The composition according to item 42, wherein the dNTP comprises dATP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution. (Item 44) Item 45. The composition of item 43, wherein the dATP is at a concentration of 0.2 mM in the aqueous solution. 45. The composition according to any one of items 42 to 44, wherein the dNTP comprises dGTP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution. (Item 46) Item 47. The composition of item 45, wherein the dGTP is at a concentration of 0.2 mM in the aqueous solution. 47. The composition according to any one of items 42 to 46, wherein the dNTP comprises dCTP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution. (Item 48) Item 49. The composition of item 47, wherein the dCTP is at a concentration of 0.2 mM in the aqueous solution. 49. The composition according to claim 42, wherein the dNTP comprises dTTP at a concentration of 0.2 mM to 0.6 mM in the aqueous solution. (Item 50) 50. The composition according to any one of items 42 to 49, wherein the dNTP comprises dUTP at a concentration of 0.2 mM to 0.6 mM in the aqueous solution. (Item 51) 51. The composition of any one of items 1 to 50, wherein the at least one polymerase comprises a polymerase present in the aqueous solution at a concentration of about 0.20 U / ul to about 0.72 U / ul in the aqueous solution. (Item 52) 51. The composition of any one of items 1 to 50, wherein the at least one polymerase comprises a polymerase present in the aqueous solution at a concentration selected from 0.25 U / ul, 0.30 U / ul, 0.32 U / ul, 0.4 U / ul, 0.5 U / ul, 0.45 U / ul, and 0.72 U / ul. (Item 53) 53. The composition of any one of items 1 to 52, wherein the at least one polymerase comprises a polymerase that is a hot-start polymerase. (Item 54) 54. The composition of claim 53, wherein the hot-start polymerase is a recombinant Taq DNA polymerase bound by an antibody that specifically blocks the polymerase activity of the polymerase. (Item 55) 54. The composition of claim 53, wherein the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase, wherein the chemical modification inhibits the polymerase activity of the polymerase. (Item 56) 56. The composition of any one of items 1 to 55, wherein the at least one polymerase comprises a reverse transcriptase present in the aqueous solution at a concentration of about 0.1 U / ul to about 0.6 U / ul. (Item 57) 57. The composition of any one of items 1 to 56, wherein the reverse transcriptase is AMV reverse transcriptase. (Item 58) 57. The composition of any one of items 1 to 56, wherein the reverse transcriptase is MMLV reverse transcriptase. (Item 59) 59. The composition according to any one of items 1 to 58, wherein the aqueous solution further comprises an RNase inhibitor. (Item 60) 60. The composition of claim 59, wherein the RNase inhibitor is present in the aqueous solution at a concentration of about 0.12 U / ul to about 0.20 U / ul. (Item 61) 61. The composition according to any one of items 1 to 60, wherein the aqueous solution further comprises a chelating agent. (Item 62) 62. The composition of claim 61, wherein the chelating agent is selected from the group consisting of EDTA, EDDS, MGDA, EGTA, and DTPA. (Item 63) Item 63. The composition of item 62, wherein the chelating agent is EDTA and is present in the aqueous solution at a concentration of 1.5 mM to 2.0 mM. (Item 64) The composition according to any one of items 1 to 62, wherein the detergent is an ionic detergent. 65. The composition of claim 64, wherein the detergent is a cationic detergent. (Item 66) Item 65. The composition of item 64, wherein the detergent is an anionic detergent. (Item 67) 67. A dry form of the composition according to any one of items 1 to 66. (Item 68) 1. A dry composition comprising at least one polymerase, a filler, an organic buffer, one or more inorganic salts, and a detergent, wherein the one or more inorganic salts are present in the dry composition in an amount by weight that is 0.350% or less of the total weight of the dry composition. (Item 69) Item 69. The dry composition according to item 68, wherein the one or more inorganic salts are present in the dry composition in an amount by weight that is from about 0.311% to about 0.024% of the total weight of the dry composition. (Item 70) 69. The dry composition of claim 68, 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 71) 71. The dry composition according to items 68 to 70, wherein the dry composition further comprises at least one oligonucleotide useful for performing a molecular assay. (Item 72) 72. The dry composition of claim 71, wherein the at least one oligonucleotide is selected from the group consisting of an amplification oligonucleotide, a detection probe oligonucleotide, a target capture probe oligonucleotide, an adapter oligonucleotide, and combinations thereof. (Item 73) 73. The dry composition of claim 71 or 72, wherein the at least one oligonucleotide is a detection probe oligonucleotide, and the detection probe oligonucleotide further comprises at least one label. (Item 74) 74. The dry composition of claim 73, wherein the label is selected from the group consisting of a fluorescent molecule, a quencher molecule, a luminescent molecule, and combinations thereof. (Item 75) 75. The dry composition of item 74, wherein the label is a fluorescent or luminescent molecule. (Item 76) 76. The dry composition of claim 74 or 75, wherein the detection probe is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. (Item 77) 77. The dry composition of any one of items 71 to 76, wherein the at least one oligonucleotide comprises a target capture probe oligonucleotide. (Item 78) 78. The dry composition of claim 77, wherein the target capture probe oligonucleotide has a target-hybridizing portion that specifically hybridizes to a target nucleic acid under stringent conditions. (Item 79) 79. The dry composition according to any one of items 71 to 78, wherein the dry composition comprises oligonucleotides for performing a multiplex molecular assay. (Item 80) 80. The composition of any one of items 71 to 79, wherein the at least one oligonucleotide comprises an adaptor oligonucleotide configured to form a hairpin. (Item 81) 81. The composition of any one of items 71 to 80, wherein the aqueous solution comprises oligonucleotides for performing a nucleic acid sequencing assay. (Item 82) Item 69. The dry composition of item 68, wherein the bulking agent is trehalose, raffinose, or a combination thereof. (Item 83) 83. The dry composition of claim 82, wherein the bulking agent is trehalose. (Item 84) 84. The dry composition according to any one of items 68 to 83, wherein the composition further comprises deoxynucleotide triphosphates (dNTPs). (Item 85) 85. The dry composition of any one of items 68 to 84, wherein the at least one polymerase comprises a hot-start polymerase. (Item 86) 86. The dry composition of claim 85, wherein the hot-start polymerase is a recombinant Taq DNA polymerase bound to an antibody that specifically inhibits polymerase activity. (Item 87) 86. The dry composition of item 85, wherein the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase. (Item 88) 88. The dry composition of any one of items 68 to 87, wherein the at least one polymerase comprises a reverse transcriptase. (Item 89) 89. The dry composition of claim 88, wherein the reverse transcriptase is AMV reverse transcriptase. (Item 90) 89. The dry composition of claim 88, wherein the reverse transcriptase is MMLV reverse transcriptase. (Item 91) 91. The dry composition according to any one of items 68 to 90, wherein the composition further comprises an RNase inhibitor. (Item 92) 92. The dry composition according to any one of items 68 to 91, wherein the composition further comprises a chelating agent. (Item 93) Item 93. The dry composition of item 92, wherein the chelating agent is selected from the group consisting of EDTA, EGTA, DTPA, EDDS, and MGDA. (Item 94) 94. The dry composition according to any one of items 68 to 93, wherein the detergent is an ionic detergent. (Item 95) 95. The dry composition of claim 94, wherein the detergent is a cationic detergent. (Item 96) Item 96. The dry composition of item 95, wherein the detergent is an anionic detergent. (Item 97) 98. A method of forming a mixture for use in performing a nucleic acid-based amplification reaction, the method comprising combining a reconstituted solution and the dry composition of any one of items 68 to 97, wherein the reconstituted solution comprises at least one inorganic salt. (Item 98) Item 98. The method of item 97, wherein the reconstitution solution comprises an inorganic salt concentration of less than 1 mM. (Item 99) 99. The method of claim 97 or 98, wherein the reconstitution solution comprises an inorganic salt selected from the group consisting of sodium ions, potassium ions, magnesium ions, manganese ions, chloride ions, and combinations thereof. (Item 100) 99. The method of claim 97, wherein the reconstitution solution comprises MgCl at a concentration of about 3.8 mM to about 4.4 mM, KCl at a concentration of about 50 mM to about 80 mM, or both. (Item 101) 101. The method of any one of items 97 to 100, wherein the reconstitution solution comprises 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, absolute ethanol at a concentration of about 0.20% v / v to about 0.30% v / v, or a combination thereof. (Item 102) Item 102. The method of item 101, wherein the concentration of methylparaben in the reconstituted solution is 0.016% weight / volume. (Item 103) Item 102. The method of item 101, wherein the concentration of the propylparaben in the reconstituted solution is 0.008% weight / volume. (Item 104) 102. The method of claim 101, wherein the concentration of absolute ethanol is present in the reconstituted solution at about 0.26% v / v. (Item 105) 67. A method for preparing a dry composition for use in performing a molecular assay, the method comprising the steps of: (i) freezing the aqueous solution of any one of items 1 to 66, thereby forming a frozen form of said aqueous solution; and (ii) exposing said frozen form from step (i) to freeze-drying conditions, thereby forming a dry composition. (Item 106) Item 106. The method of item 105, wherein the dry composition is exposed to a humid environment, the humid environment having an absolute humidity level greater than 2.3 grams of water per cubic meter of air. (Item 107) Item 107. The method of item 106, wherein the humid environment has a temperature of 25°C. (Item 108) Item 109. The method of item 105, wherein the dry composition is exposed to the humid environment for up to 3 hours. 109. The method according to any one of items 105 to 108, further comprising storing the dry composition in a sealed container. (Item 110) 109. The method of any one of items 105 to 109, wherein the aqueous solution is a single unit dose. (Item 111) 111. The method of any one of items 105 to 110, wherein the aqueous solution is present in the wells of a multi-well plate. (Item 112) 67. A method for preparing a dry composition for use in performing a nucleic acid-based amplification reaction, the method comprising the step of drying the aqueous solution of any one of items 1 to 66 using a drying method selected from the group consisting of dehydration, drying, freeze-drying, and spray-drying, thereby forming a dry composition. (Item 113) Item 113. The method according to item 112, wherein the drying method is freeze-drying and the dry composition is a freeze-dried composition. (Item 114) 114. The method according to any one of items 112 to 113, further comprising storing the dry composition in a sealed container. (Item 115) 115. The method of any one of items 112 to 114, wherein the aqueous solution is a single unit dose. (Item 116) 116. The method of any one of items 112 to 115, wherein the aqueous solution is present in the wells of a multi-well plate. (Item 117) 97. A kit for use in performing a molecular assay, the kit comprising a first container containing the dry composition according to any one of items 68 to 96 and a second container containing a reconstitution solution, the reconstitution solution comprising at least one inorganic salt, suitably MgCl. (Item 118) Item 118. The kit of item 117, wherein the reconstitution solution comprises MgCl2 at a concentration of about 3.8 mM to about 4.4 mM. (Item 119) 119. The kit of claim 117 or 118, wherein the first container is a multiwell plate containing one or more wells. (Item 120) 120. The kit of claim 119, wherein each of the one or more wells contains a dry single unit dose pellet containing a weight percent of inorganic salt relative to the weight of the pellet of 0.311% or less. (Item 121) 121. The kit of any one of items 117 to 120, wherein the first and second containers are incorporated into a device suitable for automated transfer of the reconstitution solution from the second container to the first container. (Item 122) 97. Use of a mixture for performing a molecular assay, the mixture being a combination of a reconstituted solution and the dry composition according to any one of items 68 to 96, wherein the reconstituted solution comprises at least one inorganic salt. (Item 123) 123. The use according to item 122, wherein the molecular assay is a multiplex molecular assay. (Item 124) 124. The use according to item 122 or 123, wherein the molecular assay is a nucleic acid amplification assay. (Item 125) 124. Use according to item 122 or 123, wherein the molecular assay is a nucleic acid detection assay. (Item 126) 124. Use according to item 122 or 123, wherein the molecular assay is a nucleic acid amplification and detection assay. (Item 127) 124. The use according to item 122 or 123, wherein the molecular assay is a nucleic acid sequencing assay. [Brief explanation of the drawings]

[0033] [Figure 1] Figure 1 shows the activity determined in samples stored dry at various temperatures after lyophilization with or without magnesium. [Figure 2] The effect of room temperature storage time (storage of bulk reagent prior to lyophilization) on the activity recovered from lyophilized pellets is shown. Bulk reagent was stored under a number of conditions, dried, and used in nucleic acid amplification and detection reactions to identify influenza A from samples. [Figure 3-1] 3A, 3B, and 3C are histogram plots showing relative fluorescence unit (RFU) data for amplification and detection assays performed using reconstituted forms of dry single unit dose (SUD) pellet compositions consisting of bulk reagents without KCl and with or without MgCl2, incubated at room temperature or on ice for a set period of time. [Figure 3-2] 3A, 3B, and 3C are histogram plots showing relative fluorescence unit (RFU) data for amplification and detection assays performed using reconstituted forms of dry single unit dose (SUD) pellet compositions consisting of bulk reagents without KCl and with or without MgCl2, incubated at room temperature or on ice for a set period of time. [Figure 4]Figure 1 shows bioanalyzer gel-like images showing the effect of incubating pre-dried bulk reagents with or without MgCl2 on subsequent nucleic acid multiplex amplification assays for influenza A, influenza B, and respiratory syncytial virus (RSV). Bulk reagents were prepared without MgCl2 and then dried to produce a dried composition without MgCl2. MgCl2 was added prior to the amplification reaction. Reagents incubated with MgCl2 in the pre-dried bulk reagent exhibited nonspecific low-molecular-weight amplification (smearing), followed by the formation of low-molecular-weight by-products indicative of primer dimers and other spurious events, which result in lower amplification efficiency of the intended nucleic acid target. Reagents incubated without MgCl2 in the pre-dried bulk reagent exhibited stronger distinct target bands and less by-product formation compared to conditions in which MgCl2 was present in the pre-dried bulk reagent, demonstrating improved stability in the bulk formation master mix by omitting MgCl2. Lane 1 is the result obtained after 90 minutes on an ice-cold plate with 2.5 mM MgCl2 in the master mix, lane 2 is the result obtained after 90 minutes on an ice-cold plate without MgCl2 in the master mix, lane 3 is the result obtained after 90 minutes at room temperature on a pre-chilled plate with 2.5 mM MgCl2 in the master mix, lane 4 is the result obtained after 90 minutes at room temperature on a pre-chilled plate without MgCl2 in the master mix, lane 5 is the result obtained after 180 minutes on an ice-cold plate with 2.5 mM MgCl2 in the master mix, lane 6 is the result obtained after 180 minutes on an ice-cold plate without MgCl2 in the master mix, lane 7 is the result obtained after 180 minutes at room temperature with 2.5 mM MgCl2 in the master mix, and lane 8 is the result obtained after 180 minutes at room temperature without MgCl2 in the master mix. [Figure 5]Influenza A was used as a marker to demonstrate the long-term stability of the bulk formulation without MgCl. The amplification response, measured as relative fluorescence units (RFU), of bulk reagent incubated at room temperature for 3.75 and 7.75 hours was compared to a fresh liquid control to confirm the stability of the bulk reagent at room temperature for more than 7.75 hours when MgCl is omitted from the master mix. [Figure 6] Figure 1 shows the average residual moisture of the lyophilized reagent measured as the relative absorbance of the three formulations determined by Fourier transform near infrared (FT-nIR) spectroscopy at a spatial wavelength of 5170 cm (A). [Figure 7] 1 shows the effect of KCl concentration on residual moisture, measured as absorbance. DETAILED DESCRIPTION OF THE INVENTION

[0034] definition The term "about" indicates insubstantial variation in the amount of a component of a composition that does not have any significant effect on the activity or stability of the composition.

[0035] "Bulking agents" provide a matrix for the storage 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 product "cake" or other structure, which can prevent loss of protein from the vial during drying and increase protein stability.

[0036] Chelating agents are used to remove Mg, which is necessary for enzyme activity. 2+ ions or Mn 2+ It is an agent that sequesters divalent ions, including divalent ions such as ions.

[0037] The terms "freeze drying," "lyophilized," and "freeze-dried" refer to a process in which the material to be dried is first frozen, and then the ice or frozen solvent is removed by sublimation in a vacuum environment.

[0038] The term "stringency" with respect to nucleic acid hybridization (including "stringent hybridization conditions" or "stringent conditions") refers to conditions under which a particular oligonucleotide can hybridize to its target nucleic acid relative to other nucleic acids present in a test sample. It will 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 degree of hybridization specificity desired. Suitable hybridization conditions are well known in the art of probes, amplification oligonucleotides, target capture oligonucleotides, inhibitors, and other oligonucleotides, and can be predicted based on sequence composition or determined by using 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).

[0039] An amplification oligomer is a primer or promoter-primer capable of supporting template-dependent replication of a target nucleic acid. An amplification oligomer pair is a pair of such oligomers that supports template-dependent replication of opposite strands of a template. Multiplex amplification is the simultaneous amplification of multiple amplification oligomer pairs.

[0040] Probes are oligonucleotides that can hybridize to amplification products to reveal the presence or amount of amplification product. Such probes often incorporate a molecule that provides a fluorescent or other detectable signal, in which case they are referred to as detectably labeled probes.

[0041] A primer-probe set is a combination of primers and probes configured to generate and detect amplification products from a template nucleic acid.

[0042] As used herein, "label" refers to a moiety or compound attached directly or indirectly to a probe or dNTP that is detectable or provides a detectable signal. Direct labeling can occur through a bond or interaction that links the label to the probe, including covalent or non-covalent interactions, such as hydrogen bonding, hydrophobic and ionic interactions, or the formation of chelate or coordination complexes. Indirect labeling can occur through the use of a bridging moiety or "linker," such as a binding pair member, antibody, or additional oligomer, that can be labeled either directly or indirectly and amplify the detectable signal. Labels include any detectable moiety, such as a radionuclide, a ligand (e.g., biotin, avidin), an enzyme or enzyme substrate, a reactive group, or a chromophore (e.g., a dye, particle, or bead that provides a detectable color), a light-emitting compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent label), or a fluorophore. Labels can be detectable in homogeneous assays in which bound labeled probes in a mixture exhibit a detectable change, such as instability or gradual degradation characteristics, that differ from unbound labeled probes. Methods for synthesizing, attaching labels to nucleic acids, and detecting labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; U.S. Patent Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). Two or more labels, and two or more types of labels, can be present on a particular probe, or detection can use a mixture of probes, each labeled with a compound that generates a detectable signal (e.g., U.S. Patent Nos. 6,180,340 and 6,350,579).

[0043] "Reconstitution time" is the time required to rehydrate a dry formulation with a solution to produce a solution. Preferably, but not necessarily depending on the formulation, the reconstituted solution is free of particles and turbidity to the naked eye.

[0044] Relative fluorescence units (RFU) are a measure of the amplification product and, by implication, the nucleic acid analyte in the sample that gave rise to the amplification product.

[0045] Ct refers to the number of cycles required to reach the exponential phase in real-time PCR. Ct is inversely related to the amount of analyte in the sample.

[0046] Positive, when referring to an assay reaction result, refers to experimental data generated from a sample that exceeds a threshold (such as an RFU value). The threshold is set by the user and is typically determined based on empirical data obtained for a given assay. Typically, for nucleic acid amplification assays, the threshold is set so that a positive sample exceeds the threshold and enters the logarithmic growth phase of the assay. A positive value refers to the percentage of a single sample that exceeds the threshold or multiple samples that exceed the threshold. For example, if the multiple samples are 12 samples and the number of exceeding samples is determined to be 6, then the positive is "50%." The threshold is often set to exclude background values.

[0047] "Single unit dose" or "SUD" refers to the volume of reaction mixture used to perform a molecular assay on a single sample. A single unit dose can be in liquid or dry form. By way of example, a single unit dose can be a dry pellet containing reagents useful for amplifying a single sample in a single container.

[0048] LOD is the limit of detection for an analyte. LOD+1 is the LOD+1 logarithm detected by the user. In other words, LOD+1 is 10 times the number of analytes that are the LOD.

[0049] Ranges of values herein include all whole numbers and, where practical, all fractional numbers therein. For example, a pH value range from pH 2.0 to pH 5.0 includes all whole numbers and fractional numbers therein, and a length range for an oligonucleotide from 23 to 30 contiguous nucleotides will include only all whole numbers therein.

[0050] Whenever the disclosure refers to compositions comprising particular components, the disclosure should be understood to also disclose compositions consisting of or consisting essentially of the specified components.

[0051] (Detailed explanation) I. General The present disclosure is based in part on the insight that the instability of conventional lyophilized kits for performing molecular assays, such as nucleic acid amplification, is due to the presence of inorganic salts. These salts can lead to undesired hybridization products or other by-products before, during, and after drying. These salts also make the dried composition hygroscopic, causing the composition to absorb water from its surrounding environment. Thus, the salt content in the dried composition necessitates limited exposure to moisture, refrigerated or deep-frozen storage, and / or storage in the presence of a desiccant. The presence of water and salts can cause the polymerase components of the dried composition to prematurely lose activity and promote hybridization of nucleic acids to each other. The presence of salts can also reduce the ability to prepare dry reagents, for example, by lyophilization. Conversely, the absence of salts in enzyme-containing compositions is known to result in denaturation of enzyme components, and therefore compositions without these enzyme salts are also undesirable. The present disclosure overcomes these problems by drying bulk reagents for processing nucleic acid-based reaction mixtures from bulk reagents that are essentially free of inorganic salts. These salts are provided after reconstitution of the dried composition. Contrary to the expectation that inorganic salts are required for polymerase stability, it has been found that nucleic acid-based reaction mixtures dried essentially free of inorganic salts can be stored above freezing for extended periods while fully or substantially retaining activity for reconstitution.

[0052] II. Bulk reagents and dried pellets. A bulk reagent (sometimes referred to as a pre-lyophilized mixture, solution, aqueous solution, or composition) according to the present disclosure typically contains a polymerase, nucleotides used in nucleic acid-based amplification reactions, an organic buffer, preferably Tris, and a bulking agent such as trehalose or raffinose, or a combination thereof. The bulk reagent may or may not contain one or more nucleic acids. The bulk reagent may further contain a reverse transcriptase, a chelating agent, and an RNase inhibitor. The term bulk reagent refers to an aqueous solution as described above, which may be divided into two or more aliquots having substantially the same concentrations of reagents. The aliquots of the bulk reagent may also be referred to as bulk reagents. Regardless of context, the bulk reagent is an aqueous solution as described herein.

[0053] Such bulk reagents are essentially free of inorganic salts, meaning that the concentrations of individual and collective inorganic salts are less than 7 mM, and preferably less than 1 mM. Preferably, the Mg2+ concentration is less than 1 mM, less than 0.5 mM, less than 0.1 mM, or less than 0.05 mM. Preferably, the Na+ concentration is less than 1 mM, less than 0.5 mM, less than 0.1 mM, or less than 0.05 mM. Preferably, the K+ concentration is less than 1 mM, less than 0.5 mM, less than 0.1 mM, or less than 0.05 mM. Preferably, the Cl- concentration is less than 1 mM, less than 0.5 mM, less than 0.1 mM, or less than 0.05 mM.

[0054] Preferably, the concentration of Mg2+ is less than 1 mM and the concentration of Na+ is less than 1 mM. Preferably, the concentration of Mg2+ is less than 0.5 mM and the concentration of Na+ is less than 0.5 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM and the concentration of Na+ is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.05 mM and the concentration of Na+ is less than 0.05 mM.

[0055] Preferably, the concentration of Mg2+ is less than 1 mM and the concentration of K+ is less than 1 mM. Preferably, the concentration of Mg2+ is less than 0.5 mM and the concentration of K+ is less than 0.5 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM and the concentration of K+ is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.05 mM and the concentration of K+ is less than 0.05 mM.

[0056] Preferably, the concentration of Mg2+ is less than 1 mM and the concentration of Cl- is less than 1 mM. Preferably, the concentration of Mg2+ is less than 0.5 mM and the concentration of Cl- is less than 0.5 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.05 mM and the concentration of Cl- is less than 0.05 mM.

[0057] Preferably, the Na+ concentration is less than 1 mM and the K+ concentration is less than 1 mM. Preferably, the Na+ concentration is less than 0.5 mM and the K+ concentration is less than 0.5 mM. Preferably, the Na+ concentration is less than 0.1 mM and the K+ concentration is less than 0.1 mM. Preferably, the Na+ concentration is less than 0.05 mM and the K+ concentration is less than 0.05 mM.

[0058] Preferably, the concentration of Na+ is less than 1 mM and the concentration of Cl- is less than 1 mM. Preferably, the concentration of Na+ is less than 0.5 mM and the concentration of Cl- is less than 0.5 mM. Preferably, the concentration of Na+ is less than 0.1 mM and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Na+ is less than 0.05 mM and the concentration of Cl- is less than 0.05 mM.

[0059] Preferably, the concentration of K+ is less than 1 mM and the concentration of Cl- is less than 1 mM. Preferably, the concentration of K+ is less than 0.5 mM and the concentration of Cl- is less than 0.5 mM. Preferably, the concentration of K+ is less than 0.1 mM and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of K+ is less than 0.05 mM and the concentration of Cl- is less than 0.05 mM.

[0060] Preferably, the concentration of Mg2+ is less than 1 mM, the concentration of Na+ is less than 1 mM, and the concentration of K+ is less than 1 mM. Preferably, the concentration of Mg2+ is less than 0.5 mM, the concentration of Na+ is less than 0.5 mM, and the concentration of K+ is less than 0.5 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM, the concentration of Na+ is less than 0.1 mM, and the concentration of K+ is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM, the concentration of Na+ is less than 0.1 mM, and the concentration of K+ is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.05 mM, the concentration of Na+ is less than 0.05 mM, and the concentration of K+ is less than 0.05 mM.

[0061] Preferably, the concentration of Na+ is less than 1 mM, the concentration of K+ is less than 1 mM, and the concentration of Cl- is less than 1 mM. Preferably, the concentration of Na+ is less than 0.5 mM, the concentration of K+ is less than 0.5 mM, and the concentration of Cl- is less than 0.5 mM. Preferably, the concentration of Na+ is less than 0.1 mM, the concentration of K+ is less than 0.1 mM, and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Na+ is less than 0.1 mM, the concentration of K+ is less than 0.1 mM, and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Na+ is less than 0.05 mM, the concentration of K+ is less than 0.05 mM, and the concentration of Cl- is less than 0.05 mM.

[0062] Preferably, the concentration of Mg2+ is less than 1 mM, the concentration of K+ is less than 1 mM, and the concentration of Cl- is less than 1 mM. Preferably, the concentration of Mg2+ is less than 0.5 mM, the concentration of K+ is less than 0.5 mM, and the concentration of Cl- is less than 0.5 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM, the concentration of K+ is less than 0.1 mM, and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM, the concentration of K+ is less than 0.1 mM, and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.05 mM, the concentration of K+ is less than 0.05 mM, and the concentration of Cl- is less than 0.05 mM.

[0063] Preferably, the concentration of Mg2+ is less than 1 mM, the concentration of Na+ is less than 1 mM, and the concentration of Cl- is less than 1 mM. Preferably, the concentration of Mg2+ is less than 0.5 mM, the concentration of Na+ is less than 0.5 mM, and the concentration of Cl- is less than 0.5 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM, the concentration of Na+ is less than 0.1 mM, and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM, the concentration of Na+ is less than 0.1 mM, and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.05 mM, the concentration of Na+ is less than 0.05 mM, and the concentration of Cl- is less than 0.05 mM.

[0064] Preferably, the concentration of Mg2+ is less than 1 mM, the concentration of Na+ is less than 1 mM, the concentration of K+ is less than 1 mM, and the concentration of Cl- is less than 1 mM. Preferably, the concentration of Mg2+ is less than 0.5 mM, the concentration of Na+ is less than 0.5 mM, the concentration of K+ is less than 0.5 mM, and the concentration of Cl- is less than 0.5 mM. Preferably, the concentration of Mg2+ is less than 0.1 mM, the concentration of Na+ is less than 0.1 mM, the concentration of K+ is less than 0.1 mM, and the concentration of Cl- is less than 0.1 mM. Preferably, the concentration of Mg2+ is less than 0.05 mM, the concentration of Na+ is less than 0.05 mM, the concentration of K+ is less than 0.05 mM, and the concentration of Cl- is less than 0.05 mM.

[0065] Nucleotides for incorporation into amplification reactions are typically provided as dNTPs. Exemplary concentrations for dNTPs are 0.1-0.3 mM dATP, 0.1-0.3 mM dGTP, 0.1-0.3 mM dCTP, 0.2-0.6 mM dTTP, 0.2-0.6 mM dUTP, and preferably about 0.2 mM dATP, about 0.2 mM dGTP, about 0.2 mM dCTP, and 0.4 mM dTTP or dUTP. Nucleotides for incorporation into amplification reactions can also be provided as labeled dNTPs, such as those useful in sequencing reactions.

[0066] Such mixtures can be customized for different types of amplification, including PCR, RT-PCR, and transcription-mediated amplification, through the selection of enzymes and other components.

[0067] DNA polymerase enzymes can be commercially available or user-prepared. One example of a polymerase enzyme is Taq polymerase, available from Qiagen (Germantown, MD, cat#201203). Another example of Taq polymerase is available commercially as GoTaq® G2 Flexi DNA Polymerase (Promega, Madison, WI, cat#M7801). Other commercially available DNA polymerases include, but are not limited to, Tth DNA polymerase (e.g., Sigma-Aldrich, St. Louis, MO, cat#11480022001) and chimeric DNA polymerases such as Phusion® High-Fidelity DNA Polymerase (NEB, Ipswich, MA, cat#M0530S). Also commercially available are hot-start DNA polymerase enzymes. For example, Taq polymerase is commercially available as GoTaq® Hot Start Polymerase (Promega, cat#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 that during the initial heating step of the PCR reaction, the antibody is denatured and polymerase activity is restored. Various antibodies can be used with hot start methods, e.g., TAQSTART antibody (Clontech Laboratories, Mountain View, CA, cat#R028A). Similarly, other hot start polymerase enzymes are available, including chemically mediated hot start polymerases. Equivalent polymerases and antibodies are available from various commercial sources or can be prepared by the user.

[0068] Reverse transcriptases can be commercially available or user prepared. Examples of commercially available reverse transcriptases include, but are not limited to, MMLV (Moloney Murine Leukemia Virus) reverse transcriptase and SuperScript® III Reverse Transcriptase. Examples of reverse transcriptases include MMLV RT (Sigma-Aldrich, cat#M1302), AMV Reverse Transcriptase (NEB, Ipswich, MA, cat#M0277S), and GoScript® Reverse Transcriptase (Promega, cat#A50003). GoScript Reverse Transcriptase comprises a set of reverse transcriptases and reagents for the synthesis of single-stranded cDNA optimized for quantitative PCR amplification. Equivalent reverse transcriptases and reagents are available from a variety of commercial sources or can be prepared by the user.

[0069] Exemplary concentrations of DNA polymerase enzyme in a single unit dose are 0.01 to 1.0 U / ul. For example, 0.32 U / ul, or 0.4 U / ul, or 0.72 U / ul, or 0.32 to 0.4 U / ul, or 0.4 to 0.72 U / ul, or 0.05 to 0.3 U / ul, or 0.8 to 1.0 U / ul. One unit of DNA polymerase is defined as the amount of enzyme required to catalyze the incorporation of 10 nanomoles of dNTPs into acid-insoluble material in 30 minutes at 74°C. Exemplary concentrations of reverse transcriptase in a single unit dose are 0.01 U / ul to 1.0 U / ul. One unit of reverse transcriptase is defined as the amount of enzyme required to catalyze the transfer of 1 nmol of deoxynucleotides into acid-precipitable material in 10 minutes at 37°C.

[0070] A preferred organic buffer is Tris. Alternative organic buffers that can be incorporated into the bulk reagents of the present disclosure include phosphate, citrate, acetate, CHES, histidine, and Good's buffers, such as HEPES, MES, MOPS, tricine, and glycinamide, as well as buffer combinations. Other organic buffers include succinate, citrate, gluconate, phosphate, etc. Preferred buffers are effective in the pH range of about 5.5 to about 7.0 or about 6.0 to about 7.5, preferably about 6.5. Examples of buffers that control pH within this range include succinate (e.g., sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

[0071] Preferred fillers are trehalose or raffinose, or a combination thereof. Other fillers that can be used include 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, e.g., Chow, WO 2014 / 099198). The bulking agent is preferably present at a concentration of 0.16M to 0.32M, or alternatively 0.04 to 0.12M, 0.08 to 0.16M, 0.12 to 0.20M, 0.16 to 0.24M, 0.20 to 0.28M, 0.24 to 0.32M, 0.28 to 0.36M, 0.32 to 0.40M, or any combination of the above ranges, for example, 0.08 to 0.24M.

[0072] Bulk reagents can include one or more nucleic acids, such as, for example, amplification oligomers (e.g., primers, T7 promoter oligonucleotides), capture probes, detection probes, TaqMan probes, hairpin detection probes, adapters, hairpin adapters, positive control templates, and negative control templates.

[0073] Optional additional components of the bulk reagents include RNase inhibitors, PCR reagents, detergents, zwitterionic detergents, anionic detergents, cationic detergents, non-ionic detergents, surfactants, primers, probes, templates, chelators, methylparaben, and propylparaben. Exemplary concentrations for methylparaben are 0.01 to 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 ranges bounded by these values. Exemplary concentration ranges for propylparaben are 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 bounded by these values. One unit is defined as the amount of RNasin® Ribonuclease Inhibitor required to inhibit 5 ng of ribonuclease A activity by 50%. Activity is measured by inhibition of the hydrolysis of cytidine 2',3'-cyclic monophosphate by ribonuclease A.

[0074] The chelating agent includes one or more of EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), EDDS (ethylenediamine-N,N'-disuccinic acid), MGDA (methylglycine diacetic acid), and DTPA (diethylenetriaminepentaacetic acid). Exemplary concentrations for the chelating agent are 1.0 mM to 2.5 mM.

[0075] RNase inhibitor proteins, both natural and recombinant, are 50 kDa proteins that inhibit the RNase A family and human placental RNase by non-covalently binding to RNases in a 1:1 ratio. See Botella-Estrada et al. (2001) Cancer Gene Ther. 8:278; Polakowski et al. (1992) EXS. 61:428. RNase inhibitor proteins can be either recombinant or natural proteins. Exemplary concentrations of RNase inhibitors are about 0.04 U / ul to about 0.4 U / ul.

[0076] Bulk reagents can contain low concentrations of detergents, including ionic (cationic or anionic), nonionic, and zwitterionic detergents available from a number of commercial sources (e.g., Geno Technology, Inc., St. Louis, MO). Examples include, but are not limited to, lithium lauryl sulfate, amprolium hydrochloride, benzalkonium chloride, choline p-toluenesulfonate salt, dodecyltrimethylammonium chloride, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, ethylhexadecyldimethylammonium bromide, hexadecylpyridinium chloride, hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, hexadecyltrimethylammonium p-toluenesulfonate, Luviquat™, methylbenzethonium chloride, myristyltrimethylammonium bromide, N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane liquid, oxyphenonium bromide, tetraheptylammonium bromide, tetrakis(decyl)ammonium bromide, tricaprylylmethylammonium chloride, amidosulfobetaine-16, tridodecylmethylammonium chloride, trimethyloctadecylammonium bromide, Nonidet Examples include P-40 (registered trademark), Tween-20 (registered trademark), Tween-80 (registered trademark), Brij-35 (registered trademark), and Triton X-100 (registered trademark).

[0077] Exemplary volumes of bulk reagent include about 1 ul, about 5 ul, about 10 ul, about 20 ul, about 24 ul, about 50 ul, about 100 ul, about 200 ul, about 300 ul, about 400 ul, about 500 ul, about 600 ul, about 700 ul, about 800 ul, about 900 ul, about 1,000 ul (1 mL), about 2 mL, about 5 mL, about 10 mL, about 20 mL, about 50 mL, etc. Exemplary volumes of a single unit dose include about 1 ul to about 1 mL. The reconstituted dry composition can be formed in a lower, or larger volume than the same volume of liquid used to form the dry composition. The lower volume can be about 90%, about 80%, about 60%, about 40%, about 20%, about 10%, or about 5% of the bulk reagent. The larger volume can be about 120%, 140%, 160%, 180%, 200% (2x), about 4x, about 6x, about 8x, about 10x, about 20x the bulk reagent.

[0078] The sample to be analyzed can be added to the bulk reagent either before, simultaneously with, or after reconstitution. In a preferred embodiment, the entire dry composition after reconstitution is used to combine with the sample, where the relative volumes of reconstituted solution / sample can be, for example, about 9.9 / 0.1, 9.8 / 0.2, 9.5 / 0.5, 9 / 1, 8 / 2, 7 / 3, 6 / 4, 5 / 5, etc.

[0079] Unless otherwise specified, the concentration of a reagent in the bulk reagent can be, for example, 0.0% (reagent free), 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. Ranges of reagent "about," "below," "above," and including any two of the above concentrations are also provided.

[0080] Exemplary bulk reagent compositions have 0.1-0.3 mM, and more preferably 0.2 mM, of dATP, dGTP, and dCTP, and 0.2-0.6 mM, and more preferably 0.4 mM, of dUTP or dTTP, and 0.3-0.8 U / μL of polymerase. In some compositions, the polymerase is a hot-start Taq polymerase. In some compositions, the polymerase is GoTaq® MDx Hot Start Polymerase. Some compositions also include RNAsin® RNAase Inhibitor at 0.12-0.20 U / μL. Some compositions also include EDTA, optionally at 1.5-2.0 mM. Such compositions also include trehalose at 0.16-0.32 M, EDTA at 1.5-2.0 mM, and the polymerase is Taq at 0.3-0.45 U / μL.

[0081] The present disclosure provides reagents for PCR reactions, including real-time PCR reactions. (Real-time PCR Handbook, Life Technologies (2014); Kutyavin et al. (2000) Nucleic Acids Res. 28:655; Afonina et al. (2002) Biotechniques. 32:940). In real-time PCR, magnesium salts are typically used at a final concentration of 3-6 mM (Real-time PCR Handbook, supra). In some embodiments, the present disclosure provides reagents for multiplex PCR reactions, i.e., multiple primer pairs are provided for amplifying and detecting multiple targets. The present disclosure provides primers and probes for PCR reactions. The primers and / or probes comprise a target-hybridizing sequence and may further comprise one or more of a non-target-hybridizing sequence, a nucleotide analog, a detectable moiety, and a non-nucleotide linker (see, for example, WO2010 / 151566 and WO2013 / 126793). Thermocyclers are available (Applied Biosystems ProFlex® PCR System and Veriti® Thermal Cycler). Gel scanners for quantitating PCR products are available (Agilent® 2100 Bioanalyzer®, Bio-Rad® Densitometer).

[0082] After the bulk reagent is formed, it can be left at room temperature for a significant period of time before drying. The period can be up to 8 hours before the drying step is initiated, or alternatively, up to 1 hour, up to 2 hours, up to 4 hours, up to 6 hours, up to 10 hours, up to 12 hours, or up to 14 hours before the drying step is initiated. The inclusion of salts in the bulk reagent can result in undesired hybridization products and other by-products during this incubation period. Such undesired hybridization and by-products are reduced or eliminated in accordance with the present disclosure by forming a bulk reagent composition having an inorganic salt concentration of 7 mM or less, e.g., essentially free of inorganic salts.

[0083] The presence of inorganic salts in bulk reagents results in one or more of the following undesirable properties: Nucleic acids can hybridize together, and hybridization is stimulated by the presence of inorganic salts such as potassium, sodium, manganese, magnesium, and / or chloride. Furthermore, in the presence of inorganic salts such as manganese and magnesium, undesirable enzymatic activity, such as polymerase processivity and / or triphosphate depletion, can occur. Such undesirable activity can occur with non-hot-start and hot-start enzymes. Nucleic acid hybridization and enzymatic activity in the presence of salts can initiate the formation of undesirable by-products. Furthermore, inorganic salts are hygroscopic and will draw moisture into the dried pellet. Rehydration of the dried pellet reduces storage stability, enzymatic stability, and allows for the formation of additional spurious by-products.

[0084] The dried pellet can contain reagents to provide one single unit dose (SUD), or optionally, two or more SUDs. A single unit dose is a collection of reagents necessary to perform an amplification and / or detection reaction on only a single sample. A single unit dose may refer to a liquid reagent or a dried pellet. It should be noted that a single unit dose, as referred to herein, need not contain all of the reagents necessary to perform an amplification and / or detection reaction on a single sample. A single unit dose may lack the reagents necessary to perform an amplification and / or detection reaction. Similarly, a single unit dose may contain an insufficient amount of reagent to perform an amplification and / or detection reaction. By way of example only, a dried single unit dose pellet may contain sufficient units of Taq polymerase to perform an amplification reaction but may not contain magnesium. In certain embodiments, EDTA may be added to the dried single unit dose pellet to chelate excess divalent ions (e.g., magnesium and / or manganese) that may be present in the pellet or added by the reconstitution solution. In such instances, magnesium and / or manganese can then be added to the dry single unit dose pellet, e.g., via a reconstitution solution. Also by way of example only, the dry single unit dose may contain an insufficient amount of dNTPs to perform an amplification reaction. In such instances, the remainder of the dNTPs can be added to the dry single unit dose pellet, e.g., via a reconstitution solution. These examples are non-limiting, and one of skill in the art, armed with the present disclosure, will readily generate SUDs and dry pellet SUDs with a variety of compositions.

[0085] In a preferred embodiment, the bulk reagent contains an inorganic salt content of 7 mM or less, more preferably 6 mM or less, more preferably 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, and more preferably 500 μM or less. Thus, a preferred concentration range for the inorganic salt in the bulk reagent is about 0 mM to 7 mM. Another preferred concentration range for the inorganic salt in the bulk reagent is about 0 mM to 6 mM. Another preferred concentration range for the inorganic salt in the bulk reagent is about 0 mM to 7 mM. Another preferred concentration range for the inorganic salt in the bulk reagent is about 0 mM to 4 mM. Another preferred concentration range for the inorganic salt in the bulk reagent is about 0 mM to 3 mM. Another preferred concentration range for inorganic salts in the bulk reagent is an inorganic salt content of about 0 mM to 2 mM. Another preferred concentration range for inorganic salts in the bulk reagent is an inorganic salt content of about 0 mM to 1 mM. Another preferred concentration range for inorganic salts in the bulk reagent is an inorganic salt content of about 0 mM to 0.5 mM. Common inorganic salts for amplification and detection reaction mixtures include one or more of sodium, potassium, manganese, magnesium, and chloride, to name a few.

[0086] In one embodiment, the bulk reagent contains 5 mM or less of inorganic salts, and the inorganic salts are present at 0.373 μg / ul or less, or 0.332 μg / ul or less, or 0.292 μg / ul or less by weight per microliter. In a further embodiment, the bulk reagent contains 5 mM or less of inorganic salts, and sodium chloride is present at 0.292 μg / ul or less, 0.146 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 5 mM or less of inorganic salts, and sodium is present at 0.115 μg / ul or less, 0.057 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 5 mM or less of inorganic salts, and potassium chloride is present at 0.373 μg / ul or less, 0.186 μg / ul or less, or 0.0 μg / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 5 mM inorganic salts, with potassium present at no more than 0.196 ug / ul, no more than 0.098 ug / ul, or no more than 0.0 ug / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 5 mM inorganic salts, with chloride present at no more than 0.355 ug / ul, no more than 0.178 ug / ul, no more than 0.089 ug / ul, or no more than 0.0 ug / ul by weight per microliter.

[0087] In another embodiment, the bulk reagent comprises 5 mM or less inorganic salts, sodium chloride is present at 0.292 ug / ul or less, 0.146 ug / ul or less, or 0.0 ug / ul by weight per microliter, sodium is present at 0.115 ug / ul or less, 0.057 ug / ul or less, or 0.0 ug / ul by weight per microliter, potassium chloride is present at 0.373 ug / ul or less, 0.186 ug / ul or less, or 0.0 ug / ul by weight per microliter, potassium is present at 0.196 ug / ul or less, 0.098 ug / ul or less, or 0.0 ug / ul by weight per microliter, and chloride is present at 0.355 ug / ul or less, 0.178 ug / ul or less, 0.089 ug / ul or less, or 0.0 ug / ul by weight per microliter.

[0088] In a further aspect, the dried pellets are made from drying a liquid bulk reagent containing 5 mM or less of inorganic salt, and the weight percent of inorganic salt relative to the weight of the pellet is 0.311% or less, 0.277% or less, or 0.244% or less. In a further aspect, a container is provided containing dry single unit dose pellets having a weight percent of inorganic salt relative to the weight of the pellet of 0.311% or less, 0.277% or less, or 0.244% or less. In a further aspect, a multiwell plate is provided comprising one or more wells, each of which contains dry single unit dose pellets containing a weight percent of inorganic salt relative to the weight of the pellet of 0.311% or less, 0.277% or less, or 0.244% or less.

[0089] In one embodiment, the bulk reagent contains 4 mM or less of inorganic salts, and the inorganic salts are present at 0.298 μg / ul or less, or 0.266 μg / ul or less, or 0.234 μg / ul or less by weight per microliter. In a further embodiment, the bulk reagent contains 4 mM or less of inorganic salts, and sodium chloride is present at 0.234 μg / ul or less, 0.117 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 4 mM or less of inorganic salts, and sodium is present at 0.092 μg / ul or less, 0.046 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 4 mM or less of inorganic salts, and potassium chloride is present at 0.298 μg / ul or less, 0.149 μg / ul or less, or 0.0 μg / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 4 mM inorganic salts, with potassium present at no more than 0.156 ug / ul, no more than 0.078 ug / ul, or no more than 0.0 ug / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 4 mM inorganic salts, with chloride present at no more than 0.284 ug / ul, no more than 0.142 ug / ul, no more than 0.071 ug / ul, or no more than 0.0 ug / ul by weight per microliter.

[0090] In another embodiment, the bulk reagent comprises 4 mM or less inorganic salts, sodium chloride is present at 0.234 ug / ul or less, 0.117 ug / ul or less, or 0.0 ug / ul by weight per microliter, sodium is present at 0.092 ug / ul or less, 0.046 ug / ul or less, or 0.0 ug / ul by weight per microliter, potassium chloride is present at 0.298 ug / ul or less, 0.149 ug / ul or less, or 0.0 ug / ul by weight per microliter, potassium is present at 0.156 ug / ul or less, 0.078 ug / ul or less, or 0.0 ug / ul by weight per microliter, and chloride is present at 0.284 ug / ul or less, 0.142 ug / ul or less, 0.071 ug / ul or less, or 0.0 ug / ul by weight per microliter.

[0091] In a further aspect, the dried pellets are made from drying a liquid bulk reagent containing 4 mM or less of inorganic salt, and the weight percent of inorganic salt relative to the weight of the pellet is 0.249% or less, 0.222% or less, or 0.195% or less. In a further aspect, a container is provided containing dry single unit dose pellets having a weight percent of inorganic salt relative to the weight of the pellet of 0.249% or less, 0.222% or less, or 0.195% or less. In a further aspect, a multiwell plate is provided comprising one or more wells, each of which contains dry single unit dose pellets containing a weight percent of inorganic salt relative to the weight of the pellet of 0.249% or less, 0.222% or less, or 0.195% or less.

[0092] In one embodiment, the bulk reagent contains 3 mM or less inorganic salts, and the inorganic salts are present at 0.224 μg / ul or less, or 0.199 μg / ul or less, or 0.175 μg / ul or less by weight per microliter. In a further embodiment, the bulk reagent contains 3 mM or less inorganic salts, and sodium chloride is present at 0.175 μg / ul or less, 0.088 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 3 mM or less inorganic salts, and sodium is present at 0.069 μg / ul or less, 0.034 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 3 mM or less inorganic salts, and potassium chloride is present at 0.224 μg / ul or less, 0.112 μg / ul or less, or 0.0 μg / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 3 mM inorganic salts, with potassium present at no more than 0.117 ug / ul, no more than 0.059 ug / ul, or no more than 0.0 ug / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 3 mM inorganic salts, with chloride present at no more than 0.213 ug / ul, no more than 0.107 ug / ul, no more than 0.053 ug / ul, or no more than 0.0 ug / ul by weight per microliter.

[0093] In another embodiment, the bulk reagent comprises no more than 3 mM inorganic salts, sodium chloride is present at no more than 0.175 ug / ul, no more than 0.088 ug / ul, or 0.0 ug / ul by weight per microliter, sodium is present at no more than 0.069 ug / ul, no more than 0.034 ug / ul, or 0.0 ug / ul by weight per microliter, potassium chloride is present at no more than 0.224 ug / ul, no more than 0.112 ug / ul, or 0.0 ug / ul by weight per microliter, potassium is present at no more than 0.117 ug / ul, no more than 0.059 ug / ul, or 0.0 ug / ul by weight per microliter, and chloride is present at no more than 0.213 ug / ul, no more than 0.107 ug / ul, no more than 0.053 ug / ul, or 0.0 ug / ul by weight per microliter.

[0094] In a further aspect, the dried pellets are made from drying a liquid bulk reagent containing 3 mM or less of inorganic salt, and the weight percent of inorganic salt relative to the weight of the pellet is 0.186% or less, 0.166% or less, or 0.146% or less. In a further aspect, a container is provided containing dry single unit dose pellets having a weight percent of inorganic salt relative to the weight of the pellet of 0.186% or less, 0.166% or less, or 0.146% or less. In a further aspect, a multiwell plate is provided comprising one or more wells, each of which contains dry single unit dose pellets containing a weight percent of inorganic salt relative to the weight of the pellet of 0.186% or less, 0.166% or less, or 0.146% or less.

[0095] In one embodiment, the bulk reagent contains 2 mM or less of inorganic salts, and the inorganic salts are present at 0.149 μg / ul or less, or 0.133 μg / ul or less, or 0.117 μg / ul or less by weight per microliter. In a further embodiment, the bulk reagent contains 2 mM or less of inorganic salts, and sodium chloride is present at 0.117 μg / ul or less, 0.058 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 2 mM or less of inorganic salts, and sodium is present at 0.046 μg / ul or less, 0.023 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 2 mM or less of inorganic salts, and potassium chloride is present at 0.149 μg / ul or less, 0.075 μg / ul or less, or 0.0 μg / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 2 mM inorganic salts, with potassium present at no more than 0.078 ug / ul, no more than 0.039 ug / ul, or no more than 0.0 ug / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 2 mM inorganic salts, with chloride present at no more than 0.142 ug / ul, no more than 0.071 ug / ul, no more than 0.036 ug / ul, or no more than 0.0 ug / ul by weight per microliter.

[0096] In another embodiment, the bulk reagent comprises no more than 2 mM inorganic salts, sodium chloride is present at no more than 0.117 ug / ul, no more than 0.058 ug / ul, or 0.0 ug / ul by weight per microliter, sodium is present at no more than 0.046 ug / ul, no more than 0.023 ug / ul, or 0.0 ug / ul by weight per microliter, potassium chloride is present at no more than 0.149 ug / ul, no more than 0.075 ug / ul, or 0.0 ug / ul by weight per microliter, potassium is present at no more than 0.078 ug / ul, no more than 0.039 ug / ul, or 0.0 ug / ul by weight per microliter, and chloride is present at no more than 0.142 ug / ul, no more than 0.071 ug / ul, no more than 0.036 ug / ul, or 0.0 ug / ul by weight per microliter.

[0097] In a further aspect, the dried pellets are made from drying a liquid bulk reagent containing 2 mM or less of inorganic salt, and the weight percent of inorganic salt relative to the weight of the pellet is 0.124% or less, 0.111% or less, or 0.097% or less. In a further aspect, a container is provided containing dry single unit dose pellets having a weight percent of inorganic salt relative to the weight of the pellet of 0.124% or less, 0.111% or less, or 0.097% or less. In a further aspect, a multiwell plate is provided comprising one or more wells, each of which contains dry single unit dose pellets containing a weight percent of inorganic salt relative to the weight of the pellet of 0.124% or less, 0.111% or less, or 0.097% or less.

[0098] In one embodiment, the bulk reagent contains 1 mM or less of inorganic salts, and the inorganic salts are present at 0.075 μg / ul or less, or 0.066 μg / ul or less, or 0.058 μg / ul or less by weight per microliter. In a further embodiment, the bulk reagent contains 1 mM or less of inorganic salts, and sodium chloride is present at 0.058 μg / ul or less, 0.029 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 1 mM or less of inorganic salts, and sodium is present at 0.023 μg / ul or less, 0.011 μg / ul or less, or 0.0 μg / ul by weight per microliter. In a further embodiment, the bulk reagent contains 1 mM or less of inorganic salts, and potassium chloride is present at 0.075 μg / ul or less, 0.037 μg / ul or less, or 0.0 μg / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 1 mM inorganic salts and potassium is present at no more than 0.039 ug / ul, no more than 0.020 ug / ul, or 0.0 ug / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 1 mM inorganic salts and chloride is present at no more than 0.071 ug / ul, no more than 0.036 ug / ul, no more than 0.018 ug / ul, or 0.0 ug / ul by weight per microliter.

[0099] In another embodiment, the bulk reagent contains 1 mM or less inorganic salts, sodium chloride is present at 0.058 ug / ul or less, 0.029 ug / ul or less, or 0.0 ug / ul by weight per microliter, sodium is present at 0.023 ug / ul or less, 0.011 ug / ul or less, or 0.0 ug / ul by weight per microliter, potassium chloride is present at 0.075 ug / ul or less, 0.037 ug / ul or less, or 0.0 ug / ul by weight per microliter, potassium is present at 0.039 ug / ul or less, 0.020 ug / ul or less, or 0.0 ug / ul by weight per microliter, and chloride is present at 0.071 ug / ul or less, 0.036 ug / ul or less, 0.018 ug / ul or less, or 0.0 ug / ul by weight per microliter.

[0100] In a further aspect, the dried pellets are made from drying a liquid bulk reagent containing 1 mM or less of inorganic salt, and the weight percent of inorganic salt relative to the weight of the pellet is 0.062% or less, 0.055% or less, or 0.049% or less. In a further aspect, a container is provided containing dry single unit dose pellets having a weight percent of inorganic salt relative to the weight of the pellet of 0.062% or less, 0.055% or less, or 0.049% or less. In a further aspect, a multiwell plate is provided comprising one or more wells, each of which contains dry single unit dose pellets containing a weight percent of inorganic salt relative to the weight of the pellet of 0.062% or less, 0.055% or less, or 0.049% or less.

[0101] In one embodiment, the bulk reagent contains 500 uM or less of inorganic salts, and the inorganic salts are present at 0.037 ug / ul or less, or 0.033 ug / ul or less, or 0.029 ug / ul or less by weight per microliter. In a further embodiment, the bulk reagent contains 500 uM or less of inorganic salts, and sodium chloride is present at 0.029 ug / ul or less, 0.015 ug / ul or less, or 0.0 ug / ul by weight per microliter. In a further embodiment, the bulk reagent contains 500 uM or less of inorganic salts, and sodium is present at 0.011 ug / ul or less, 0.006 ug / ul or less, or 0.0 ug / ul by weight per microliter. In a further embodiment, the bulk reagent contains 500 uM or less of inorganic salts, and potassium chloride is present at 0.037 ug / ul or less, 0.019 ug / ul or less, or 0.0 ug / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 500 uM inorganic salts and potassium is present at no more than 0.020 ug / ul, no more than 0.010 ug / ul, or 0.0 ug / ul by weight per microliter. In further embodiments, the bulk reagent comprises no more than 500 uM inorganic salts and chloride is present at no more than 0.036 ug / ul, no more than 0.018 ug / ul, no more than 0.009 ug / ul, or 0.0 ug / ul by weight per microliter.

[0102] In another embodiment, the bulk reagent contains 500 uM or less of inorganic salts, sodium chloride is present at 0.029 ug / ul or less, 0.015 ug / ul or less, or 0.0 ug / ul by weight per microliter, sodium is present at 0.011 ug / ul or less, 0.006 ug / ul or less, or 0.0 ug / ul by weight per microliter, potassium chloride is present at 0.037 ug / ul or less, 0.019 ug / ul or less, or 0.0 ug / ul by weight per microliter, potassium is present at 0.020 ug / ul or less, 0.010 ug / ul or less, or 0.0 ug / ul by weight per microliter, and chloride is present at 0.036 ug / ul or less, 0.018 ug / ul or less, 0.009 ug / ul or less, or 0.0 ug / ul by weight per microliter.

[0103] In a further aspect, the dried pellets are made from drying a liquid bulk reagent containing 500 uM or less of inorganic salt, and the weight percent of inorganic salt relative to the weight of the pellet is 0.031% or less, 0.028% or less, or 0.024% or less. In a further aspect, a container is provided containing dry single unit dose pellets having a weight percent of inorganic salt relative to the weight of the pellet of 0.031% or less, 0.028% or less, or 0.024% or less. In a further aspect, a multiwell plate is provided comprising one or more wells, each of which contains dry single unit dose pellets containing a weight percent of inorganic salt relative to the weight of the pellet of 0.031% or less, 0.028% or less, or 0.024% or less.

[0104] In one embodiment, the bulk reagent comprises about 5 mM to about 500 uM of inorganic salt, and the inorganic salt is present at about 0.373 ug / ul to about 0.029 ug / ul by weight per microliter. In a further embodiment, the bulk reagent comprises about 5 mM to about 500 uM of inorganic salt, and sodium chloride is present at about 0.292 ug / ul to about 0.029 ug / ul by weight per microliter. In a further embodiment, the bulk reagent comprises about 5 mM to about 500 uM of inorganic salt, and sodium is present at about 0.115 ug / ul to about 0.006 ug / ul by weight per microliter. In a further embodiment, the bulk reagent comprises about 5 mM to about 500 uM of inorganic salt, and potassium chloride is present at about 0.373 ug / ul to about 0.019 ug / ul by weight per microliter. In a further embodiment, the bulk reagent comprises about 5 mM to about 500 uM inorganic salt, with potassium present at about 0.196 ug / ul to about 0.010 ug / ul by weight per microliter. In a further embodiment, the bulk reagent comprises about 5 mM to about 500 uM inorganic salt, with chloride present at about 0.355 ug / ul to about 0.009 ug / ul by weight per microliter. In a further embodiment, the bulk reagent comprises about 5 mM to about 500 uM inorganic salt, with the inorganic salt present at about 0.373 ug / ul to about 0.029 ug / ul by weight per microliter, and sodium chloride present at about 0 ug / ul by weight per microliter. In a further embodiment, the bulk reagent comprises about 5 mM to about 500 uM of inorganic salt, the inorganic salt being present at about 0.373 ug / ul to about 0.029 ug / ul by weight per microliter, and potassium chloride being present at about 0 ug / ul by weight per microliter. In a further embodiment, a dried pellet is made from drying a liquid bulk reagent comprising 5 mM to 500 uM of inorganic salt, and the weight percent of the inorganic salt relative to the weight of the pellet is 0.311% to 0.024%.

[0105] In a further embodiment, the bulk reagent comprises from about 5 mM to about 500 uM inorganic salts, wherein sodium chloride is present at 0.292 ug / ul to about 0.029 ug / ul by weight per microliter, sodium is present at 0.115 ug / ul to about 0.006 ug / ul by weight per microliter, potassium chloride is present at about 0.373 ug / ul to about 0.019 ug / ul by weight per microliter, potassium is present at 0.196 ug / ul to about 0.010 ug / ul by weight per microliter, and chloride is present at 0.355 ug / ul to about 0.009 ug / ul by weight per microliter.

[0106] In a further embodiment, the bulk reagent comprises from about 5 mM to about 500 uM inorganic salt, the inorganic salt being present at about 0.373 ug / ul to about 0.029 ug / ul by weight per microliter, sodium chloride being present at about 0 ug / ul by weight per microliter, and potassium chloride being present at about 0 ug / ul by weight per microliter.

[0107] In a further aspect, the dried pellets are made from drying a liquid bulk reagent comprising 5 mM to 500 uM of inorganic salt, wherein the weight percent of the inorganic salt relative to the weight of the pellet is 0.311% to 0.024%, the weight percent of sodium chloride relative to the weight of the pellet is about 0%, and / or the weight percent of potassium chloride relative to the weight of the pellet is about 0%. In a further aspect, a container is provided containing dry single unit dose pellets having a weight percent of the inorganic salt relative to the weight of the pellet of about 0.311% to 0.024%, the weight percent of sodium chloride relative to the weight of the pellet is about 0%, and / or the weight percent of potassium chloride relative to the weight of the pellet is about 0%. In a further aspect, a multi-well plate is provided comprising one or more wells, each of the one or more wells containing dry single unit dose pellets containing a weight percent of inorganic salt relative to the weight of the pellet of about 0.311% to 0.024%, wherein the weight percent of sodium chloride relative to the weight of the pellet is about 0%, and / or the weight percent of potassium chloride relative to the weight of the pellet is about 0%.

[0108] In one embodiment, a multi-well plate is provided that includes one or more wells. In one aspect, the one or more wells include walls constructed from a material that has low moisture permeability, thermal conductivity, light transparency, low autofluorescence, or a combination thereof. In one aspect, the one or more wells include cone-shaped walls. In one aspect, the one or more wells include walls configured to fit into a PCR thermal cycler to perform a PCR amplification reaction on a reaction mixture contained within the well. In one aspect, the wells include walls configured to fit into a thermally conductive tube receiving area of a device to perform a PCR, TMA, or other nucleic acid amplification reaction. In one aspect, the one or more wells include a heating block (e.g., available from Southern Labware, Cummings under product SKU BSH100G). The multi-well plate includes walls configured to fit into a dry block (see dry block for use with a digital dry block heater available from GA). In one embodiment, one or more wells of the multi-well plate include an opening for access to the chamber of the well. In one embodiment, one or more wells each include a cap sealing the opening of the associated well. In one embodiment, the opening of each of the one or more wells is sealed with a cap that is a low moisture permeable foil. In one embodiment, the opening of each of the one or more wells is sealed with a cap that is a low moisture permeable elastomeric material. In one embodiment, the multi-well plate includes one or more wells as described herein, wherein the chamber of the well contains a dry single unit dose pellet comprising a polymerase enzyme and an inorganic salt, wherein the weight percent of the inorganic salt relative to the weight of the pellet is about 0.311% to 0.024%. In one embodiment, a multi-well plate comprises one or more wells as described herein, wherein the chambers of the wells contain dry single unit dose pellets comprising a reverse transcriptase enzyme and an inorganic salt, wherein the weight percentage of the inorganic salt relative to the weight of the pellet is between about 0.311% and 0.024%. In one embodiment, a multi-well plate comprises one or more wells as described herein, wherein the chambers of the wells contain dry single unit dose pellets comprising a polymerase enzyme, a reverse transcriptase enzyme, and an inorganic salt, wherein the weight percentage of the inorganic salt relative to the weight of the pellet is between about 0.311% and 0.024%.

[0109] The reaction mixture can be collected and the reaction can be performed in an automated sampling and handling instrument, such as the Hologic® Panther Instrument (Hologic, Inc., MA), a robotic device equipped with pipettors, mixers, incubators, and wash stations that can perform multiple assays simultaneously, e.g., using PCR reactions, transcription-mediated amplification, and target capture hybridization.

[0110] III. DRYING APPARATUS AND 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 offered by a number of companies (e.g., Biopharma Technology Ltd., Winchester, Hampshire, Great Britain, and BioPharma Solutions Sterile Contract Manufacturing, Baxter Healthcare Corp, Deerfield, IL). Guidance on lyophilization is available from a number of sources (e.g., L. Rey, J.C. May (eds.) (2010) Freeze Drying / Lyophilization of Pharmaceuticals and Biological Products, 3 rd ed. Informa Healthcare, NY or Methods in Enzymology, Vol. 22, Pages 33-39, Academic Press, New York (1971), or Freeze-Drying, E. W. Flosdorf, Rheinhold, New York (1949). Optionally, the oxygen content can be reduced during freeze-drying (Phillips et al. (2001) Biologics. 19:219).

[0111] A variety of containers are suitable for drying. The container must be sealed and able to withstand external pressure when stored under partial vacuum. The container must be made of a material that allows adequate 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.

[0112] Samples can be dried in separate containers or multi-specimen receptacles. A multi-specimen receptacle refers to a contiguous receptacle capable of containing at least two specimens, allowing them to be simultaneously but separately stored and manipulated. Standard formats for multi-specimen receptacles include 6, 24, 96, 384, or 1536 wells. The volume of each well in an exemplary 96-well format is approximately 300-400 microliters, with a working volume of approximately 75-200 microliters. The volume generally varies inversely with the number of wells, typically within the range of 1 nL to 10 mL per well, although other sizes are also contemplated. Exemplary wells may have flat, round, or V-bottoms, among others. As used herein, a container is also referred to as a well. As used herein, a multi-specimen receptacle is also referred to as a multi-well plate. Additionally, a well may also be referred to as a reaction well. The term reaction well does not require that any reaction actually occur in the reaction well. Rather, the term is used to refer to a vessel or well that contains reagents and in which no reaction, a partial reaction, or a complete reaction may occur.

[0113] In some embodiments herein, the multiwell plate can be lyophilized to form a dry composition from an aqueous solution. Lyophilization can occur in a nest device (see co-pending International Patent Application No. PCT / US2016 / 045166). The nest is a container for holding the multiwell plate, and the nest includes a vent that can be closed by a mechanism operable from outside the sealed lyophilization chamber. The nest containing the multiwell plate is placed in the lyophilization chamber with one or more vents in an open position. The chamber is then sealed, and a lyophilization atmosphere is applied throughout the chamber, including the space within the nest. The one or more vents are then closed, thereby sealing the nest. The seal on the lyophilization chamber is then opened, and the nest containing the multiwell plate is removed. The nest can then be relocated, and the multiwell plate can be placed therein and stored until an operator is ready to use the lyophilized composition therein or reseal the multiwell plate containing the lyophilized specimen for further storage or sale. The wells of the multi-well plate can then be sealed to substantially prevent moisture ingress from the ambient air. Small amounts of moisture ingress into the sealed multi-well plate can be prevented by storing the sealed multi-well plate in a pouch containing a desiccant. Similarly, lyophilization can be performed in separate containers, and lyophilization can be performed in nests.

[0114] Other drying methods include spray drying, fluidized bed drying, dehumidifiers, and batch contact drying, in which the filter cake is dried at low temperatures under vacuum to a free-flowing dry product (N.P. Cheremisinoff (2000) Handbook of Chemical Processing Equipment, Butterworth Heinemann, Boston, MA). Dehumidifiers are available from Bry Air, Inc., Sunbury, Ohio, and DST Seibu Giken, Wyoming, PA. Rotary dryers, conical dryers, and tray dryers are available (McGill (AirPressure LLC, Columbus, Ohio). In one embodiment, a vacuum dryer removes moisture by exposing the material to reduced pressure, and heat is used sufficient to replace that lost through evaporation. Desiccants include silica gel desiccants, molecular sieve desiccants such as aluminosilicate and synthetic zeolites, and bentonite desiccants.

[0115] The reaction mixtures are preferably dried in the same container in which they will be reconstituted for use.

[0116] Lyophilized or otherwise dried formulations have a low water content, for example, 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, less than 0.01% by weight, or less than 5% to less than 0.01% by weight, less than 4% to less than 0.01% by weight, less than 3% to less than 0.01% by weight, less than 2% to less than 0.01% by weight, less than 1.0% to less than 0.01% by weight, less than 0.5% to less than 0.01% by weight, less than 0.2% to less than 0.01% by weight, less than 0.1% to less than 0.01% by weight, less than 0.05% to less than 0.01% by weight, less than 0.02% to less than 0.01% by weight.

[0117] IV.Save The freeze-dried or other dried composition is stored before use. The storage period can include the time the dried composition is exposed to ambient air and stored at room temperature. Such a period can be up to 3 hours, or alternatively, any range, such as up to 1.0 hours, up to 1.5 hours, up to 2.0 hours, up to 2.5 hours, up to 3.5 hours, up to 4.0 hours, up to 5.0 hours, up to 6.0 hours, up to 8.0 hours, or 90 to 180 minutes. The absolute humidity during such storage can be at least 2.3 grams of water per cubic meter of air at 25°C, or alternatively, greater than 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3.0 grams of water per cubic meter of air. Alternatively, the relative humidity can be, for example, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% relative humidity. In certain embodiments, the relative humidity may be about 10% and the storage time may be up to 8 hours. In certain embodiments, the absolute humidity may be about 2.3 grams per cubic meter of air at 25° C. and the storage time may be up to 8 hours.

[0118] Storage can also include longer periods during which the dry composition is sealed and substantially prevented from contact with ambient air outside the seal. This storage time can be long, for example, at least one week, at least one month, at least six months, at least one year, or at least two years. Periods of one month to two years are exemplary.

[0119] Storage temperatures for long-term storage or long-term or short-term stability studies include, 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 under liquid nitrogen, -4 to -2°C, -6 to -2°C, -8 to -2°C, -10 to -2°C, -20°C, -40°C, -60°C, and -80°C. Preferably, storage is above freezing, within the range of about 4 to 8°C. Accelerated degradation studies can be performed at about 25°C, about 30°C, about 35°C, or about 40°C for periods of, for example, 1 hour, 2 hours, 4 hours, 24 hours, 2 days, 4 days, 8 days, 1 month, etc. Conditions for storage or, alternatively, stability testing, can involve varying temperatures, e.g., from above to below freezing.

[0120] The absence of inorganic salts reduces the loss of enzyme activity and the formation of by-products during storage of the bulk reagent before drying, during short-term storage of the dry composition before sealing, and during long-term storage after sealing. Preferably, the enzyme activity after all storage periods is at least 99%, at least 98%, at least 95%, at least 90%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20% of the value immediately before storage began, and ranges bounded by these percentages relative to the value before storage began, or alternatively, the value of a control sample stored under optimal conditions.

[0121] V. Reconstruction A preferred reconstitution solution provides 3.8-4.4 mM MgCl2 and 50-80 mM KCl in water. The reconstitution solution may also contain, among other ingredients, 0.012-0.020% methylparaben, 0.006-0.010% propylparaben, and / or 0.26% absolute ethanol.

[0122] The reconstitution time is less than 1 second, less than 2 seconds, less than 5 seconds, less than 10 seconds, less than 15 seconds, less than 20 seconds, less than 50 seconds, or less than 60 seconds (1 minute) after contacting the dry composition with an aqueous solution appropriate for the dry composition's intended use, which contact can optionally be facilitated by shaking, tapping, vortexing, rocking, aspirating with a pipette tip, or collapsing or crushing a malleable vial. An exemplary reconstitution time is 2 to 10 seconds. Reconstitution time can be measured with a reconstitution solution at either refrigerator temperature (approximately 4°C), ambient temperature (approximately 23°C), or warm (approximately 37°C). Typically, the dry composition is removed from the refrigerator and is cold before the addition of the reconstitution solution. The environment (room) for either of these procedures is typically an ambient temperature of about 23°C. The time at which a substance is determined to be reconstituted can be, for example, the time at which a substance is determined 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 is a measure of complete solubilization. Alternatively, complete solubilization can be determined by optical devices, such as machines that measure light scattering.

[0123] VI. Stability of the Composition The stability of a composition is typically assessed by activity (i.e., rate or yield of amplification) or by-product formation 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 an absolute or relative measure. If relative, the baseline for comparison can be the bulk reagent mixture before drying and reconstitution or a control reconstituted mixture different from the one being tested in a specified manner (e.g., the presence of magnesium or other salts or ions). Activity can be assessed by the rate of real-time amplification or the final yield or hit rate of amplified products. By-products can be analyzed by one or more of gel electrophoresis, gel scanner, agarose gel, capillary electrophoresis, etc.

[0124] The activity of the reconstituted amplification mixture (corrected as necessary for any differences due to different volumes of reconstitution relative to the volume of reagents before drying) is preferably within 75, 80, 85, 90, or 95%, or indistinguishable within experimental error from that of the reagents before drying. By-products present in the reconstituted amplification mixture (corrected as necessary for any differences due to different volumes of reconstitution relative to the volume of reagents before drying) are preferably less than 20, 15, 10, 5, 4, 3, 2, or 1% by weight, or the average moles of the original compounds present in the bulk reagents before drying. Preferably, the by-products are below the detection limit.

[0125] VII. Kit The dry composition described above can be provided in a kit. Such a kit can contain the dry composition in a container, such as a tube. In some embodiments, the kit contains a multi-well plate containing one or more wells. Some kits contain multiple dry compositions provided in separate containers. Some kits include one or more multi-well plates containing multiple dry compositions in one or more sealed well members of the multi-well plate.

[0126] Some kits also include a reconstitution solution in a container separate from the dry composition. The reconstitution solution may be provided in bulk for dispensing aliquots into individual dry composition containers, or may be provided in the form of one or more unit doses, each for combination with a single container containing the dry composition.

[0127] Optionally, the container containing the dry composition and the container containing the reconstitution solution can be separated by a fragile material. The fragile material can be aluminum foil, polypropylene, polyester, polyvinyl chloride (PVC), polyethylene, or other similar materials. The barrier can include one, two, three, or more layers, each having the same composition, or each having a different composition, such as a foil layer in contact with a PVC layer. Films can be obtained, for example, from Dow Chemical Co., Midland, MI, or Arkema, Inc., King of Prussia, PA. Perforations in the fragile material allow the reconstitution solution to contact the freeze-dried composition.

[0128] The kit can be designed to be compatible with a thermocycler or incubator so that the enzymatic reaction occurs directly within the kit compartment, avoiding the need to transfer the composition to a different reaction vessel or container holding such a vessel.

[0129] The kits can be adapted for the introduction of user-supplied reagents into containers within the kit, e.g., via septum-piercing ports, hoses, syringes (see US2014 / 0121515 and US2014 / 0276356), or alternatively, user-supplied reagents, such as nucleic acid templates, can be mixed with the reagents of the present disclosure in user-supplied containers. One or more of the kit compartments can be supplied empty and used as a mixing chamber. [Example]

[0130] Example 1. Bulk Reagents Examples 1-3 demonstrate preparing bulk reagents, drying single unit dose volumes of bulk reagents in containers to obtain SUD dried pellets, and reconstituting the dried pellets to obtain SUD amplification and detection mixtures. Tables 1 and 2 disclose exemplary bulk reagent components for drying. The two tables also disclose exemplary single unit dose concentrations. Master Mix 1 was a 2X master mix containing 0.4 mM each of dATP, dGTP, dCTP, and dTTP, 0.8 mM dUTP, BSA, and substantially no inorganic salts. The Taq polymerase in Table 1 was in a glycerol-free Tris buffer containing cationic detergent.

[0131] 2X Master Mix 2 contains 0.4 mM dATP, dGTP, dCTP, 0.8 mM dUTP, 0.74 units / µL GoTaq® MDx Hot Start The solution contains Polymerase, Tris, non-acetylated BSA, and glycerol-free buffer, 0.48 M trehalose.

[0132] The 50X GoScript RT mix is as follows: 20 U / μL GoScript®, 8 units / μL RNasin® Plus RNase Inhibitor from Table 2, 10 U / μL GoScript®, 8 units / μL RNasin® Plus from Table 1.

[0133] GoScript® RT Custom is a concentrated solution of 160 U / μL GoScript RT, glycerol-free, and without RNase inhibitors.

[0134] Stabilizers, deamidation inhibitors, antioxidants, detergents, and surfactants can be included, such as: fatty acid esters of polyethoxylated sorbitan (e.g., polysorbate 20 or polysorbate 80), and poloxamer 188. [Table 1] [Table 2] [Table 3]

[0135] The 10X oligonucleotide mix in each of the bulk amplification reagents for Tables 1-3 contained a collection of primers and probes for carrying out the amplification and detection reactions in the examples below. Those skilled in the art will understand how to prepare primer and probe mixtures for amplification reactions (see, e.g., Innis, Michael A. et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990)). For the examples below, primers and probes were present in the bulk reagents at bulk concentrations of about 8 uM to about 12 uM.

[0136] Example 2. Freeze-drying In this example, the bulk reaction mixture is dried using a lyophilizer. 24 microliters of bulk amplification reagent, as generally described in Example 1, is added to a container and then loaded into a lyophilization chamber. For the examples herein, 24 μl represents the amount of bulk reagent used to perform amplification and detection reactions on a single sample (also referred to as a single unit dose or SUD). In this example, a multiwell plate (specifically a 12-well plate) was used for both the lyophilization reaction and the storage of the lyophilized pellets present in the wells of the multiwell plate. Each of the 12 wells of the 12-well plate received a 24 μl aliquot of the bulk reagent mixture. The reagents in each well represented a single unit dose for performing amplification and detection reactions on the target nucleic acid. The lyophilization cycle was initiated (approximately 36 hours run). After the lyophilization cycle, the 12-well plate was collected and transferred to a location where the individual containers of the 12-well plate were sealed. The containers were sealed with metal foil over the container openings. The metal foil was a low moisture permeable foil. The sealed 12-well plate is then pouched with a desiccant. The 12-well plate is directly loaded into the device for use in the amplification reaction. The dry composition in each container is reconstituted manually, or, in the case of a device that is equipped with a reconstitution solution and programmed to automate the reconstitution of the dry composition, the dry composition is reconstituted by the device. The sample is combined with the reagents, and the amplification and detection reaction is carried out.

[0137] Example 3. Reconstitution Solution. This example describes one reconstitution solution. The purpose of the reconstitution solution is to rehydrate the dried pellet in preparation for performing amplification and detection reactions on samples using the reconstituted pellet. 24 μl of reconstitution buffer was dispensed into each of 12 wells of the 12-well plate from Example 2 by pre-punching the foil cover over the well and then dispensing the buffer into the well. Table 4 discloses the reconstitution solutions used in these examples and provides both the bulk reconstitution solution concentrations and the final assay concentrations. After reconstitution of the dried material, target nucleic acid from the sample was added to the well and PCR amplification and detection reactions were performed. [Table 4]

[0138] Example 4. Adverse effects of inorganic salts on bulk reagents This example illustrates the adverse effect of inorganic salts on bulk reagents. Two bulk reagent mixtures were prepared generally according to Example 1 and Table 5. The difference between bulk reagent A and bulk reagent B in Table 5 was the presence or absence of MgCl in the reaction mixture. [Table 5]

[0139] Liquid bulk reagents A and B were each prepared on ice. Bulk reagents A and B were then each separately aliquoted into multiple wells of a multiwell plate (specifically, 12-well plates were used here). These 12-well plates, containing either 12 aliquots of bulk reagent A or 12 aliquots of bulk reagent B, were then divided into four different incubation conditions: (1) 90-minute incubation on ice, (2) 90-minute incubation at room temperature, (3) 180-minute incubation on ice, or (4) 180-minute incubation at room temperature. Thus, one 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, one 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 period.

[0140] After the incubation period, the aliquoted amplification reactions in the 12-well plate were then lyophilized to obtain a substantially dry composition, each of which represented a dry single unit dose for a triplex amplification and detection reaction to identify one or more of influenza A, influenza B, and respiratory syncytial virus B in the sample.

[0141] The dry compositions were reconstituted with a buffer containing 65 mM KCl, 0.02% and 0.01% w / v methyl and propyl parabens, respectively, and 0.33% v / v absolute ethyl alcohol. The reconstitution solution for the dry compositions made from bulk reagent A also contained 2.5 mM MgCl.

[0142] All three influenza A, influenza B, and respiratory syncytial virus B positive samples were combined into a reconstitution reaction mixture at three times their LoDs, such that all components of the reconstitution mix were at approximately 80% of their bulk reagent concentrations. These positive samples were extracted virus in negative plasma combined with transport medium and serially diluted to the desired concentrations (except for the RSVB sample serial dilution, which differed by 10-fold). As shown for Table 6, primer and probe mixes were designed to specifically detect each of the three viral targets, i.e., influenza A, influenza B, or respiratory syncytial virus B, in separate fluorescence channels despite being single-molecule reactions.

[0143] Samples were analyzed using a real-time PCR-compatible thermal cycler (ABI 7500FAST, Applied Biosystems, Carlsbad, CA). The results are shown below (Table 6, Figures 3A-C). The percent positive values in the table represent the number of samples with RFU values above the threshold as a percentage of the 12 samples tested. In designing and setting up the assay, it is preferable that the amount of virus (viral particles per assay) be sufficient to produce at least 95% positivity for the particular assay designated as the positive control.

[0144] [Table 6]

[0145] These results indicate that the bulk reagent (solution without MgCl and KCl before lyophilization) is stable at room temperature for at least 180 minutes. After reconstitution and combination with sample, the dried SUD pellet from bulk reagent A resulted in a more robust amplification and detection reaction than that produced by the dried SUD pellet from bulk reagent B. When bulk reagent B was incubated for as short a time as 90 minutes at room temperature or even on ice, followed by drying and reconstitution to produce an amplification reaction mixture, it produced a relatively lower signal and more numerous small by-products in the amplification reaction than bulk reagent A under the same conditions. Bulk reagents containing little to no inorganic salts are useful for drying to produce dry compositions containing components for amplification reactions, including polymerase enzyme components, dNTPs, and nucleic acids.

[0146] Example 5. Stability of dried pellets with and without salt This example compares the stability of single unit-dose dried pellets containing salt with single unit-dose dried pellets containing no salt (less than 5 mM inorganic salt in this example). The single unit-dose pellets were made by drying bulk reagents generally as described above. Immediately after synthesis, bulk reagent A and bulk reagent B were each aliquoted into separate multiwell reaction plates (12 wells) and dried using a lyophilizer. After lyophilization, the multiwell plates containing the dried pellets were placed in a nitrogen gas environment with a relative humidity of approximately 5%, and the multiwell plates were sealed by covering the well openings with foil. The sealed plates were placed in aluminum pouches containing desiccant, and the pouches were then sealed. The sealed pouches containing the dried pellets in the multiwell plates were stored at 4°C for 8 days.

[0147] After 8 days, the pouch-packaged multiwell plates were transferred to one of three conditions: Condition #1--Pouch-packaged multiwell plates containing dried pellets from one subset of bulk reagent A and one subset of bulk reagent B were removed from their pouches and placed in a 15°C environment with 70% relative humidity; Condition #2--Pouch-packaged multiwell plates containing dried pellets from one subset of bulk reagent A and one subset of bulk reagent B were removed from their pouches and placed in a 45°C environment with 15% relative humidity (accelerated stability); Condition #3--Pouch-packaged multiwell plates containing dried pellets from one subset of bulk reagent A and one subset of bulk reagent B were placed at 4°C (the multiwell plates were in pouches with desiccant, so humidity was 0 percent). The plates were left under these conditions for a further 30 days.

[0148] At the end of the incubation period, the dried pellets from each condition were reconstituted in buffer containing 100 mM KCl and sufficient MgCl for a final concentration of 2.5 mM and tested for influenza A target amplification and detection reactions using a real-time PCR thermal cycler (ABI PRISM 7000, Applied Biosystems, Carlsbad, CA). Briefly, influenza A targets were extracted at LOD 10^0 (+ / - 1 log) in negative pools along with matched liquid controls. Results are shown in Table 7 and Figure 1. [Table 7]

[0149] These results show that single amplification reaction dried pellets containing less than 5 mM inorganic salts have higher RFU values after storage at a number of different temperature and humidity conditions compared to single amplification reaction dried pellets containing more than 5 mM inorganic salts.

[0150] Example 6. Effect of holding time before filling cartridge with reagent Table 8 discloses results from a stability study in which all lyophilized samples were prepared from the same bulk reagent. This example describes the stability of dried pellets prepared generally as described above. Bulk reagent containing polymerase enzyme, dNTPs, and nucleic acids was prepared without MgCl2 and without KCl. The bulk reagent was divided into four separate portions of equal composition, each of which was either (1) dried after 45 minutes of incubation at room temperature, (2) dried after 4 hours of incubation at room temperature, (3) dried after 8 hours of incubation at room temperature, or (4) dried directly without incubation.

[0151] The resulting dry composition was then transferred to an aluminum pouch containing 1.5 g of desiccant pillows, and the pouch was then sealed. The sealed pouch was stored at 5°C under conditions simulating accelerated stability equivalent to 22.5 months. After the accelerated stability incubation, the dry composition was reconstituted and the resulting amplification reaction was used in an assay for amplification and detection of influenza A samples (TCID 50 / mL=1). The results in Table 8 show that there is no perturbation of amplification and detection assay performance when the bulk solution containing neither MgCl nor KCl is incubated at room temperature for up to 8 hours before drying. The reconstituted dried composition produces robust amplification and detection reactions, providing reproducible results even when tested on samples containing low viral titers. [Table 8]

[0152] This study demonstrates that varying bulk holding times for the liquid lysis reagent during container filling do not reduce enzyme activity (Figure 2). To test the stability of the bulk reagent during container filling, a study was conducted in which the bulk mix was exposed to ambient temperature for up to 8 hours before lyophilization. Separate FluA / B / RSV bulk liquid lysis reagent mixes were prepared and incubated at room temperature for 45 minutes, 4 hours, and 8 hours before being used to generate lyophilized pellets. These pellets were then pouched and exposed to 45°C for 22 days to simulate accelerated stability equivalent to a 22.5-month shelf life at 5°C before being tested at low viral titers. The results (Figure 2) demonstrate no disruption of assay performance when the liquid lysis bulk is stored at ambient conditions for up to 8 hours.

[0153] The respective RFUs for the histogram bars are 824,895, 806,570, 855,772, and 1,162,967. The Ct (time of emergence) values were as follows: for the liquid control (mean Ct = 34.3), 45 minutes (mean Ct = 34.5), 4 hours (mean Ct = 34.4), and 8 hours (mean Ct = 33.9). The respective standard deviations for the Ct values were 0.39, 0.58, 0.32, and 0.60 (Figure 2).

[0154] Example 7. Reconstitution Time Course Study This relates to the experiment shown in Table 9. Experimental setup to determine whether incubating dried pellets without KCl but with 2.5 mM MgCl in reconstitution solution affects assay activity. Briefly, dried pellet SUDs without KCl but with 2.5 mM MgCl were prepared by lyophilization of bulk reagents, generally as described above. After lyophilization, the dried pellet SUDs were sealed under nitrogen at 5% relative humidity, pouched with desiccant, and stored at 4°C for 27 days. After 27 days of incubation, the dried pellets were reconstituted and used to detect Flu-A from samples; all assays had final concentrations of 100 mM KCl and 2.0 mM MgCl in the reaction mixture.

[0155] The KCl-free / 2.5 mM MgCl2 SUD was reconstituted with 125 mM KCl and incubated on ice for 0, 5, 10, or 15 minutes before being transferred to a pre-chilled PCR plate and target added. The plate was sealed and the samples were spun for 1 minute. The plate was then transferred to a pre-heated ABI 7500 FAST instrument (Applied Biosystems, Carlsbad, CA). The assay was run on the ABI under a 54-minute thermal profile with N=4 replicates and a threshold set at 25,000. Table 9 discloses the results. [Table 9] Example 8. Effect of magnesium chloride on stabilization This example investigated the stabilization effect of removing MgCl from a bulk reagent master mix containing buffer, trehalose, EDTA, nucleotide triphosphates, primers, probes, and enzymes. Removal of MgCl from the bulk reagent was found to stabilize the formulation from 90 minutes to 7.75 hours at room temperature prior to lyophilization, as measured by activity after reconstitution with water and MgCl. The addition of EDTA to the lyophilized pellets is useful for chelating excess MgCl in the reconstitution buffer.

[0156] Primers, probes, and triphosphates can be provided in a buffered master mix, to which enzymes are added prior to the amplification reaction. For some amplification reactions, it may be desirable to have the enzymes in the final master mix and lyophilize it in a "ready-to-use" form with a universal reconstitution solution of water and MgCl2. This avoids the need to add the mix prior to the amplification reaction. Magnesium acts as a complexing agent (catalyst) for the polymerization reaction. In the absence of target, nonspecific amplification can occur, which can deplete the reaction mix of necessary triphosphates. Holding the materials at 2-8°C and loading them into a pre-cooled freeze dryer can be used to slow the reaction rate, which can slow the depletion of triphosphates from the reaction mix. However, even at 2-8°C, the stability of the reaction mix may only last 90 minutes. Also, compliance with these limitations during routine manufacturing requires the use of a cooling system to maintain the bulk solution temperature at the desired temperature, as well as a pre-cooling step.

[0157] Removal of MgCl2 from the bulk reagent containing buffer, trehalose, triphosphate, EDTA, primers, probes, and enzymes and placement in a reconstitution solution containing water and MgCl2 was considered a way to prevent or minimize nonspecific amplification, even though it acts as a catalyst for the polymerization reaction. Addition of EDTA solution to a master mix containing buffer, trehalose, triphosphate, primers, probes, and enzymes could be used to chelate excess magnesium in the universal reconstitution for each analyte. Paired amplification reactions with and without MgCl2 were performed under wet chemistry reaction conditions to assess the effect on stability. The results are shown in Figure 4. A study without magnesium in the bulk reagent containing buffer, trehalose, triphosphate, EDTA, primers, probes, and enzymes demonstrated room temperature stability for 180 minutes (see lanes 6 and 8, which show the same bands on the bioanalyzer gel compared to the nonspecific droplets in lanes 5 and 7, where MgCl2 is present). A follow-up study is shown in Figure 2, where target nucleic acid amplification was performed under three different conditions. (1) A fresh liquid control containing buffer, trehalose, triphosphate, EDTA, primers, probe, MgCl2, and enzyme, with the enzyme added immediately before initiating the PCR reaction; (2) Lyo 67 RT solution containing buffer, trehalose, triphosphate, EDTA, primers, probe, and enzyme for 3.75 hours. The solution was then lyophilized. After lyophilization, the pellet was reconstituted in water and MgCl2 immediately before initiating the PCR reaction; and (3) Lyo 67 RT solution containing buffer, trehalose, triphosphate, EDTA, primers, probe, and enzyme for 7.75 hours. The solution was then lyophilized. After lyophilization, the pellet was reconstituted in water and MgCl2 immediately before initiating the PCR reaction. This experiment demonstrated the stability of the bulk reagent containing buffer, trehalose, triphosphate, EDTA, primers, probe, and enzyme for 7.75 hours at room temperature before lyophilization without nonspecific amplification.

[0158] Removal of MgCl2 from the bulk reagents results in a room temperature stable master mix containing buffer, trehalose, triphosphate, EDTA, primers, probes, and enzymes.

[0159] Example 9 Effect of potassium chloride (KCl) on sublimation during freeze-drying. This example investigated the effect of potassium chloride (KCl) on sublimation during lyophilization. PCR requires KCl for PCR amplification. The results of this study showed that low concentrations of KCl (<10 mM, 0.391 μg / μL potassium) did not significantly affect lyophilization. Conversely, KCl concentrations as high as 126 mM (4.92 μg / μL potassium) inhibited water removal from the pellet during lyophilization. Residual water is considered an "impurity" that adversely affects stability. Eliminating or minimizing the amount of KCl in the lyophilization bulk reagent is desirable in certain embodiments.

[0160] The effectiveness of freeze-drying in the presence of three concentrations of KCl was investigated by measuring the residual moisture content after freeze-drying, as higher levels of residual moisture are known to inhibit the sublimation process.

[0161] Fourier transform near-infrared (FT-nIR) spectroscopy was used to measure the relative water content in the lyophilized bulk reagent. The water-OH bond is located at 5170 cm -1 (A 5170 ) is known to absorb energy in the nIR spatial wavelengths.

[0162] Three aqueous bulk reagent formulations were prepared, the compositions of each of which are shown in Table 10. [Table 10]

[0163] 1.45 mL of each formulation was filled into two 10 mL glass vials for each KCl concentration. The vials were partially stoppered with butyl stoppers. All vials were lyophilized together. At the end of lyophilization, the vials were sealed under anhydrous nitrogen gas. All sealed vials were removed from the lyophilizer and crimp seals were applied. The vials were then measured for residual moisture content by FT-nIR spectroscopy. To account for lag trends in moisture levels over time, measurements were taken over a 10-day period. The time points were: day 0, day 1, day 3, day 7, and day 10. Residual moisture content was measured by FT-nIR spectroscopy and was measured at 5170 cm -1 nIR absorbance parameters were obtained at 1000 kJ / µL. For each formulation, the absorbance parameters were averaged and then normalized to the average parameters obtained from the reference sample, Formulation 1 (0 µg / µL KCl). The following calculations were used:

number

[0164] Tables 11 and 12 show the FT-nIR absorbance results. [Table 11] [Table 12]

[0165] Figures 6 and 7 show the effect of increasing the concentration of KCl. The graphs show that the presence of 6.3 mM KCl did not significantly increase the normalized absorbance parameter. Thus, from these data, low concentrations of KCl did not significantly inhibit water removal during freeze-drying. Conversely, 126 mM KCl (a 20-fold increase) induced a 2-fold increase in the normalized absorbance parameter, indicating a higher residual moisture level compared to the reference. Thus, higher concentrations of KCl affect water removal.

[0166] Lyophilization of aqueous bulk reagents tolerates small amounts of KCl (approximately 6.3 mM in this example). Higher concentrations of KCl can affect the hygroscopicity of the dried pellet, causing it to absorb water from the environment and thus resulting in a less robust reaction mixture.

[0167] Example 10: Determination of post-lyophilization stability of lyophilized bulk reagents Ambient and controlled glovebox experiments using lyophilized aliquots of bulk reagents showed that minimal salt / no salt formulations exposed to 10% relative humidity for less than 8 hours resulted in acceptable assay performance. Formulations containing salt and / or exposed to higher relative humidity after lyophilization did not result in acceptable assay performance.

[0168] The purpose of this example was to determine the post-lyophilization stability of lyophilized bulk reagent sealed in multi-well plates and sealed to protect from ambient moisture (in pouches) under ambient humidity conditions and controlled 10% relative humidity conditions. In addition to the specific experiments described here, several additional studies using additional time points and formulation variations were completed.

[0169] Multiwell plates were filled with aliquots of bulk reagent while kept on ice at 2-8°C. Sufficient amounts of all necessary components were mixed to create a bulk reagent with a volume sufficient to fill 30 multiwell plates and a 5% excess. Mixing was performed according to the following instructions: nuclease-free water, 2X glycerol-free GoTaq Master Mix (formulation shown below), 1.2M trehalose, 10X probe / primer mix, and 50X Promega RT (0.5% glycerol formulation for lyophilization) were added in this order. Multiple multiwell plate wells were filled with 24 μL of bulk reagent per well within 1 h. The multiwell plate was placed on ice and pre-equilibrated at 2-8°C before filling the tubes. Liquid bulk reagent was dispensed into the bottom of each tube. The multiwell plate was kept on ice during the 1-h filling window. The filled multiwell plate was loaded into the lyophilization chamber within 30 min after filling was completed. The filled multiwell plates were then exposed to lyophilization conditions. After the lyophilization process, the multiwell plates were covered / stoppered before being removed from the lyophilization chamber. Two multiwell plates were transferred to the lab bench. The remaining multiwell plates, which served as the T=0 samples, were transferred to a glove box pre-equilibrated to 10% relative humidity and were uncovered / stoppered and then heat-sealed after various times as shown in Table 13. A 6-second sealing time and a 169°C sealing temperature were used. Within 5 minutes, the sealed multiwell plates were transferred to ziplock foil pouches containing desiccant pillows and then heat-sealed. For the other multiwell plates on the bench, they were uncovered / stoppered and exposed to ambient atmosphere for various times as shown in Table 13 below.

[0170] The relative humidity and temperature at the time of exposure, sealing, and pouching were recorded. After exposure, the multiwell plates were heat-sealed in foil stock using a 6-second sealing time and a sealing temperature of 169°C. The heat-sealing process was performed under the exposure conditions using the same sealing time / temperature parameters described above (i.e., multiwell plates exposed to laboratory atmosphere were sealed in the laboratory, and multiwell plates exposed to dehumidified conditions were sealed under dehumidified conditions). Within 5 minutes, the sealed multiwell plates were transferred to Ziploc foil pouches containing desiccant pillows, and the pouches were heat-sealed. All sealed pouches were then stored at 2-8°C.

[0171] Two multiwell plate runs were performed per time point. After lyophilization under ambient conditions, the multiwell plates were sealed for 0, 4, and 8 hours (see Table 13). After lyophilization under 10% relative humidity conditions, the multiwell plates were sealed for 0, 4, 8, and 24 hours (see Table 13). The number of conditions times two multiwell plates per time point is 2*3 (ambient=6) and 2*4 (10% relative humidity glove box=8), respectively.

[0172] A salt-free formulation was prepared using 2X GoTaq Master Mix, glycerol-free, 10X probe / primer, trehalose, and 50X RT. For example, for a 30-well plate, add 449 μL of nuclease-free water, 4.81 ml of 2X GoTaq Master Mix (B, no salt) (1.25X final), 1.28 ml of 1.2 M trehalose (0.2 M final), 963 μL of 10X probe / primer mix (1.25X final), and 193 μL of 50X RT (1.25X final) = 7.70 ml of salt-free mix. Dispense 24 μL per tube into 300 tubes using a repeat pipette.

[0173] A salt-containing formulation was prepared using 2X GoTaq Master Mix, glycerol-free, 10X probe / primer, trehalose, and 50X RT. For example, for a 38-well plate, add 560 μL of nuclease-free water, 6.00 ml of 2X GoTaq Master Mix (B, salt-containing) (1.25X final), 1.60 ml of 1.2 M trehalose (0.2 M final), 1.20 ml of 10X probe / primer mix (1.25X final), and 240 μL of 50X RT (1.25X final) = 9.60 ml of salt-containing mix. Dispense 24 μL per 380 tubes using a repeat pipette.

[0174] The multiwell plates were tested for performance according to Table 13. In Step A of Table 13, the salt-free formulation was filled into the multiwell plate and lyophilized. After lyophilization, the multiwell cartridge was sealed after exposure to the conditions for the indicated period. In Step B of Table 13, the salt-free formulation was filled into the multiwell cartridge and lyophilized. After lyophilization, the multiwell cartridge was sealed after exposure to the conditions for the indicated period. In Step C of Table 13, the salt-containing formulation was filled into the multiwell cartridge and lyophilized. After lyophilization, the multiwell cartridge was sealed after exposure to the conditions for the indicated period. [Table 13]

[0175] Ambient and glove box temperatures were approximately 25°C. The results of this and related experiments showed that exposure of salt-free formulations to 10% relative humidity for less than 8 hours resulted in acceptable assay performance. In this example, exposure to 10% relative humidity (2.3 g / m at 25°C) with minimal salt / no salt formulations was 3 ) for a period of less than 8 hours results in acceptable assay performance.

[0176] The present disclosure is not limited by the disclosed compositions, reagents, methods, diagnostics, laboratory data, etc., nor is the disclosure limited by any preferred embodiment disclosed herein. Any embodiment, step, feature, aspect, etc. can be used in combination with any other, unless otherwise apparent from the context. All references cited herein are incorporated by reference to the same extent as if each individual patent and published patent application, as well as figures, drawings, sequence listings, compact discs, etc., were specifically and individually indicated to be incorporated by reference.

Claims

1. At least one polymerase; a trehalose filler; Detergent and an organic buffer solution; at least one oligonucleotide useful for conducting a molecular assay; deoxynucleotide triphosphates (dNTPs); A chelating agent; less than 0.1 mM magnesium ions; wherein said at least one polymerase comprises a reverse transcriptase present in said aqueous solution at a concentration of from about 0.1 U / μl to about 0.6 U / μl.

2. The composition described in claim 1, wherein the at least one oligonucleotide is selected from the group consisting of an amplification oligonucleotide, a detection probe oligonucleotide optionally containing at least one label, a target capture probe oligonucleotide, an adapter oligonucleotide, and combinations thereof.

3. The composition described in claim 1 or 2, wherein the aqueous solution contains oligonucleotides for performing a multiplex molecular assay.

4. A composition described in any one of claims 1 to 3, wherein the filler is present at a concentration of about 0.16M to about 0.32M.

5. A composition described in any one of claims 1 to 4, wherein the aqueous solution contains from about 0.196 μg / μl of potassium ions to about 0.010 μg / μl of potassium ions.

6. The composition described in any one of claims 1 to 5, wherein the dNTPs include dATP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution, dGTP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution, dCTP at a concentration of 0.1 mM to 0.3 mM in the aqueous solution, dTTP at a concentration of 0.2 mM to 0.6 mM in the aqueous solution, or dUTP at a concentration of 0.2 mM to 0.6 mM in the aqueous solution.

7. The at least one polymerase comprises a polymerase that is a hot-start polymerase, and optionally: the hot-start polymerase is a recombinant Taq DNA polymerase bound by an antibody that specifically blocks the polymerase activity of the polymerase; or the hot-start polymerase is a chemically modified recombinant Taq DNA polymerase, wherein the chemical modification inhibits the polymerase activity of the polymerase; The composition according to any one of claims 1 to 6.

8. A composition described in any one of claims 1 to 7, wherein the reverse transcriptase is AMV reverse transcriptase or MMLV reverse transcriptase.

9. The aqueous solution further comprises an RNase inhibitor, and optionally: the RNase inhibitor is present in the aqueous solution at a concentration of about 0.12 U / μl to about 0.20 U / μl; The composition according to any one of claims 1 to 8.

10. The chelating agent is selected from the group consisting of EDTA, EDDS, MGDA, EGTA, and DTPA, preferably the chelating agent is EDTA and is present in the aqueous solution at a concentration of 1.5 mM to 2.0 mM; The composition according to any one of claims 1 to 9.

11. The composition described in claim 1, having an inorganic salt concentration of 7 mM or less.

12. A dry form of the composition described in any one of claims 1 to 11.

13. A method of forming a mixture for use in performing a nucleic acid-based amplification reaction, said method comprising: Combining the reconstituted solution and the dried form of the composition of claim 12. wherein the reconstituted solution comprises at least one inorganic salt, and optionally the reconstitution solution comprises MgCl 2 at a concentration of about 3.8 mM to about 4.4 mM, KCl at a concentration of about 50 mM to about 80 mM, or both; method.

14. A method for preparing a dry composition for use in performing a molecular assay, said method comprising: (i) freezing the aqueous solution of any one of claims 1 to 11, thereby forming a frozen form of the aqueous solution; (ii) exposing said frozen form from step (i) to freeze-drying conditions, thereby forming a dry composition; A method comprising:

15. A kit for use in performing a molecular assay, said kit comprising: a first container containing a dry form of the composition of claim 12; a second container containing a reconstitution solution; wherein said reconstitution solution comprises at least one inorganic salt, suitably MgCl 2 .

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