Non-aqueous compositions for storage and / or stabilization of nucleic acid and methods of use thereof

Abstract

The present application provides a non-aqueous storage composition for use in storage or stabilization of nucleic acid from or in biological samples. The non-aqueous storage composition comprises a non-aqueous solvent that comprises: dimethylsulfoxide, a glycol, glycerol or a mixture of two or more thereof; one or more surfactant; a buffer to provide a pH in the range of from about 4 to about 8; and, optionally, a chelator. Also provided herein are methods and kits for storage or stabilization of nucleic acid from or in biological samples. Further provided herein are methods and kits for concentration of nucleic acid or other analyte(s) of interest from a liquid biological sample using the non-aqueous storage composition.

Description

NON-AQUEOUS COMPOSITIONS FOR STORAGE AND / OR STABILIZATION OF NUCLEIC ACID AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 678,791, filed August 2, 2024, the contents of which is hereby incorporated by reference in its entirety.FIELD

[0002] The present application pertains generally to compositions and methods for the stabilization of nucleic acid from or in biological samples. More particularly, this application pertains to stabilization of nucleic acid for extended periods at ambient temperature using a substantially water-free or non-aqueous composition.BACKGROUND

[0003] Compositions comprising denaturing agents (e.g. detergents or surfactants), chelating agents, salts, nuclease inhibitors and buffering agents, dissolved in aqueous solvents (typically water), are commonly used to stabilize nucleic acid (DNA and RNA) in collected biological samples (U.S. Patent Nos. 7,482,116; 9,523,115; 10,619,187; 11,572,581; 10,435,735; 11,198,899; U.S. Application Nos. 17 / 995,138; 63 / 208212). It has been observed that aqueous compositions stored within off-the-shelf plastic labware, and even specialized biological sample collection devices (see, e.g., www.dnagenotek.com), tend to evaporate with time, due to their intrinsic volatility and high vapor pressure. This evaporative loss is accelerated when storage at ambient temperature exceeds room temperature (23°C±3°C), which limits the shelf-life of medical devices comprising these compositions, both before and after sample collection.

[0004] There are many strategies, processes, and common practices that can be used to mitigate evaporative mass loss of conventional aqueous solvents, including the use of a protective film barrier to prevent evaporation from biological sample collection devices, enclosing the volatile liquid in a tube with a well-sealed cap, foil packaging used as a vapor barrier to prevent evaporation, lyophilization of chemical compounds, and many others. However, such strategies used to mitigate evaporation fail to address the root cause ofsolvent volatility and evaporation still occurs, even when such preventative measures are employed or incorporated in sample collection devices for the stabilization of biomolecules. Thus, even with these measures, such sample collection devices have a limited shelf-life due to evaporation of the aqueous storage compositions.

[0005] Thus, there remains a need for a means of overcoming the failure mode of evaporative mass loss leading to the limited shelf-life of conventional solvents, such as ethanol and water, typically used in common nucleic acid stabilization reagents.

[0006] The above information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.SUMMARY

[0007] An object of the present application therefor is to provide non-aqueous compositions for storage, stabilization and / or concentration of nucleic acid, and methods of use thereof.

[0008] In accordance with one aspect of the present invention, there is provided a composition comprising: (a) a non-aqueous solvent that comprises dimethylsulfoxide, a glycol, glycerol or a mixture of two or more thereof; (b) one or more surfactant; (c) a buffer to provide a pH in the range of from about 4 to about 8, or from about 4.6 to about 5.7; and (d) optionally, a chelator.

[0009] In some embodiments, the non-aqueous solvent is a glycol having the structure of Formula II where:R1is absent or R1is a bivalent straight-chain, branched or cyclic hydrocarbon with 1 to 20, preferably 1 to 10, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'-,-CR'=CR"-, or - CY'=CY"- in such a manner that O and / or S atoms are not linked directly to one another, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L; where each R2is independently H , F , Cl , CN , or straight-chain, branched or cyclic alkyl with 1 to 20, preferably 1 to 10, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'-,-CR'=CR"-, or -CY'=CY"- in such a manner that O and / or S atoms are not linked directly to one another, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;L is F, Cl, -CN, -NC, -NCO, -NCS, -OCN, -SCN, -R', -OR', -SR', -C(=O)R'-, -C(=O)- OR'-, -NH2, -NHR', -NR'R", -SO3R', -SO2R', -OH, -NO2, -CF3, or optionally substituted silyl, carbyl or hydrocarbyl with 1 to 30 C atoms that is optionally substituted and optionally comprises one or more hetero atoms;R' and R" are each independently H or a straight-chain or branched alkyl with 1 to 6 C atoms; andY' and Y" are each independently H, F, Cl or CN.

[0010] In particular embodiments, the glycol is propylene glycol, diethylene glycol, triethylene glycol, 1,2-butanediol, N-methyl diethanolamine, 2,2-thiodiethanol or a combination thereof.

[0011] In some embodiments, the one or more surfactant in the composition comprises a surfactant compound of Formula IIII where:R3is CH, — O— , aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L; each R4is independently H or a straight-chain, branched or cyclic alkyl with 1 to 20, preferably 10 to 18, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'-, -SiR'R"-, -CR'=CR"-, -CY'=CY"- or -C=C- in such a manner that O and / or S atoms are not linked directly to one another , and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L, and wherein at least one R4is not H; each R', R", Y', Y", and L is as defined in relation to the compound of Formula I;M is an ammonium compound, H+, Li+, Na+, K+, Ca2+, or Mg2+, preferably Li+, Na+, or Mg2+; and n is an integer between 1 and 2, and n is equal to the oxidation state of M.

[0012] In some embodiments, the one or more surfactant comprises sodium dodecyl sulfate(SDS), lithium dodecyl sulfate, alkylbenzene sulfonic acid (e.g., dodecylbenzene sulfonicacid) or a combination of SDS with another surfactant, such as docusate sodium sulfate, orTween 20 or a combination of alkylbenzene sulfonic acid with another surfactant, such asTween 20.

[0013] In some embodiments, the buffer in the composition comprises a carboxylate ofFormula III or a salt thereof,where:R5is a straight-chain, branched or cyclic alkyl with 1 to 8, preferably 1 to 6, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, — C(=O)— , - C(=O)-O-, -O-C(=O)-, -C(=O)-OH, -C(=O)-OM, -NR6-, -NR'-, -CR'=CR"-, -CY'=CY"- or — C=C— in such a manner that O and / or S atoms are not linked directly to one another, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I, C(=O)-OH, C(=O)-O_, or CN, and in which one or more CH2, or CH3 groups are optionally replaced by an aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;R6is a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof; and each R', R", Y', Y", and L is as defined in relation to the compound of Formula I.

[0014] In some embodiments, the buffer in the composition comprises an acetate buffer, such as lithium acetate, magnesium acetate, tetraethyl ammonium acetate, sodium acetate, ammonium acetate, or a combination thereof, preferably, lithium acetate or magnesium acetate, or a combination thereof.

[0015] In some embodiments, the composition comprises a chelator. The chelator can comprise a compound of Formula IVFormula IV where: each R7is independently a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof; andR8is a bivalent straight-chain, branched or cyclic hydrocarbon with 1 to 12, preferably 1 to 8, C atoms, in which one or more CH2 groups are optionally replaced by -CR'R"-, -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR9-, -NR'-, -CR =CR"-, - CY'=CY"- or -C=C- in such a manner that O and / or S atoms are not linked directly to one another , and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by an aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;R9is a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof; and each R', R", Y', Y", and L is as defined in relation to the compound of Formula I.

[0016] In some embodiments, the compound of Formula IV is a compound of Formula IVawhere:where R10and R11, together with the carbon atoms to which they are attached, form a cycloalkyl, such as a cyclohexyl, as in, for example cyclohexanediaminetetraacetic acid (CDTA).

[0017] In some embodiments, the chelator comprises ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetra acetic acid (EGTA), CDTA, 2,2',2",2"'-(l,2- propanediyldinitrilo)tetraacetic acid (PDTA), l,3-diamino-2-hydroxypropane-N,N,N',N'- tetraacetic acid or hydroxyethylethylenediaminetriacetic acid (HEDTA).

[0018] In some embodiments, the composition further comprises one or more additive, such as an antioxidant, denaturing agent, antimicrobial agent, reducing agent, colorant, bittering agent, enzyme inhibitor (such as a protease, RNase inhibitor, or DNase inhibitor), emulsifying agent, defoamer or antifoaming agent, viscosity modifier, or coating additive. In some examples, the one or more additive comprises one or more denaturing agent, such as, 5-su Ifosal icy lie acid, urea, or a combination thereof.

[0019] In some embodiments, the composition comprises or consists of a liquid, gel, solid, semi-solid, slurry, film (coating), emulsion, powder, lyophilizate, cream or suspension.

[0020] In accordance with some aspects of the present application, there is provided a use of the composition as defined above for stabilizing nucleic acid (DNA, RNA or a combination thereof) in or from a biological sample.

[0021] In accordance with some aspects of the present application, there is provided a use of the composition as defined above for concentrating nucleic acid (DNA, RNA or a combination thereof) and / or another analyte of interest from a liquid biological sample.

[0022] In accordance with other aspects of the present application, there is provided a kit for stabilizing and / or concentrating nucleic acid (DNA, RNA or a combination thereof) contained in a biological sample at ambient temperature, the kit comprising: (a) a sample container having a resealable closure for receiving the biological sample; (b) the nonaqueous composition as defined herein; and (c) instructions for use. The kit optionally further includes a means to transfer the biological sample, or a portion thereof, into the sample container; and / or a mixing or homogenization means, optionally contained within the sample container.

[0023] In accordance with other aspects of the present application, there is provided a method of stabilizing nucleic acid contained in a biological sample at ambient temperature comprising the steps of: (a) obtaining a biological sample comprising nucleic acid; (b) mixing the biological sample with the non-aqueous storage composition as defined herein; and (c) storing the mixture from step (b) at ambient temperature.

[0024] In some embodiments, in the method of stabilizing nucleic acid the step (c) storage is for a time sufficient to permit phase separation of the mixture into an upper and a lower phase, and wherein the method additionally comprises collecting the phase containing concentrated nucleic acid.

[0025] In accordance with other aspects of the present application, there is provided a method of concentrating nucleic acid or other analyte of interest contained in a liquid biological sample comprising the steps of: (a) obtaining the liquid biological sample comprising nucleic acid; (b) mixing the biological sample with the composition according to any one of claims 1 to 13; (c) incubating the mixture from step (b) at an incubation temperature for an incubation time sufficient to permit phase separation of the mixture into an upper and a lower phase; and (d) collecting the phase containing the concentrated nucleic acid.

[0026] In some embodiments, the incubation temperature is from about 35°C to about - 80°C, from about 20°C to about -20°C, or from about 0°C to about -20°C, or about -20°C.

[0027] In some embodiments, the incubation time in step (c) is from about 1 minute to about 1 week, or the incubation time is at least one minute, at least 5 minutes, at least 15 minutes, at least 1 hour, at least 4 hours, at least 8 hours, at least 1 day, at least 2 days, at least 5 days or at least a week before step (d).

[0028] In some embodiments of the method of concentrating, the nucleic acid or other analyte(s) of interest is concentrated by at least 2 fold, at least 5 fold, at least 10 fold or greater than 10 fold over the concentration of the nucleic acid or other analyte(s) of interest in the biological sample.

[0029] In some embodiments, the method of concentrating additionally comprises: (i) storing the collected phase containing the concentrating nucleic acid or other analyte(s) ofinterest; (ii) extracting the nucleic acid from the collected phase; or (iii) analyzing the nucleic acid in the collected phase.

[0030] In some embodiments, the biological sample used in the storage or concentration method is a saliva sample, a buccal sample, a sputum sample, a blood sample, a plasma sample, a serum sample, a urine sample, a fecal sample, a rectal swab sample, a throat swab sample, a vaginal sample, a swab containing a biological material, a tissue sample, a skin sample, an environmental sample, a surface sample, a forensic sample, a soil sample, a sewage sample, a wastewater sample, or a water sample. Optionally, the nucleic acid is from a human, an animal, a bacterium, a virus, a parasite or plant.BRIEF DESCRIPTION OF FIGURES

[0031] For a better understanding of the application as described herein, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:

[0032] Figure 1 graphically illustrates evaporative mass loss percentage from non-aqueous storage compositions according to embodiments of the present application (see Table 1), in comparison to conventional aqueous compositions (water, aqueous ethanol) or an aqueous formulation (Oragene®) after storage at 50°C for 35 days in a closed container.

[0033] Figure 2 depicts average UV-VIS absorbance across non-aqueous storage compositions according to embodiments of the present application (n=5 per composition) following storage at room temperature (23°C±3°C) for 42 days. Full UV-VIS absorbance spectrum from 220-900 nm using the Molecular Devices SpectraMax® M2 Microplate Reader (Catalog No. 89429-532) and a Greiner Bio-One UV-Star® half area microplate.

[0034] Figure 3 graphically depicts calculated percent (%) mass loss of composition 5 (Table 7), compared to the commercial aqueous transport chemistry compositions, Aptima® urine transport medium and Cobas® PCR Media after storage at 50°C for 35 days.

[0035] Figure 4 shows a comparison of average (n=3) UV-VIS absorbance measured across composition 5 (Table 7), Aptima® urine transport medium (n=3) and Roche Cobas® PCR Media (n=3) following storage at 50°C±2°C for 35 days (T35). Data are represented as T35subtracted by the baseline TO (T35 - TO) from 220 - 900 nm, run on the Molecular Devices SpectraMax® M2 Microplate Reader using a Greiner Bio-One UV-Star® half area microplate.

[0036] Figure 5 shows the results of TapeStation™ (Agilent) DNA analysis of samples following storage, using non-aqueous storage compositions comprising various surfactants (sodium dodecyl sulfate, 4-dodecylbenzene sulfonic acid and lithium dodecyl sulfate), for 25 days at room temperature after mixing with two male (Ml, M2) and two female (Fl, F2) urine samples at a 5:1 ratio, compared to respective unstabilized urine samples at baseline TO.

[0037] Figure 6 shows the results of RNA analysis of samples following storage, using nonaqueous storage compositions comprising various surfactants (sodium dodecyl sulfate, 4- dodecylbenzene sulfonic acid and lithium dodecyl sulfate), for 25 days (T25) at room temperature (23°C±3°C) after mixing with two male (Ml, M2) and two female (Fl, F2) urine samples at a 5:1 ratio, compared to commercially available Aptima®(Hologic) urine preservative and a control composition without surfactant at baseline (TO).

[0038] Figure 7 shows the results of RNA analysis of samples following storage, using nonaqueous storage compositions comprising various mixtures of surfactants (Table 8), for 7 days at room temperature (23°C±3°C) after mixing with two male (Ml, M2) and two female (Fl, F2) urine samples at a 5:1 ratio.

[0039] Figure 8 shows the results of RNA analysis of samples following storage, using nonaqueous storage compositions comprising various mixtures of surfactants (Table 8), for 7 days at room temperature (23°C±3°C) after mixing with two male (Ml, M2) and two female (Fl, F2) urine samples at a 5:1 ratio.

[0040] Figure 9 shows the results of RNA analysis of saliva samples from 4 donors (DI, D2, D3, D4) following storage with a non-aqueous storage composition ( I D#4; Table 8), mixed with a saliva sample at a 3:1 ratio, for 5 days at room temperature (23°C±3°C), compared to each donor's respective unstabilized saliva sample at baseline (TO).

[0041] Figure 10 shows the results from RNA analysis of pooled urine samples stored in non-aqueous storage compositions containing propylene glycol, 100 mM of CDTA, 229 mM of sodium dodecyl sulfate, 50 mM of 5 sulfosalicylic acid, and various acetate buffers at a500 mM concentration. The samples were a male pooled (MP) urine sample and a female pooled (FP) urine sample, volumes of which were combined with the storage compositions at a 10:1 ratio. The RNA analysis was performed after storage at 37°C±2°C for 3 days (T3) and compared to results from a sample stored with Aptima® urine transport medium and unstabilized urine at baseline (TO).

[0042] Figure 11 shows DNA analysis of the male pooled (MP) and female pooled (FP) urine samples stored in non-aqueous storage compositions containing propylene glycol, 100 mM of CDTA, 229 mM of sodium dodecyl sulfate, 50 mM of 5-su Ifosa I icy I ic acid, and various acetate buffers at a 500 mM concentration. Volumes of the pooled samples were combined with the storage compositions at a 5:1 ratio. The DNA analysis was performed after storage at 37°C±2°C for 3 days (T3) and compared to results from a sample stored with Aptima® urine transport medium and unstabilized urine at baseline (TO).

[0043] Figure 12 shows results of RNA analysis for an effective pH range within a composition comprising of propylene glycol, 50 mM 5-su Ifosa licy lie acid, 500 mM magnesium acetate tetrahydrate, 100 mM of CDTA and 229 mM of SDS with pH adjusted using acetic acid. The composition was mixed with two male pooled (MP1, MP2) urine samples and one female pooled (FP1) urine sample at a 5:1 ratio and held for 3 days (T3) at 37°C±2°C.

[0044] Figure 13 shows results of DNA analysis for an effective pH range within a composition comprising of propylene glycol, 50 mM 5-su Ifosa licy lie acid, 500 mM magnesium acetate tetrahydrate, 100 mM of CDTA and 229 mM of SDS with pH adjusted using acetic acid. The composition was mixed with two male pooled (MP) urine samples and one female pooled (FP) urine sample at a 5:1 ratio and held for 3 days (T3) at 37°C±2°C.

[0045] Figure 14 shows results of RNA analysis for an effective pH range within a composition comprising of propylene glycol, 500 mM magnesium acetate tetrahydrate, 100 mM of HEDTA and 229 mM of SDS with pH adjusted using acetic acid across a range from about 4.33-5.40 after urine was mixed at a 10:1 ratio and stored at room temperature for 33 days; compared to an unstabilized urine sample.

[0046] Figure 15 shows results from RNA analysis for a study of an effective pH range from 4.39-5.2 across 4 donors (2 males, 2 females) within compositions comprising propyleneglycol, a range of 39-95 mM of HEDTA, 350-802 mM of magnesium acetate tetrahydrate and 50-381 mM of 4-DBS, after urine was mixed at a 5:1 ratio and stored for 3 days (T3) at 37°C±2°C; compared to each respective urine sample mixed at a 5:1 ratio with Aptima® urine transport medium.

[0047] Figure 16 shows results from DNA analysis for a study of an effective pH range from 4.39-5.2 across 4 donors (2 males, 2 females) within a composition comprising of propylene glycol and a range of 39-95 mM of HEDTA, 350-802 mM of magnesium acetate tetrahydrate and 50-381 mM of 4-DBS, after urine is mixed at a 5:1 ratio and stored for 3 days (T3) at 37°C±2°C; compared to each respective unstabilized urine sample at baseline (TO).

[0048] Figure 17 shows the results of RNA analysis of examples of non-aqueous storage compositions containing 100 mM of CDTA ( I D#l) or 100 mM HEDTA ( I D#2) or a mixture of HEDTA and CDTA at a 50 mM concentration (I D#3) within a composition comprised of propylene glycol, 500 mM of magnesium acetate tetrahydrate, 229 mM of SDS and 50 mM of 5-su Ifosa I icy I ic acid after mixing with two male pooled urine samples and a female pooled urine sample at a 5:1 ratio and stored at 37°C±2°C for 3 days.

[0049] Figure 18 shows the results of DNA analysis of examples of non-aqueous storage compositions containing 100 mM of CDTA ( I D#l) or 100 mM HEDTA ( I D#2) or a mixture of HEDTA and CDTA at a 50 mM concentration (I D#3) within a composition comprised of propylene glycol, 500 mM of magnesium acetate tetrahydrate, 229 mM of SDS and 50 mM of 5-su Ifosa I icy I ic acid after mixing with two male pooled urine samples and a female pooled urine sample at a 5:1 ratio and stored at 37°C±2°C for 3 days (T3).

[0050] Figure 19 shows the results of RNA analysis of examples of non-aqueous storage compositions containing propylene glycol, 500 mM of magnesium acetate tetrahydrate, 229 mM of SDS and with ( I D#4) or without ( I D#5) the presence of a chelator (100 mM CDTA) after mixing with two male pooled urine samples (MP1, MP2) and a female pooled urine sample (FP1) at a 5:1 ratio and stored at 37°C±2°C for 3 days.

[0051] Figure 20 shows the results of DNA analysis of examples of non-aqueous storage compositions containing propylene glycol, 500 mM of magnesium acetate tetrahydrate, 229 mM of SDS and with ( I D#4) or without ( I D#5) the presence of a chelator (100 mM CDTA)after mixing with two male pooled urine samples and a female pooled urine sample at a 5:1 ratio and stored at 37°C±2°C for 3 days.

[0052] Figure 21 shows the results of RNA analysis of examples of non-aqueous storage compositions containing propylene glycol, magnesium acetate tetrahydrate (buffer; 350 mM-632 mM), 4-dodecylbenzene sulfonic acid or 4-DBS (surfactant; 381 mM) and a pH range of about 4.88-5.12 mixed with two male (Ml, M2) urine samples and two female (Fl, F2) urine samples at a 5:1 ratio at baseline TO and after 3 days of storage at 37°C±2°C (T3), compared to Aptima® urine transport medium.

[0053] Figure 22 shows the results of DNA analysis of examples of non-aqueous storage compositions containing propylene glycol, magnesium acetate tetrahydrate (buffer; 350 mM - 632 mM), 4-dodecylbenzene sulfonic acid (surfactant; 381 mM) and a pH range of about 4.88-5.12 mixed with two male (Ml, M2) urine samples and two female (Fl, F2) urine samples at a 5:1 ratio at baseline TO and after 3 days (T3) of storage at 37°C±2°C, compared to unstabilized urine (TO).

[0054] Figure 23 shows the results of DNA analysis of examples of non-aqueous storage compositions, as described in Table 18, mixed with a male pooled (MP) or female pooled (FP) urine samples at a 1:1 ratio, and stored for 3 days at 37°C±2°C.

[0055] Figure 24 shows the results of DNA analysis of various non-aqueous solvents at baseline (TO) after mixing with a pooled urine sample from two males and two females (n=4) at a 1:1 ratio with respective non-aqueous solvents, as summarized in Table 19; compared to the positive spike-in control.

[0056] Figure 25 shows the results of RNA analysis of various non-aqueous solvents at baseline (TO) after mixing with a pooled urine sample from two males and two females (n=4) at a 1:1 ratio with respective non-aqueous solvents, as summarized in Table 19; compared to the positive spike-in control.

[0057] Figure 26 depicts an electrophoretogram showing DNA quality at baseline (TO) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3),compared to Aptima® urine transport medium at a 1:1 ratio and the positive spike-in control material (+ Ctrl).

[0058] Figure T1 depicts an electrophoretogram showing DNA quality post freeze / thaw cycling (F / T) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3), compared to Aptima® urine transport medium at a 1:1 ratio.

[0059] Figure 28 depicts an electrophoretogram showing RNA quality at baseline (TO) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3), compared to Aptima® urine transport medium at a 1:1 ratio and the positive spike-in control material (+ Ctrl).

[0060] Figure 29 depicts an electrophoretogram showing RNA quality after freeze / thaw cycling (F / T) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3), compared to Aptima® urine transport medium at a 1:1 ratio.

[0061] Figure 30 graphically depicts P-G lobin DNA Delta Ct values (F / T - TO) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3), compared to Aptima® urine transport medium at a 1:1 ratio.

[0062] Figure 31 graphically depicts GAPDH RNA Delta Ct values (F / T - TO) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3), compared to Aptima® urine transport medium at a 1:1 ratio.

[0063] Figure 32 graphically depicts S. pyogenes DNA Delta Ct values (F / T - TO) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3).

[0064] Figure 33 graphically depicts -G lobin DNA Delta Ct values (T14 - TO) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3).

[0065] Figure 34 graphically depicts GAPDH RNA Delta Ct values (T14 - TO) within composition A at a 5:1 ratio (urine to chemistry) and composition B at both a 5:1 and 10:1 ratio (urine to chemistry) across 3 males (Ml, M2, M3) and three females (Fl, F2, F3).

[0066] Figure 35 depicts an electrophoretogram showing results from RNA analysis of a non-aqueous storage (NAS) composition compared to an aqueous storage composition (Aq) at baseline (TO) and after 3 days of storage at 37°C±2°C (T3) after mixing with two male pooled urine samples and one female pooled urine sample (n=2 each pooled sample) at a 5:1 ratio.

[0067] Figure 36 depicts an electrophoretogram showing results from DNA analysis of a non-aqueous storage (NAS) composition compared to an aqueous storage composition (Aq) at baseline (TO) and after 3 days of storage at 37°C±2°C (T3) after mixing with two male pooled urine samples and one female pooled urine sample (n=2 each pooled sample) at a 5:1 ratio.

[0068] Figure 37 depicts an electrophoretogram showing results from DNA analysis of nonaqueous storage compositions (1, 2, 4) compared to equivalent aqueous solvent-based storage compositions (3, 5) containing either magnesium acetate or lithium acetate (Table 22) mixed with two male pooled and one female pooled urine sample (n=2 each pooled sample) at a 5:1 ratio, compared to respective unstabilized urine samples after storage at 37°C for 3 days.

[0069] Figure 38 depicts an electrophoretogram showing results from RNA analysis of nonaqueous solvent compositions (1, 2, 4) compared to equivalent aqueous solvent-based compositions (3, 5) containing either magnesium acetate or lithium acetate (Table 22) mixed with two male pooled and one female pooled urine sample (n=2 each pooled sample) at a 5:1 ratio, compared to respective unstabilized urine samples after storage at 37°C for 3 days.

[0070] Figure 39 graphically depicts analysis of GAPDH RNA delta Ct values (T3 at 37°C - TO) from 2 males and 2 female urine donors (n=4) mixed with non-aqueous solvent formulations from Table 23, compared to Aptima® and unstabilized urine samples at a 5:1 ratio (urine: chemistry). Nucleic acid was extracted using two commercially available kits (QiagenQIAamp® Viral RNA Mini kit Cat No. 52904; Panel A, Zymo Quick™-DNA / RNA™ Viral Magbead Kit Cat No. R2130; Panel B).

[0071] Figure 40 graphically depicts analysis of P-G lobin DNA delta Ct values (T3 at 37°C - TO) from 2 males and 2 female urine donors (n=4) mixed with non-aqueous solvent formulations from Table 23, compared to Aptima® and unstabilized urine samples at a 5:1 ratio (urine: chemistry). Nucleic acid was extracted using two commercially available kits (Qiagen QIAamp® Viral RNA Mini kit Cat No. 52904; Panel A, Zymo Quick™-DNA / RNA™ Viral Magbead Kit Cat No. R2130; Panel B).

[0072] Figure 41 depicts a comparison of Ct values (CT, NG) from rt-PCR analysis of urine samples, from female (panel A) and male (panel B) donors, co-spiked with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) and mixed with the present composition (5:1), at baseline (TO) and after storage for 90 days at -80°C.

[0073] Figure 42 depicts real-time PCR results demonstrating the stability of CT (panel A) and NG (panel B) DNA in low and medium co-spiked FV urine samples in the present composition at baseline (B) and following exposure to a freeze / thaw (F / T) temperature cycling challenge to simulate routine laboratory processing.

[0074] Figure 43 depicts real-time PCR results demonstrating the stability of CT (panel A) and NG (panel B) DNA in low and medium co-spiked FV urine samples in the present composition at baseline (B) and following exposure to a simulated 5-day "Winter" (W) or "Summer" (S) transport profile.

[0075] Figure 44 depicts real-time PCR results demonstrating the stability of CT (panel A) and NG (panel B) DNA in low and medium co-spiked FV urine samples in the present composition at baseline (B), following exposure to a simulated 5-day "Winter" (W) transport profile plus an additional 33-day storage at 0°C (W-0) or 32°C (W-32), or following exposure to a simulated 5-day "Summer" (S) transport profile plus an additional 33-day storage at 0°C (S-0) or 32°C (S-32).

[0076] Figure 45 depicts the recovery of high molecular weight DNA from vaginal swab samples collected into the present composition and stored for 6 days at room temperature.

[0077] Figure 46 depicts the recovery of visible ribosomal RNA from vaginal swab samples collected into the present composition and stored for 6 days at room temperature.DETAILED DESCRIPTION

[0078] Definitions

[0079] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0080] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0081] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is open-ended and is used herein to mean that the list following is non- exhaustive and may or may not include any other additional suitable items or method steps, for example one or more further feature(s), component(s), step(s) and / or ingredient(s), as appropriate.

[0082] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0083] Reference throughout this specification to "one embodiment," "an embodiment," "another embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment" or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics of each embodiment may be combined in any suitable manner in one or more embodiments.

[0084] The term "and / or" as used in a phrase such as "X and / or Y" herein is intended to include "X and Y", "X or Y", "X", and "Y".

[0085] The term "aliphatic" or "aliphatic group", as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "cycloaliphatic" or "cycloalkyl"), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" refers to a monocyclic C3-C8 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment or two points of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloa Iky l)a Iky I, (cycloa lkenyl)a Iky I or (cycloalky l)alkeny I.

[0086] The term "alkoxy", as used herein, refers to straight-chain or branched alkyl group bonded to an oxygen. In some embodiments, the alkoxy group includes an alkyl having 1 to about 12 carbons, or 1 to about 8 carbons, or 1 to 6 carbons. Examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy and octoxy. By way of example, the term "Ci-Cg-alkoxy" refers to an alkoxy having 1 to 6 carbon atoms, such as, but not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy, n-butoxy, 1- methylpropoxy, 2-methylpropoxy and 1,1-dimethylethoxy. "Alkoxy" is intended to embrace all structural isomeric forms of an alkoxy group. For example, as used herein, propoxy encompasses both n-propoxy and isopropoxy, etc.

[0087] The term "alkyl", as used herein, refers to a monovalent saturated hydrocarbon of 1 to about 20, 1 to about 12, or 1 to about 8 carbons, or 1 to about 6, carbon atoms in length, such as, but not limited to, methyl, ethyl, propyl and butyl. The alkyl group may be astraight-chain, a branched-chain or cyclic ("cycloalkyl"). By way of example, the term "Ci-Ce- alkyl" as used herein refers to a saturated hydrocarbon having 1 to 6 carbon atoms. "Alkyl" is intended to embrace all structural isomeric forms of an alkyl group. For example, as used herein, propyl encompasses both n-propyl and isopropyl; butyl encompasses n-butyl, secbutyl, isobutyl and tert-butyl.

[0088] The term "alkenyl", as used herein, refers to a monovalent hydrocarbon of 1 to about 12, or 1 to about 8, carbon atoms in length that contains at least one carbon-carbon double bond. The alkenyl group may be a straight-chain, a branched-chain or cyclic ("cycloalkenyl"). By way of example, the term "Cz-Cg-alkenyl" as used herein refers to a straight-chain or branched hydrocarbon having 2 to 6 carbon atoms and containing at least one carbon-carbon double bond. "Alkenyl" is intended to embrace all structural isomeric forms of an alkenyl group.

[0089] The term "alkynyl", as used herein, refers to a monovalent hydrocarbon of 1 to about 12, or 1 to about 8, carbon atoms in length that contains at least one carbon-carbon triple bond. The alkynyl group may be a straight-chain, a branched-chain or cyclic ("cycloalkynyl"). By way of example, the term "Cz-Cg-alkynyl" as used herein refers to a straight-chain or branched hydrocarbon having 2 to 6 carbon atoms and containing at least one carbon-carbon triple bond. "Alkynyl" is intended to embrace all structural isomeric forms of an alkynyl group.

[0090] The term "ammonium compound", as used herein, refers to ammonium or derivatives of ammonium, (NH4+)Y“, in which one or more of the four hydrogens bonded to nitrogen are replaced with independently selected hydrocarbyl groups.

[0091] As used herein, the term "aryl," used alone or as part of a larger moiety, such as "arylalkyl" or "aryloxy", refers to monocyclic or bicyclic ring systems in which at least one ring in the system is aromatic. In certain embodiments, the term "aryl" refers to an aromatic ring system and exemplary groups include phenyl, biphenyl, naphthyl, phenanthrenyl, anthracyl, pyrenyl, and the like. Included within the term "aryl" are "heteroaryl" groups including one or more heteroatom, such as oxygen, sulfur and / or nitrogen, in the aromatic system, for example, pyridyl, furyl, and thienyl. In some embodiments, aryl groups have from 6 to 10 carbon atoms. A "substituted aryl" includes one or more substituent.

[0092] As used herein, the term "bivalent saturated or unsaturated, straight, branched or cyclic hydrocarbon", refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched and to bivalent cycloalkylene, cycloalkenylene and cycloalkynylene groups. The term "alkylene" refers to a bivalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CHzJn-, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. The term "alkenylene" refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. The term "alkynylene" refers to a bivalent alkynyl group. A substituted alkynylene chain is a polymethylene group containing at least one triple bond in which one or more hydrogen atoms are replaced with a substituent. The term "cycloalkylene" refers to a bivalent cycloalkyl.

[0093] As used herein, the term "glycol" is used to refer to dihydric alcohols, also known as diols, in which the two hydroxy groups are on different carbon atoms, usually but not necessarily adjacent. Non-limiting examples of glycols include propylene glycol, diethylene glycol, triethylene glycol, 1,2-butanediol, 1,4-butanediol, N-methyl diethanolamine, and 2,2- thiodiethanol. Included within the term "glycol", as used herein, are substituted glycols, which include one or more substituent (e.g., OH, F , Cl , Br , I, CN, alkyl, heteroalkyl, alkynyl, aryl or heteroaryl) on the hydrocarbon portion of the glycol and / or in which one or more carbon is replaced with a heteroatom.

[0094] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)).

[0095] The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroarylalkyl," or "heteroaryloxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 n electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Exemplary heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.

[0096] The term "heterocycle" or "heterocyclic ring", as used herein refers to fourmembered to eight-membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfur and / or nitrogen. These four-membered to eight-membered rings can be saturated, fully unsaturated or partially unsaturated. Non-limiting examples of heterocyclic rings include piperidinyl, pyrrolidinyl, pyrrolyl, pyrazolyl, pyrazolidinyl, pyridinyl, pyrimidinyl, piperazinyl, indolinyl, and the like.

[0097] As described herein, compounds employed in the present compositions may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.

[0098] The term "sample" or "biological sample" as used herein will be understood to mean any specimen that potentially contains nucleic acid of interest. The term "sample" can encompass a solution, such as an aqueous solution, cell, tissue, biopsy, powder, solid, or population of one or more of the same. The sample can be a biological sample, such as saliva, sputum, buccal swab sample, serum, plasma, blood, buffy coat, pharyngeal, nasal / nasal pharyngeal or sinus swabs or secretions, throat swabs or scrapings, urine, mucous, feces, rectal swabs, lesion swabs, chyme, vomit, gastric juices, pancreatic juices, gastrointestinal juices, semen / sperm, urethral swabs and secretions, cerebral spinal fluid, products of lactation or menstruation, egg yolk, amniotic fluid, aqueous humour, vitreous humour, cervical secretions or swabs, vaginal fluid / secretions / swabs or scrapings, bone marrow samples and aspirates, pleural fluid and effusions, sweat, pus, tears, lymph, bronchial or lung lavage or aspirates, peritoneal effusions, cell cultures and cell suspensions, connective tissue, epithelium, epithelial swabs and smears, mucosal membrane, muscle tissue, placental tissue, biopsies, exudates, organ tissue, nerve tissue, hair, skin, nails,forensic samples, plants, plant extracts, algae, soil samples, environmental sample, sewage, wastewater, foodstuff, meat-processing equipment swabs or the like.

[0099] As used herein, the term "storage composition" is used interchangeable with "stabilization composition" and is intended to reference a composition that is able to stabilize nucleic acid (e.g., DNA, RNA or a combination thereof) under ambient conditions. Ambient conditions are the average conditions of an environment and encompass room temperature conditions, i.e., about 23°C±3°C, and conditions that are more extreme, such as those that may be experienced, for example, during transport of medical devices before use or biological samples following collection.

[0100] As used herein, in reference to stabilization of nucleic acid, the term "stabilize" is used to reference the ability to store a biological material such that the nucleic acid remains viable for downstream molecular analysis. In some embodiments, the nucleic acid in the biological sample stored using the present non-aqueous storage composition is "stabilized" if there is no more than 50%, 40%, 30%, 20% or 10% of the degradation of nucleic acid that would otherwise occur in an untreated biological sample stored under the same conditions.

[0101] Surprisingly, the present inventors have discovered that certain non-aqueous solvents, such as glycols, offer a unique advantage over conventional solvents, such as water and ethanol. The inventors have found that select non-aqueous solvents can be used to mitigate or overcome solvent evaporation in nucleic acid storage compositions, thus having the potential to extend shelf-life of these compositions while also providing successful stabilization of nucleic acid, and other biomolecules, in or from biological samples. The select non-aqueous solvents identified in this application are capable of solubilizing numerous functionally active ingredients, such as salts, pH buffers, surfactants / detergents, antioxidants, nuclease inhibitors and chelators that are required for the stabilization of nucleic acid in biological samples. These non-aqueous solvents are also miscible with aqueous solutions and biological samples that contain nucleic acid, such as, but not limited to, saliva, blood and urine. These non-aqueous solvents are also compatible with other biological samples containing nucleic acid, such as, but not limited to stool, skin, soil samples, and plant materials.

[0102] Unlike ethanol, which is a flammability hazard due to its low flashpoint (14°C) and is regulated under the Transportation of Dangerous Goods (TDG, UN number 1170), these select non-aqueous solvents have a high flashpoint, which reduces their flammability making them safer for transportation. These non-aqueous solvents can also circumvent or reduce evaporation and leakage during extreme transportation conditions, such as under pressure changes associated with air freight, since these solvents have a higher viscosity and lower vapor pressure, compared to aqueous solvents. Importantly, these non-aqueous solvent-based compositions demonstrate functional performance for stabilization of nucleic acid, including DNA and RNA, under ambient conditions.

[0103] Provided herein is a composition for stabilizing nucleic acid comprising (a) a solvent that is a glycol (as defined above), dimethylsulfoxide, glycerol, or a mixture of two or more thereof; (b) one or more surfactant; (c) a buffer to provide a pH in the range of from about 4 to about 6, or from about 4.6 to about 5.7; and (d) optionally, a chelator. Each of these components are described in more detail below.

[0104] The non-aqueous storage composition of the present application can be formulated as a liquid, gel, solid, semi-solid, slurry, film (e.g., coating), emulsion, powder, lyophilizate, cream or suspension. As would be readily appreciated by the skilled person, selection of the form of the composition will be dependent on one or more factors, such as, but not limited to, specific components in the composition, biological sample type, storage conditions (before and / or after mixing with biological sample) and downstream application(s).

[0105] Non-aqueous solvent

[0106] The non-aqueous nucleic acid storage compositions of the present application comprise a non-aqueous solvent. Water may also be present in the compositions, but only in relatively small amounts in comparison to nucleic acid storage / stabilization compositions currently in use. However, it is also important that the non-aqueous solvent be miscible with water since most biological samples contain water.

[0107] In accordance with some embodiments of the present application, the nucleic acid storage composition comprises at least 55 wt% non-aqueous solvent, or at least 60 wt% non-aqueous solvent, or at least 65 wt% non-aqueous solvent. In a particular, non-limitingexample, the nucleic acid storage composition comprises from about 65.0 wt% to about 67.0 wt% non-aqueous solvent.

[0108] The non-aqueous solvent for use in a nucleic acid storage composition must have a lower volatility than water in order to avoid or minimize evaporation during storage at ambient temperatures, especially room temperature, and thereby provide an improvement in shelf-life over aqueous storage compositions.

[0109] To be suitable for use in a nucleic acid storage composition the non-aqueous solvent must also be compatible with materials typically employed in medical devices and containers for storage of nucleic acid-containing biological samples (e.g., common plastics such as polystyrene). That is the solvent must not degrade or otherwise react with these materials.

[0110] Further, to be suitable for use, the non-aqueous solvent must provide dissolution of common functional components used for the stabilization and storage of nucleic acid, at their effective concentrations.

[0111] Finally, the non-aqueous solvent used in the storage composition should also be selected while considering upstream or downstream compatibility with other chemistries and processes that may be used for isolating and / or analyzing the nucleic acid in the biological sample.

[0112] The present inventors have found glycols, glycerol and DMSO meet all of the above requirements. Accordingly, the non-aqueous storage compositions of the present application comprise one or more glycols, glycerol or DMSO, or a combination thereof as the non-aqueous solvent.

[0113] In accordance with some embodiments, the non-aqueous solvent comprises one or more glycol having the structure of Formula I:I where:R1is absent or R1is a bivalent straight-chain, branched or cyclic hydrocarbon with 1 to 20, preferably 1 to 10, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'-,-CR'=CR"-, or - CY'=CY"- in such a manner that O and / or S atoms are not linked directly to one another, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L; where each R2is independently H , F , Cl , CN , or straight-chain, branched or cyclic alkyl with 1 to 20, preferably 1 to 10, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'-,-CR'=CR"-, or -CY'=CY"- in such a manner that O and / or S atoms are not linked directly to one another, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;L is F, Cl, -CN, -NC, -NCO, -NCS, -OCN, -SCN, -R', -OR', -SR', -C(=O)R'-, -C(=O)- OR-, -NH2, -NHR', -NR'R", -SO3R', -SO2R', -OH, -NO2, -CF3, or optionally substituted silyl, carbyl or hydrocarbyl with 1 to 30 C atoms that is optionally substituted and optionally comprises one or more hetero atoms;R' and R" are each independently H or a straight-chain or branched alkyl with 1 to 6 C atoms; andY' and Y" are each independently H, F, Cl or CN.

[0114] In some embodiments, the glycol is propylene glycol, diethylene glycol, triethylene glycol, 1,2-butanediol, N-methyl diethanolamine, 2,2-thiodiethanol or a combination thereof.

[0115] In some embodiments, the non-aqueous solvent is used in the nucleic acid storage composition in combination with another solvent, which may be an aqueous solvent or another organic solvent (e.g., triethylene glycol monomethyl ether), or a combination thereof. However, as noted above, the non-aqueous solvent must be present in an amount of at least 55 wt% such that the non-aqueous solvent makes up the majority of the total solvent present in the composition.

[0116] Surfactant

[0117] The non-aqueous nucleic acid storage compositions of the present application comprise at least one surfactant. In some embodiments, surfactants suitable for use in the storage compositions are those that aid in providing nucleic acid stabilization, for example, by denaturing enzymes, particularly deoxyribonucleases and ribonucleases, present in the biological samples that would breakdown DNA and / or RNA in the sample. Alternatively, or in addition, surfactants can also function to aid in dissolution or suspension of materials in the biological samples during mixture with the storage composition, for example, through their activity as surface active agents.

[0118] In some embodiments, the non-aqueous storage composition comprises one or more non-ionic, anionic, cationic, or amphoteric surfactant, or a combination of two or more thereof. In particular embodiments, the nucleic acid storage composition comprises one or more anionic surfactant alone or in combination with a non-ionic surfactant.

[0119] In some embodiments, the non-aqueous storage composition comprises one or more surfactant having the structure of Formula II:II where:R3is CH2 , -O-, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ringatoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L; each R4is independently H or a straight-chain, branched or cyclic alkyl with 1 to 20, preferably 10 to 18, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'-, -SiR'R"-, -CR'=CR"-, -CY'=CY"- or -C=C- in such a manner that O and / or S atoms are not linked directly to one another , and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L, and wherein at least one R4is not H;L is F, Cl, -CN, -NC, -NCO, -NCS, -OCN, -SCN, -R', -OR', -SR', -C(=O)R'-, -C(=O)- OR-, -NH2, -NHR', -NR'R", -SO3R', -SO2R', -OH, -NO2, -CF3, or optionally substituted silyl, carbyl or hydrocarbyl with 1 to 30 C atoms that is optionally substituted and optionally comprises one or more hetero atoms;R' and R" are each independently H or a straight-chain or branched alkyl with 1 to 6 C atoms;Y' and Y" are each independently H, F, Cl or CN;M is an ammonium compound H+, Li+, Na+, K+, Ca2+, or Mg2+, preferably Li+, Na+, or Mg2+; and n is an integer between 1 and 2, and n is equal to the oxidation state of M.

[0120] In some embodiments, the non-aqueous storage composition comprises one or more surfactant that is sodium dodecyl sulfate (SDS), alkylbenzene sulfonic acid (e.g., dodecylbenzene sulfonic acid), lithium dodecyl sulfate. As used herein, reference to an "alkylbenzene sulfonic acid" includes reference to an alkylbenzene sulfonic acid of a particular alkyl chain and to blends of alkylbenzene sulfonic acids having varying alkyl chains. Commercially available alkylbenzene sulfonic acid is often provided as a blend oflinear and / or branched alkylbenzene sulfonic acids having a range of alkyl chain lengths (e.g., C10-C18). In some embodiments, the alkylbenzene sulfonic acid is dodecylbenzene sulfonic acid.

[0121] In some embodiments, the non-aqueous storage composition comprises a combination of SDS with another surfactant, such as, docusate sodium sulfate, or Tween 20. In other embodiments, the non-aqueous storage composition comprises a combination of alkylbenzene sulfonic acid (e.g., dodecylbenzene sulfonic acid) with another surfactant, such as Tween 20.

[0122] Buffer

[0123] The non-aqueous storage composition of the present application has a slightly acidic pH. In certain embodiments, the pH is in the range of from about 4 to about 8. The pH is maintained close to neutral or as slightly acidic, depending on the nucleic acid to be stabilized, by incorporation of a buffer. In addition, in some embodiments, the pH of the composition can be adjusted to the appropriate pH using a strong acid or base.

[0124] In embodiments in which the nucleic acid to be stabilized is DNA, the non-aqueous storage composition can have a pH within the broad range of from about 4 to about 8. In embodiments in which the nucleic acid to be stabilized is RNA, the non-aqueous storage composition can have a pH within the range of from about 4 to about 6. In embodiments in which the nucleic acid to be stabilized includes both DNA and RNA, the non-aqueous storage composition can have a pH within the range of from about 4.3 to about 5.7, or from about 4.5 to about 5.7, or from about 4.5 to about 5.2.

[0125] In some embodiments, the buffer is a carboxylate of Formula III or a salt thereof,III where:R5is a straight-chain, branched or cyclic alkyl with 1 to 8, preferably 1 to 6, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, — C(=O)— , -C(=0)-0- -0-C(=0)-, -C(=O)-OH, -C(=O)-OM, -NR6- -NR'- -CR'=CR"-, -CY'=CY"- or — C=C— in such a manner that O and / or S atoms are not linked directly to one another, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I, -C(=O)-OH, — C(=O)— O , or CN, and in which one or more CH2, or CH3 groups are optionally replaced by an aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;R6is a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof;L is F, Cl, -CN, -NC, -NCO, -NCS, -OCN, -SCN, -R', -OR', -SR', -C(=O)R'-, -C(=O)- OR-, -NH2, -NHR', -NR'R", -SO3R', -SO2R', -OH, -NO2, -CF3, or optionally substituted silyl, carbyl or hydrocarbyl with 1 to 30 C atoms that is optionally substituted and optionally comprises one or more hetero atoms;R' and R" are each independently H or a straight-chain or branched alkyl with 1 to 6 C atoms; andY' and Y" are each independently H, F, Cl or CN.

[0126] In some embodiments, the buffer is an acetate buffer, where R5is CH3. Examples of suitable acetate buffers include, but are not limited to, lithium acetate, magnesium acetate, tetraethyl ammonium acetate, sodium acetate, ammonium acetate, or a combination thereof. In specific embodiments, the buffer is lithium acetate, magnesium acetate, or a combination thereof.

[0127] Chelators

[0128] The non-aqueous storage compositions of the present application optionally comprise one or more chelator, or chelating agent. The term "chelator" or "chelating agent", as used herein, will be understood to mean a chemical that will form a soluble, stable complex with certain metal ions (e.g., Ca2+and Mg2+), sequestering the ions so that they cannot normally react with other components, such as deoxyribonucleases (DNases) or ribonucleases (RNAses) or endonucleases (e.g. type I, II and III restriction endonucleases)and exonucleases (e.g. 3' to 5' exonuclease), enzymes which are abundant in various biological samples. In some embodiments, when present, chelating agent(s) may participate in the inhibition of nucleases in biological samples.

[0129] In some embodiments, the chelator functions to contribute to buffering capacity of the composition.

[0130] In some embodiments, the storage composition comprises one or more chelator which is a compound of Formula IV:where: each R7is independently a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof; andR8is a bivalent straight-chain, branched or cyclic hydrocarbon with 1 to 12, preferably 1 to 8, C atoms, in which one or more CH2 groups are optionally replaced by -CR'R"-, -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR9-, -NR'-, -CR=CR"-, - CY'=CY"- or -C=C- in such a manner that O and / or S atoms are not linked directly to one another , and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by an aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;R9is a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof;L is F, Cl, -CN, -NC, -NCO, -NCS, -OCN, -SCN, -R', -OR', -SR', -C(=O)R'-, -C(=O)- OR-, -NH2, -NHR', -NR'R", -SO3R', -SO2R', -OH, -NO2, -CF3, or optionally substituted silyl, carbyl or hydrocarbyl with 1 to 30 C atoms that is optionally substituted and optionally comprises one or more hetero atoms;R' and R" are each independently H or a straight-chain or branched alkyl with 1 to 6 C atoms; andY' and Y" are each independently H, F, Cl or CN.

[0131] In some embodiments, the chelator is a compound of Formula IVaIVa where R10and R11, together with the carbon atoms to which they are attached, form a cycloalkyl, such as a cyclohexyl. An example of such a chelator is cyclohexanediaminetetraacetic acid (CDTA).

[0132] In some embodiments, the chelator can be, for example, ethylene glycol tetraacetic acid (EGTA), (2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), nitrilotriacetic acid (NTA), ethylenediaminetriacetic acid (EDTA), 1 ,2-cyclohexanediaminetetraacetic acid (CDTA), N,N-bis(carboxymethyl)glycine, triethylenetetraamine (TETA), tetraazacyclododecanetetraacetic acid (DOTA), desferioximine, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, and lithium citrate. These chelating agents may be used in the present non-aqueous nucleic acid storage compositions singly or in combination of two or more thereof.

[0133] Additives

[0134] The non-aqueous nucleic acid storage composition ingredients described above are sufficient to provide stabilization of nucleic acid from a wide range of biological samples and under ambient conditions. However, in some embodiments the non-aqueous storagecomposition can further comprise one or more additives, for example, to optimize the composition for particular sample types, storage conditions, composition forms and / or downstream applications.

[0135] As used herein the term "additive" is used to refer to one or more additional constituents added to the storage composition before, during or after mixture with a biological sample.

[0136] Suitable additives can be selected from, for example, antioxidants, denaturing agents, antimicrobial agents, reducing agents, colorants, bittering agents, enzyme inhibitors (such as proteases, RNase inhibitors, and DNase inhibitors), emulsifying agents, defoamers or antifoaming agents, viscosity modifiers, coating additives.

[0137] The term "antifoaming agent" or "defoamer" as used herein will be understood to mean a chemical additive that reduces or hinders the formation of foam.

[0138] The term "antimicrobial agent" as used herein will be understood to mean a substance or group of substances which reduces the rate of growth of an organism, compared to the rate of growth of the organism in their absence. A reduction in the rate of growth of an organism may be by at least 5%, more desirably, by at least 10%, even more desirably, by at least 20%, 50%, or 75%, and most desirably, by 90% or more. The definition also extends to substances which affect the viability, virulence, or pathogenicity of an organism. An antimicrobial agent can be natural (e.g., derived from bacteria), synthetic, or recombinant. An antimicrobial agent can be bacteriostatic, bactericidal, or both. An antimicrobial agent is bacteriostatic if it inhibits cell division, without affecting the viability of the inhibited cell. An antimicrobial agent is bactericidal if it causes cell death. Cell death is commonly detected by the absence of cell growth in liquid growth medium (e.g., absence of turbidity) or on a solid surface (e.g., absence of colony formation on agar). Those of skill in the art know that a substance or group of substances which is bacteriostatic at a given concentration may be bactericidal at a higher concentration. Common antimicrobial agents known in the art, include certain alcohols, Triclosan or Irgasan, and ProCiin™ 950.Optionally, the present storage composition may include an antimicrobial agent such as Triclosan.

[0139] The term "antioxidant" is used herein in its broadest sense to refer compounds that inhibit oxidation and thereby reduce damage or degradation associated with oxidation during storage of nucleic acid. Non-limiting examples include phenols such as 3,4,5- trimethoxyphenyl and 3,5-di-t-butyl-4-hydroxyphenyl, indole amines such as melatonin and flavonoids; each of which can be used in the present storage composition singly or in combination.

[0140] The term "reducing agent" is used herein in its broadest sense to refer to substances that donate electrons and in this way reduce other substances.

[0141] The term "denaturing agent" is used herein in its broadest sense to refers to a compound that can cause denaturation of proteins or other biological compounds. Nonlimiting examples include 5-su Ifosa I icy I ic acid and urea; each of which can be used in the present storage composition singly or in combination.

[0142] RNase inhibitors and DNase inhibitors can be used as additives in the present storage composition to further stabilize RNA and DNA, respectively, present in the biological sample by inhibiting the activity of RNase and DNase enzymes, respectively, already present in the biological sample or inadvertently introduced into the mixture of the storage composition and biological sample.

[0143] The term "colorant" as used herein is used to broadly reference a substance that imparts a colour to the storage composition. Colorants include a variety of dyes, organic and inorganic pigments. Examples of dyes include azo, indigoid, triphenylmethane, anthraquinone and xanthine dyes, called D&C and FD&C blue, brown, green, orange, red, yellow, etc. Organic pigments generally consist of insoluble metal salts of legal dye additives, referred to as lakes, particularly lakes of D&C and FD&C dyes; and carbon black. Inorganic pigments include iron oxide, navy blue, chromium, chromium hydroxide pigments, and mixtures thereof.

[0144] The term "bittering agent" is used herein to reference a substance that imparts a bitter taste or smell, which may be useful, for example, to make the present storage composition unpalatable so as to deter a user from ingesting the composition.

[0145] The term "emulsifying agent" is used herein to reference a substance that acts as a stabilizer for emulsions, preventing liquid emulsions from separating into oil and water phases. Non-limiting examples of emulsifying agents include, lecithin, sodium phosphates, sodium stearoyl lactylate, soy lecithin, Pickering stabilization, and DATEM (diacetyl tartaric acid ester of monoglyceride).

[0146] The term "viscosity modifier" as used herein refers to a polymer or other chemical moiety that increases the viscosity of a fluid. Examples of viscosity modifiers include chitosan, cellulosic derivatives such as methyl cellulose, gelatin, N-isopropylacrylamide polymers, poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) polymers and poly(ethylene glycol)-biodegradable polyester copolymers, alginic acid, hyaluronic acid, acacia (gum Arabic) carbomer, cetostearyl alcohol, particulates such as silica, and the like, or a combination of viscosity modifiers.

[0147] The term "coating additive" as used herein refers to a substance that facilitates or improves the formation and / or inherent properties of a film formed from the non-aqueous storage composition of the present application.

[0148] Method for Storage and Stabilization of Nucleic acid

[0149] Also provided herein is a method of stabilizing nucleic acid contained in a biological sample at ambient temperature comprising the steps of: a) obtaining a biological sample; b) contacting the biological sample with a non-aqueous storage composition, as described above, to form a mixture; and c) storing the mixture at ambient temperature.

[0150] In one embodiment of this method for storage of nucleic acid, the ambient temperature is from about 15°C to about 26°C, or is room temperature, which is about 23°C±3°C. In another embodiment, ambient temperature is between about -20°C to about 50°C, for example, about -20°C, about 37°C or about 50°C, which are temperature conditions that can be encountered in the field, such as during collection or transport.

[0151] In some embodiments, the biological sample is as defined above. For example, the sample may be a saliva sample, a buccal sample, a sputum sample, a blood sample, a plasma sample, a urine sample, a seminal fluid sample, a fecal sample, a skin sample, a soil sample, a sewage sample, an environmental sample (e.g., from an insect or plant), a wastewatersample, or a water sample. In another embodiment, the biological sample is a urine sample obtained from a mammal, such as a human. In another embodiment, the biological sample is a saliva sample obtained from a mammal, such as a human. In another embodiment, the biological sample is a feces sample obtained from a mammal, such as a human. In some embodiments, the biological sample is obtained from a swab, such as a surface swab, a buccal swab, a throat swab, a nasopharyngeal swab, a rectal swab, a vaginal swab, a skin swab, etc.

[0152] In one embodiment, in which the biological sample is saliva and the saliva sample is optionally collected using a device such as, for example, those described in WO 2007 / 068094 entitled "CONTAINER SYSTEM FOR RELEASABLY STORING A SUBSTANCE", WO 2010 / 020043 entitled "SAMPLE RECEIVING DEVICE", and WO 2010 / 130055 entitled "CLOSURE, CONTAINING APPARATUS, AND METHOD OF USING SAME".

[0153] In another embodiment, in which the biological sample is urine, and the urine sample is optionally collected using a device such as, for example, those described in WO 2014 / 037152 entitled 'LIQUID SAMPLER, KIT OF PARTS, AND METHOD FOR ASSEMBLY", WO 2021 / 069454 entitled "SMALL VOLUME LIQUID SAMPLER", and WO 2023 / 227748 entitled "LIQUID SAMPLER FOR FAST CAPTURE OF INITIAL VOLUME OF A LIQUID FLOW".

[0154] In another embodiment, in which the biological sample is a fecal sample, and the fecal sample is optionally collected using a device such as that described in WO 2015 / 172250 entitled "DEVICE FOR COLLECTING, TRANSPORTING AND STORING BIOMOLECULES FROM A BIOLOGICAL SAMPLE".

[0155] In still another embodiment, the biological sample can be collected in a standard, commercially-available laboratory or transport tube (e.g., 10 ml round- bottom tube (92 x 15.3 mm), Cat. No. 60.610; Sarstedt, or larger tube depending on the sample type and size). The tube containing the biological sample and non-aqueous composition can be sealed with an appropriate cap, and the combined sample and non-aqueous composition can be gently mixed, for example by inverting the tube.

[0156] The biological sample should preferably be mixed immediately with the non-aqueous composition at the point of collection.

[0157] As the skilled worker will appreciate, the non-aqueous storage composition described herein can be combined with the biological sample in a variety of ratios. Samples can be mixed with the non-aqueous storage composition at a ratio of 1 :1 to 1 :10 (vol / vol). The appropriate ratio can vary depending on the sample type. In some embodiments, in which the sample is a non-liquid sample (e.g., swab, feces, tissue), the sample, or a portion thereof, is added directly to the non-aqueous storage composition as is, without prior mixture or dissolution in an intermediate composition. In other embodiments, the nonliquid sample is dissolved in or mixed with an intermediate composition prior to combination with the non-aqueous storage composition. In such embodiments, the ratio of combination will be dependent on the amount of intermediate composition used.

[0158] In some embodiments of the present storage method, the nucleic acid contained in the biological sample are deoxyribonucleic acid (DNA). In another embodiment, the nucleic acid contained in the biological sample are ribonucleic acid (RNA). In yet another embodiment, the method and composition of the present application are suitable for stabilization of both DNA and RNA contained in a biological sample.

[0159] In some embodiments, the present method renders the nucleic acid stable for at least 7 days at a temperature of from about 2°C to about 30°C, or about 15°C to about 26°C, or for at least 14 days at a temperature of from about 2°C to about 30°C, or about 15°C to about 26°C, or for at least 25 days at a temperature from about 2°C to about 30°C, or about 15°C to 26°C, or for at least 33 days at a temperature from about 2°C to about 30°C, or about 15°C to 26°C, or under freeze thaw cycling conditions at a temperature from about - 20°C to about 40°C.

[0160] Methods of assessing stabilization of nucleic acid are well known to the skilled worker and / or are outlined in further detail in the Examples described below.

[0161] Method for Storage, Stabilization and Concentration of Nucleic acid

[0162] Typically, large volume liquid biological samples, which typically contain low concentrations of nucleic acid, have not been considered an ideal source of nucleic acid, and especially cell-free or circulating cell-free nucleic acid. In particular, such biological samples typically have a low abundance / concentration of target nucleic acid such that, for certain applications (e.g., diagnostics, clinical monitoring, treatment response monitoring,molecular biology, etc.), there is a particular and critical need for processing methods that can increase the concentration of target nucleic acid or other target analyte. For large volume nucleic acid-containing liquid samples or liquid biological samples with low concentrations of nucleic acid (or other analyte) such methods can facilitate the detection and analysis of the nucleic acid (or other analyte) in downstream applications. Specifically, such methods can lead to an improved signal on a biochemical or molecular assay.

[0163] A variety of purification strategies have been used in the past for the separation of nucleic acid from urine. These include precipitation, aqueous two-phase separation, and adsorption using anion-exchange columns. While these methods may be useful for processing small volumes of urine, they are especially cumbersome and labor intensive when greater volumes per individual sample are needed to be processed. Furthermore, these methods are performed separately from storage or stabilization of nucleic acid.

[0164] Accordingly, also provided herein is a method of concentrating, and optionally storing, nucleic acid or other analytes contained in a biological sample comprising the steps of: a) obtaining a liquid biological sample comprising nucleic acid; b) contacting the liquid biological sample with a non-aqueous storage composition, as described herein, to form a mixture; c) incubating the mixture at an incubation temperature for an incubation time sufficient to permit phase separation of the mixture into an upper and a lower phase; and d) collecting the phase containing the concentrated nucleic acid / analyte(s).

[0165] In some embodiments, the phase containing the concentrated nucleic acid / analyte(s) is the lower phase.

[0166] In some embodiments, the method comprising storing the mixture prior to collection of the phase containing the concentrated nucleic acid. In other embodiments, the method comprises storing the collected nucleic acid-containing phase.

[0167] Phase separation of the mixture will initiate at the cloud point, which is the temperature at which a solution, or binary or multicomponent mixture, becomes turbid due to phase separation. At the cloud point, the phase separation can be a liquid-liquid phaseseparation, to form an emulsion, or a liquid-solid phase transition to form either a stable sol (colloid) or a suspension. The emulsion will partition into separate layers over time and the sol or suspension will result in the formation of a precipitate over time.

[0168] Cloud point temperature and time required for phase separation will vary depending on the components in the non-aqueous storage composition and / or the biological sample. For example, the total ion concentration, pH and / or presence of impurities in the mixture can influence the cloud point and / or time required for phase separation. Use of the nonaqueous storage composition of the present application permits tailoring of the phase separation for concentration of nucleic acid / analytes from the biological sample.

[0169] In some embodiments, phase separation will occur spontaneously upon mixing, for example when the cloud point is at or above the temperature at which the biological sample is contacted with the non-aqueous storage composition and mixed. In this embodiment, the storage temperature can be maintained at ambient temperature, or it can be adjusted. In other embodiments, the cloud point is below the temperature at which the biological sample is contacted with the non-aqueous storage composition and mixed. In this embodiment, the temperature is adjusted to an appropriate storage temperature that is at or below the cloud point, to facilitate phase separation and concentration of nucleic acid / analytes.

[0170] The present method for concentrating nucleic acid employs a cloud point extraction, also known as micelle-mediated extraction or coacervative extraction, which is a phase partitioning extraction technique that exploits the phase separation of surfactants from aqueous solutions upon reaching their cloud point. This results in the formation of a surfactant-rich phase (coacervate) that concentrates target analytes (e.g., nucleic acid), facilitating their separation and analysis. Depending on the components of the biological sample and of the non-aqueous storage composition, this surfactant-rich phase can be either the upper or lower phase.

[0171] In one embodiment of this method for concentration, and optional storage, of nucleic acid, the incubation temperature is from about 35°C to about -80°C, from about 20°C to about -20°C, or from about 0°C to about -20°C, or about -20°C.

[0172] In one embodiment of this method for concentration, and optional storage, of nucleic acid, the mixture is incubated at the incubation temperature for an incubation time of from about 1 minute to about 1 week prior to collection. In some embodiments, the incubation time is at least one minute, at least 5 minutes, at least 15 minutes, at least 1 hour, at least 4 hours, at least 8 hours, at least 1 day, at least 2 days, at least 5 days or at least a week. The incubation time can be selected to permit both phase separation and partitioning of the nucleic acid and / or analyte(s) of interest into one of the two phases.

[0173] In one embodiment of this method for concentration, and optional storage, of nucleic acid, the ambient temperature for obtaining the biological sample or storing the nucleic acid-containing mixture or phase (layer) is from about 15°C to about 26°C, or is room temperature, which is about 23°C±3°C. In another embodiment, ambient temperature is between about -20°C to about 50°C, for example, about -20°C, about 37°C or about 50°C, which are temperature conditions that can be encountered in the field, such as during collection or transport, or during storage in the laboratory.

[0174] In some embodiments, large volume biological samples or biological samples with low concentrations of nucleic acid is as defined above and may be a plasma sample, a urine sample, a sewage sample, a wastewater sample, or a water sample. In some embodiments, the biological sample is a urine sample obtained from a mammal, such as a human. In some embodiments, the biological sample is obtained from extraction of a swab, such as a surface swab, a buccal swab, a throat swab, a tongue swab or scrapping, a nasopharyngeal swab, a rectal swab, a vaginal swab, a skin swab, etc.

[0175] In some embodiments, in which the biological sample is urine, and the urine sample is optionally collected using a device such as, for example, those described in WO 2014 / 037152 entitled 'LIQUID SAMPLER, KIT OF PARTS, AND METHOD FOR ASSEMBLY", WO 2021 / 069454 entitled "SMALL VOLUME LIQUID SAMPLER", and WO 2023 / 227748 entitled "LIQUID SAMPLER FOR FAST CAPTURE OF INITIAL VOLUME OF A LIQUID FLOW".

[0176] In still another embodiment, the biological sample can be collected in a standard, commercially-available laboratory or transport tube (e.g., 10 mL round-bottom tube (92 x 15.3 mm), Cat. No. 60.610; Sarstedt, or larger tube depending on the sample type and size). The tube containing the biological sample and non-aqueous composition can be sealed withan appropriate cap, and the combined sample and non-aqueous composition can be gently mixed, for example by inverting the tube.

[0177] The biological sample should preferably be mixed immediately with the non-aqueous composition at the point of collection.

[0178] As the skilled worker will appreciate, the non-aqueous storage composition described herein can be combined with the biological sample in a variety of ratios. Samples can be mixed with the non-aqueous storage composition at a ratio of 1 :1 to 1 :10 (vol / vol). The appropriate ratio can vary depending on the sample type. In other embodiments, a nonliquid sample is dissolved in or mixed with an intermediate composition prior to combination with the non-aqueous storage composition. In such embodiments, the ratio of combination will be dependent on the amount of intermediate composition used.

[0179] In some embodiments of the present storage and concentration method, the nucleic acid contained in the biological sample is deoxyribonucleic acid (DNA). In another embodiment, the nucleic acid contained in the biological sample is ribonucleic acid (RNA). In yet another embodiment, the method and composition of the present application are suitable for stabilization of both DNA and RNA contained in a biological sample.

[0180] In some embodiments, the present method renders the nucleic acid stable for at least 7 days at a temperature of from about 2°C to about 30°C, or about 15°C to about 26°C, or for at least 14 days at a temperature of from about 2°C to about 30°C, or about 15°C to about 26°C, or for at least 25 days at a temperature from about 2°C to about 30°C, or about 15°C to 26°C, or for at least 33 days at a temperature from about 2°C to about 30°C, or about 15°C to 26°C, or under freeze thaw cycling conditions at a temperature from about -20°C to about 40°C. The stored composition can be either the full composition or it can be the collected, nucleic acid-containing phase. In either embodiment, the nucleic acid is stabilized.

[0181] Methods of assessing stabilization of nucleic acid are well known to the skilled worker and / or are outlined in further detail in the Examples described below.

[0182] In some embodiments, the present method provides at least a 2 fold concentration, or at least a 5 fold concentration or at least a 10 fold concentration of the biological sample,with the nucleic acid and / or other analyte(s) of interest being distributed to the smaller volume phase (coacervate). In some embodiments, the present method can provide concentration of the nucleic acid and / or other analyte(s) of interest of greater than 10 fold following distribution into the smaller volume phase (coacervate).

[0183] Methods of assessing concentration of nucleic acid are well known to the skilled worker and / or are outlined in further detail in the Examples described below.

[0184] Furthermore, since the nucleic acid and / or analyte(s) is concentrated in the coacervate containing the surfactant of the non-aqueous storage composition, stability of the nucleic acid can be further improved. In particular, partitioning of the nucleic acid in the coacervate means that it is concentrated together with components of the non-aqueous storage composition that function to support nucleic acid stability. This is entirely unlike previously known methods for concentrating nucleic acid.

[0185] In addition to providing concentration of the nucleic acid, the present method partitions the nucleic acid from the biological sample in a phase separate from water- soluble inhibitor compounds. As used herein, the term "inhibitor compounds" is intended to refer to compounds that inhibit or have a negative effect on downstream applications, such as PCR or other detection / quantification / amplification methods.

[0186] In some embodiments, the method comprises isolating the smaller volume, , concentrated nucleic acid-containing layer from the larger volume layer, and storing the concentrated nucleic acid-containing layer. Such a method can be particularly useful in situations, such as biobanking, where there is a need to minimize the size of stored samples in order to reduce overall space requirements.

[0187] In some embodiments, following collection of the concentrated nucleic acidcontaining layer, the nucleic acid is extracted from the nucleic acid-containing layer. In other embodiments, the nucleic acid-containing layer is used directly in a downstream detection or quantification application.

[0188] Kit

[0189] Methods for storing and stabilizing nucleic acid and for storing, stabilizing and concentrating nucleic acid using the non-aqueous storage composition described herein areconveniently practiced by providing the non-aqueous storage composition used in the form of a kit. Such a kit preferably contains a sample container for collecting the biological sample. The sample container has a resealable closure to facilitate collection and secure storage of the sample. The sample container may be included in the kit with an appropriate amount of the non-aqueous storage composition. Alternatively, the kit comprises a separate container including the non-aqueous storage composition to be added to the sample container in an appropriate amount.

[0190] The kit also includes instructions for use based on the storage method or storage and concentration method described herein.

[0191] In some embodiments, the kit further comprises a means to transfer the biological sample, or a portion thereof, into the sample container; and / or a mixing or homogenization means, optionally contained within the sample container.

[0192] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.EXAMPLES

[0193] Summary of Examples:1) Evaluation of Evaporation of Biological Sample Preservatives and Solvents;2) Solvent solubility evaluation and feasibility for the collection of biological specimens;3) Composition Stability at Room Temperature;4) Surfactant Evaluation in Non-Aqueous Storage Compositions;5) Acetate Buffer Evaluation in Non-Aqueous Storage Compositions;6) Effective pH Range in Non-Aqueous Storage Compositions;7) Effect of Chelators on Nucleic Acid Stability in Non-Aqueous Storage Compositions;8) Contribution of Non-Aqueous Solvent on Nucleic Acid Stabilization;9) Non-aqueous storage composition comprising 4-DBS and Tween 20 under freeze / thaw cycling or ambient storage at room temperature; and10) Specific Ion Interaction in Non-aqueous Solvent Formulations.11) Storage of Urine Samples Containing Bacterial Nucleic Acid12) Storage of Nucleic Acid from Urine Samples Under Freeze / Thaw Cycle and Extreme Conditions13) Concentration of Nucleic Acid from Urine Samples14) Stabilization of Nucleic acid from Vaginal Swab Samples

[0194] EXAMPLE 1: Evaluation of Evaporation of Biological Sample Preservatives and Solvents

[0195] The purpose of this Example was to investigate and quantify the evaporative mass loss (%) of compositions of the present invention, compared to conventional aqueous solvents (e.g., water, ethanol) and commercial aqueous chemistry formulations or preservatives, such as Oragene® in saliva collection kits (DNA Genotek, Inc.). Evaporative loss of each solvent or chemistry was monitored using screw cap transport tubes (Axygen; Catalogue No. 14-222-651) stored in a monitored oven set to 50°C±2°C.

[0196] These tubes were weighed with a Mettler Toledo™ precision balance for their total mass (g), before 2 mL of the present compositions (Table 1), conventional aqueous solvents (water and 23% aqueous ethanol) or Oragene® were dispensed into the bottom of each tube. These tubes were re-capped and weighed again to get the total mass after the compositions were dispensed. These capped tubes were placed into a Binder BD730 Oven at 50°C±2°C and monitored for evaporative mass loss (g) after storage for up to 35 days. The temperature of 50°C±2°C was employed to accelerate the anticipated effect of longer-term storage at ambient temperatures in lab or clinical settings, which is typically ambient room temperature (between about 20°C to about 26°C), and during more extreme ambient conditions that may be experienced, for example during transport of medical devices before use or biological samples following collection.

[0197] The compositions according to particular embodiments of the present application that were studied in the present Example are summarized in Table 1. The results are summarized in Figure 1.Table 1: Compositions of present invention used for evaporation testing; combination of solvents (1:1 ratio by volume) are indicated in compositions 4 - 7.

[0198] As shown in Figure 1, there was only an average of 2.29% mass loss across the nonaqueous compositions 1-7 following storage at 50°C±2°C for 35 days. In contrast, there was significantly greater evaporative mass loss observed from sealed tubes containing water (27%), 23% ethanol (45%) and Oragene® (19%). These results demonstrate that the nonaqueous compositions of this application can be stored for prolonged periods of time without significant evaporative loss, leading to improved shelf-life of potential products, including medical devices for sample storage.

[0199] EXAMPLE 2: Solvent solubility evaluation and feasibility for the collection of biological specimens

[0200] In this example, various non-aqueous solvents of interest (Table 2) with chemical properties including a low vapor pressure, a high boiling point, and a high flash point with various chemical structures including alcohols, polar aprotics, esters, ketones, phenols, and polyols were screened for properties required for the stabilization of nucleic acid and collection of biological specimens in medical devices. Specifically, solvents including: triacetin (CAS# 102-76-1), cyrene (CAS# 53716-82-8), dimethyl sulfoxide (DMSO) (CAS# 67- 68-5), triethylene glycol (CAS# 112-27-6), 1,5-pentanediol (CAS# 111-29-5), propylene glycol (CAS# 57-55-6), 2-methyl-l,3-propanediol (CAS# 2163-42-0), dipropylene glycol (CAS# 25265-71-8), propylene carbonate (CAS# 108-32-7), y-valerolactone (CAS# 108-29-2),glycerol (CAS# 56-81-5), tri(propylene glycol) methyl ether, mixture of isomers (CAS# 25498- 49-1), diethylene glycol monobutyl ether (CAS# 112-34-5), 2,2,4-trimethyl-l,3-pentanediol monoisobutyrate (CAS# 25265-77-4), 1,2 butanediol (CAS# 584-03-2), 1,3 butanediol (CAS# 107-88-0), triethylene glycol monomethyl ether (CAS# 112-27-6), l-phenoxy-2-propanol (CAS# 770-35-4), tripropionin (CAS# 139-45-7), tri(propylene glycol) butyl ether, mixture of isomers (CAS# 55934-93-5), triethyl citrate (CAS# 77-93-0), ethyl acetoacetate (CAS# 141- 97-9), di(propylene glycol) methyl ether, mixture of isomers (CAS# 34590-94-8) and diethyl malonate (CAS# 105-53-3) were evaluated for plastic compatibility with polystyrene, miscibility with water (1:1 ratio), mixing with a urine sample (1:1 ratio) via inversion and mixing with a saliva sample (1:1 ratio) via inversion. This evaluation narrowed in on solvents with chemical structures of glycols, as defined herein, as suitable for mixing with a biological sample (urine or saliva) within a plastic container and suitable for the collection of biological samples and use in stabilization of nucleic acid in medical devices.

[0201] Next, solvents were evaluated for the dissolution of common chemical components used for the stabilization of nucleic acid from a biological sample (Table 3). Solvents were evaluated for dissolution of trans-l,2-cyclohexanediaminetetraacetic acid monohydrate (chelator, 300 mM; CAS# 125572-95-4), sodium acetate (pH buffer, 750 mM; CAS# 127-09- 3), trans-l,2-cyclohexanediaminetetraacetic acid monohydrate and sodium acetate in combination (300 mM and 750 mM respectively), sodium dodecyl sulfate (surfactant, 6% w / v; CAS# 151-21-3) and sodium chloride (salt, 10% (w / v); CAS# 7647-14-5). Interestingly, propylene glycol (CAS#57-55-6) as a solvent, and other structurally similar glycols including triethylene glycol (CAS#112-27-6), triethylene glycol monomethyl ether (CAS#112-27-6), 2- methyl -1,3 propanediol (CAS#2163-42-0), and dipropylene glycol (CAS# 25265-71-8) were identified as being: suitable for feasible mixing with a biological sample; compatible with common plastics (polystyrene) used in medical devices for the collection of a biological specimen; and able to dissolve common chemical components used for the stabilization of nucleic acid collected from a biological specimen.

[0202] Following the initial evaluation for the feasibility of solvents in the stabilization of nucleic acid in biological samples (Table 2 and Table 3) a broader solvent solubility evaluation was performed (Table 4) for the dissolution of various functional chemical components that could be used for the stabilization of nucleic acid. Specifically, triacetin,diacetin, dimethyl sulfoxide (DMSO), triethylene glycol, 1,5-pentanediol, propylene glycol, 2- methyl-l,3-propanediol, dipropylene glycol, poly(ethylene glycol) M(n) 300, diethylene glycol monobutyl ether, 1,2 butanediol, 1,3 butanediol, triethylene glycol monomethyl ether and di(propylene glycol) methyl ether, or a mixture of isomers were evaluated for the dissolution of Bicine (200mM; CAS# 150-25-4), sodium citrate tribasic dihydrate (200 mM; CAS# 6132-04-3), Tris (200 mM; CAS# 77-86-1), betaine (6% (w / v); CAS# 107-43-7), aurin tricarboxylic acid (ATA) (50 mM; CAS# 4431-00-9), ascorbic acid (250 mM; CAS# 50-81-7), 0 - cyclodextrin (200 mM; CAS# 7585-39-9), lithium sulfate (500 mM; CAS# 10102-25-7), polyacrylic acid (5% (w / v); CAS# 9033-79-8) and lithium chloride (IM; CAS# 7447-41-8) were tested.

[0203] Lastly, the present inventors selected particular solvents of interest, including DMSO, glycerol and glycols (Table 5), to evaluate the dissolution of additional functional components which could be used for the stabilization of nucleic acid. Specifically, tripropylene glycol methyl ether (CAS# 25498-49-1), propylene glycol (CAS# 57-55-6), tripropylene glycol butyl ether (CAS# 55934-93-5), (±)-l,3-butanediol (CAS# 107-88-0), 1,4- butanediol (CAS# 110-63-4), 1,5-pentanediol (CAS# 111-29-5), 2-methyl-l,3-propanediol (CAS# 2163-42-0), diethylene glycol (CAS# 111-46-6), triethylene glycol (CAS# 112-27-6), tetra ethylene glycol (CAS# 112-60-7), polyethylene glycol (Mn300) (CAS# 25322-68-3), diethylene glycol monobutyl ether (CAS# 112-34-5), dipropylene glycol monomethyl ether (CAS# 34590-94-0), triethylene glycol monomethyl ether (CAS# 112-35-6), N-(2- hydroxyethyl)lactamide (CAS# 5422-34-4), N-methyl diethanolamine (CAS# 105-59-9), 2-2 thiodiethanol (CAS# 111-48-8), glycerol (CAS# 56-81-5), DMSO (CAS# 67-68-5), 1,2 butanediol (CAS# 584-03-2) and water (aqueous control, CAS#7732-18-5) were evaluated for the dissolution of guanidine acetic acid (200 mM; CAS# 352-97-6), calcium acetate monohydrate (200 mM; CAS# 5743-26-0), potassium acetate (500 mM; CAS# 127-08-2), lithium acetate (500 mM; CAS# 546-89-4), sodium hexane sulfonate (5% (w / v); CAS# 2832- 45-3), 1-octanesulfonic acid sodium salt (5% (w / v); CAS# 5324-84-5), sodium 1- nonanesulfonate (5% (w / v); CAS# 35192-74-6), N-acetyl-L-cysteine (200 mM; CAS# 616-91- 1), pentaerythritol tetra kis (3,5-di-tert-butyl-4-hydroxyhydrocinnamate) (200 mM; CAS# 6683-19-8), L-glutathione reduced (200 mM; CAS# 70-18-8), EGTA (50mM; CAS# 67-42-5), boric Acid (5% (w / v); CAS# 10043-35-3), D(-)fructose (20% (w / v); CAS# 57-48-7).- M -

[0204] The results of these studies demonstrate that glycols, as defined herein, glycerol and DMSO are all suitable for use as a non-aqueous solvent in a non-aqueous nucleic acid storage composition. In addition, these studies provide insight into the additional components that can be incorporated in the non-aqueous storage composition, as further demonstrated in the following Examples.Table 2: Solvent feasibility assessment for compatibility with plastics (polystyrene) and mixing with water, urine and saliva as a biospecimen sample type.Table 3: Solvent solubility of various formulation components used for nucleic acid stabilization in biological samples. N = no solubility, Y = successful solubility, PS = partial solubility.Table 4: Solvent solubility of various formulation components used for nucleic acid stabilization in biological samples. N = no solubility, Y = successful solubility, PS = partial solubility.Table 5: Solvent selection comprised of glycols, as defined herein, glycerol or DMSOfor dissolution of components which are useful to facilitate nucleic acid stability.

[0205] EXAMPLE 3: Composition Stability at Room Temperature

[0206] During the testing of numerous compositions according to embodiments of the present application, for properties such as nucleic acid stabilization, it was observed that certain compositions became visibly yellow after approximately 42 days storage at room temperature, which, for the purpose of this Example, was 23°C±3°C (see Table 6). Yellowing was confirmed by performing UV-VIS absorbance scans of each composition under investigation.

[0207] Specifically, 170 pL of each composition (Table 6) was dispensed into separate wells of a UV-Star® half area 96-well microplate (Greiner Bio-One; Catalog No. 655801). A full UV- VIS absorbance scan from 220 - 900 nm was done using a Molecular Devices SpectraMax M2® Microplate Reader (Catalog No. 89429-532). Averages of the absorbance wavelengths across 5 technical replicates were plotted across the full UV-VIS absorbance wavelengths (Figure 2).Table 6: Colour change in non-aqueous compositions over time.

[0208] Triethylene glycol and triethylene glycol monomethyl ether compositions demonstrated a significant change to their absorption spectra, which was visible to the naked eye during storage at room temperature (23°C±3°C) for 42 days (Table 6 and Figure 2). The absorption spectra of compositions 1-4 demonstrated a varying level of relative absorption between 250-400 nm, where composition 4 had the most pronouncedabsorbance change with the appearance of a peak at 300 nm, indicative of a chemical change in the formulation (Figure 2). Propylene glycol demonstrated minimal to no colour change during storage at room temperature for 42 days with no obvious peak at 300 nm (Table 6 and Figure 2). This data suggests that, under the conditions studied, propylene glycol may be preferred, compared to other glycols.

[0209] Medical devices are not always maintained at room temperature (23°C±3°C) and often experience extreme temperatures during transport, for example to a patient, healthcare clinic, or processing laboratory. The chemical stability of composition 5, which contains the preferred solvent, propylene glycol, was tested (Table 7). Evaporative mass loss (%) and changes to the absorption spectra during long-term storage (35 days) at elevated temperature (50°C) of composition 5 were evaluated.Table 7: Formulation details of composition 5.

[0210] Specifically, composition 5 was tested in comparison to commercially available aqueous transport media chemistry, the Aptima® urine transport medium (Hologic; Catalog No. 105575) and the Cobas® PCR Media (Roche Diagnostics, Catalog No. 5170486190). A volume of 1 mL of each chemistry was aliquoted into empty Falcon tubes (Sarstedt; Catalog No. 62.554.205) and weighed at baseline. Next, 170 pL of composition 5, Aptima® urine transport media and the Cobas® PCR Media was dispensed as triplicates into a UV-Star® half area 96-well microplate (Greiner Bio-One; Catalog No. 655801) and a full UV-VIS absorbance scan was performed at baseline and at the evaporation endpoint at 35 days (T35), scanning from 220-900 nm with scanning every 5 nm using a Molecular Devices SpectraMax M2® Microplate Reader. Composition 5 (Table 7) demonstrated the least change in absorption spectra or mass loss over the 35-day period, compared to Aptima® urine transport medium and Cobas® PCR Media (Figures 3 and 4).

[0211] EXAMPLE 4: Surfactant Evaluation in Non-Aqueous Storage Compositions

[0212] Numerous surfactants (each at 229 mM concentration; see Table 8) were evaluated for DNA (Figure 5) and RNA (Figures 6 and 9) stability in compositions comprised of propylene glycol (non-aqueous solvent), magnesium acetate tetrahydrate (buffer; 500 mM), and HEDTA (chelating agent; 100 mM). In the compositions where mixtures of surfactants were evaluated (Figures 7 and 8), the secondary surfactants were tested at a concentration of 2.56% (w / v) (Table 8).Table 8: Summary of surfactants and additives evaluated for nucleic acid stability in urine samples held for 7 days at room temperature. N = no stability, Y = stability, PS = partial stability.*Each composition comprised the listed surfactant at a 229 mM concentration in propylene glycol as the solvent, together with 500 mM magnesium acetate tetrahydrate, and 100 mM HEDTA.“Mixtures of surfactants were evaluated in combination with SDS with secondary surfactants at a concentration of 2.56% (w / v). pH was adjusted using acetic acid to about 5 - 5.5, excluding formulation #4.***urea and 5-sulfosalicylic acid were included in these compositions as additives.

[0213] For each sample, 30 mL of urine was collected into an empty urine collection cup from 2 male and 2 female donors. Urine from the 4 donors was mixed with each formulation (Table 8) at a 5:1 ratio using 500 pL of urine and 100 pL of the respective formulation in a deep well plate. Next, 200 pL of this mixture was transferred to a new deep well plate and spiked with 45 ng / pL Francisella philomiragia extracted RNA (RNA Extracted from Francisella philomiragia with DNase treatment using the QIAGEN Powermicrobiome® Kit on the QiaCube®) and 6 ng / pL of Escherichia coli gDNA (DNA Extracted from Escherichia coli using the QIAGEN Powerfecal® Pro Kit on the QiaCube®). 50 pL of sample was transferred to another deep well plate and placed at 37°C±2°C for 3 days. Remaining sample volume was held at room temperature (23°C±3°C) for future timepoints.

[0214] Next, 50 pL of this sample was extracted at baseline (TO), after 3 days (T3) storage at 37°C, after 7 days (T7) of storage at room temperature (RT), as well as best performing formulations extracted after 25 days (T25) of storage at room temperature using the spike and recovery (SAR) extraction method. In SAR (spike and recovery), a known amount of analyte is added (spiked) into the natural test sample matrix. An assay is then performed to measure the response (recovery) of the spiked sample matrix compared to an identical spike in a control. In this case, the analyte is a known DNA or RNA and the sample matrix is nonaqueous storage composition in combination with a biological sample. The control can be a DNA or RNA spiked in a biological sample (without dilution in a storage composition) or in a previously known storage composition.

[0215] Extracted samples were run on the Agilent TapeStation™ for DNA or RNA analysis across the above storage conditions (Table 8 and Figures 5-9).

[0216] As summarized in Table 8, several surfactants stabilized both DNA and RNA in compositions of this invention, compared to control compositions without a surfactant (ID#21, Table 8). Compositions containing various surfactants including: Sodium Dodecyl Sulfate (SDS), 4-Dodecylbenzene Sulfonic Acid (4-DBS), Lithium Dodecyl Sulfate (LDS), or compositions containing SDS combined with other anionic surfactants (Tween 20) or nonionic surfactants such as Docusate Sodium Salt were particularly effective at stabilizing nucleic acid. In addition, naturally occurring components in urine, such as urea, were also effective at preserving nucleic acid (DNA and RNA) for 7 days at room temperature 23°C±3°C when included in the composition in addition to SDS. In addition, TapeStation analysis showed that both RNA (Figure 6) and DNA (Figure 5) were preserved by compositions that contained SDS, 4-DBS or LDS, following extended storage of urine samples for 25 days (T25) at room temperature 23°C±3°C.

[0217] Interestingly, when SDS was mixed with a zwitterionic secondary surfactant, namely Cocamidopropyl Betaine (CAPB), nucleic acid stability was not necessarily ensured. After 7 days (T7) at room temperature, urine samples mixed with a composition containing a mixture of SDS and Docusate Sodium Salt or SDS plus Tween 20 showed intact RNA for all 4 donors. However, RNA was degraded in samples mixed with a composition containing a mixture of SDS and CAPB in one of four donor samples after 7 days storage at room temperature (Figure 7). Surprisingly, high molecular weight DNA was preserved with this same composition of SDS and Docusate Sodium Salt for all four donors, while partial DNA degradation was observed with compositions containing a mixture of SDS and CAPB or SDS and Tween 20 for some donors (Figure 8).

[0218] For comparison with a commercially available preservative, Aptima® urine transport medium (Hologic; Catalog No. 105575) was tested with the same urine samples. 100 pL of the Aptima® urine transport medium was mixed with 500 pL of urine samples (Ml, M2, Fl, F2) to make a 5:1 ratio. 50 pL of mixture was extracted at baseline (TO) and after 25 days (T25) of storage at room temperature using the SAR extraction method.

[0219] The manufacturer's recommended ratio of urine to Aptima® urine transport medium is 1:1. However, in order to accurately compare the effect of the non-aqueous storage compositions of the present application to the effect of the Aptima® urine transport medium, the ratio used in the present example was normalized to 5:1 by volume to ensure consistency and avoid extraneous effects resulting from over dilution of the samples.

[0220] Extracted samples were run on the Agilent TapeStation™ for DNA or RNA analysis. At T25, while high molecular weight DNA was present in all samples (Figure 6), RNA, particularly from female donors, was partially degraded (Figure 5).

[0221] To demonstrate the performance of the present non-aqueous storage compositions in stabilizing nucleic acid from an alternate biological fluid, stability of nucleic acid (DNA and RNA) was tested in saliva. For the saliva samples, 4 donors each provided 2 mL of unstabilized saliva into empty 15 mL Falcon tubes. Saliva from 4 donors was mixed with the present compositions at a 3:1 (saliva to chemistry) ratio using 300 pL of saliva and 100 pL of composition in a deep well plate. Next, 200 pL of this mixture was transferred to a new deep well plate and spiked with 45 ng / pL of Francisella philomiragia extracted RNA (extracted as described above) and 6 ng / pL of Escherichia coli gDNA (extracted as described above). At baseline (TO) and after 5 days (T5) at room temperature, nucleic acid were extracted from 50 pL of samples and run on the Agilent Tapestation™. Compositions with the surfactant 4- DBS (ID# 4, Table 8) preserved spiked RNA for 5 days at room temperature (Figure 5).However, RNA was rapidly degraded even at TO when surfactant was absent from the spiked saliva samples (Figure 9).

[0222] Finally, at baseline (TO) and after 5 days at RT (T5), extracted DNA served as template for Human P-globin qPCR using 2X iTaq™ Universal SYBR® Mastermix (Table 9). Table 9 shows a summary of qPCR Delta Ct values for saliva samples mixed with the composition in the presence of the surfactant 4-DBS, compared to unstabilized saliva. Ct values were unchanged for samples mixed with the composition with 4-DBS, indicating DNA is preserved for at least 5 days at room temperature. However, the large Delta Ct values for the unstabilized saliva indicates that DNA degrades in saliva samples stored at room temperature (Table 9).Table 9: qPCR Delta Ct value summary (T5 - TO) when saliva samples were mixed with a storage composition at a 3:1 ratio and held at room temperature (23°C±3°C) for 5 days, compared to respective non-diluted, unstabilized saliva samples from each of the 4 donors

[0223] The present Example demonstrates that a variety of different surfactants can be successfully included in the non-aqueous storage compositions of the present application. In particular, SDS, 4-DBS and LDS, were found to be effective in storage compositions for stabilization of both DNA and RNA. Furthermore, mixtures of surfactants that include SDS in combination with another anionic or cationic surfactant can be successfully included in the non-aqueous storage compositions of the present application for stabilization of both DNA and RNA.

[0224] EXAMPLE 5: Acetate Buffer Evaluation in Non-Aqueous Storage Compositions

[0225] In this example, various acetate buffering agents were incorporated in examples of non-aqueous storage compositions of the present application and assessed in terms of their impact on nucleic acid (DNA and RNA) stability. Urine samples were mixed with the compositions and nucleic acid stability was determined following a three-day exposure to elevated temperature 37°C(±2°C). Elevated temperatures were used to provide accelerated conditions to allow extrapolation to longer-term storage at standard room temperature conditions and / or to simulate more extreme ambient conditions that may be experienced, for example, during transport.

[0226] A volume of urine (30 mL) was collected into an empty urine collection cup from 2 male and 2 female donors. Next, urine from female donors and male donors was pooled to create pooled male (MP) and pooled female (FP) samples. Each pooled urine sample was mixed with composition 1-7 (Table 10) at a 10:1 or 5:1 ratio (by volume) in a deep well plate. 100 pL of mixed sample was transferred to a new deep well plate and spiked with 40 ng / pL Francisella philomiragia extracted RNA (extracted as described above) and 40 ng / pL of Escherichia coli gDNA (extracted as described above). Next, 50 pL of spiked sample wasextracted at baseline (TO) and remaining sample placed at 37°C±2°C for 3 days (T3) and extracted using a Spike and Recovery (SAR) extraction method. Extracted samples were run on the Agilent Tapestation for RNA (Figure 10) and DNA (Figure 11) analysis. For comparison with a commercially available preservative, Aptima® urine transport medium (Hologic; Catalog No. 105575) was tested with the same pooled urine samples (MP, FP) at the same ratios (10:1 or 5:1 by volume) using the methodology as outlined above.

[0227] As noted above in Example 4, the manufacturer's recommended ratio of urine to Aptima® urine transport medium is 1:1. However, in order to accurately compare the effect of the non-aqueous storage compositions to the effect of the Aptima® urine transport medium, the ratio used in the present example was changed to 10:1 or 5:1 by volume to ensure consistency and avoid extraneous effects resulting from over dilution of the sample. It is also noted here that an additional potential advantage of the present non-aqueous storage composition is the fact that a lower chemistry volume is required than is recommended and used with existing storage compositions, such as the Aptima® urine transport medium.Table 10: Summary of acetate buffers evaluated for nucleic acid stability in compositions comprised of propylene glycol, 50 mM of 5-sulfosalicylic acid, 229 mM of sodium dodecyl sulfate, 100 mM of CDTA and pH was adjusted using acetic acid. PS = partial stability.

[0228] Incorporation of different acetate buffers in the non-aqueous storage composition of the present application were found to be useful for nucleic acid stability in biological samples, including urine samples. Except for zinc acetate, all acetate buffers tested stabilized both RNA and DNA under stressed conditions, i.e., elevated temperature. As a negative control, both spiked RNA and DNA in urine, without combination with a storage composition, immediately (TO) degraded in urine. Of further note is the finding that the two ratios tested (10:1 and 5:1) were found to function similarly.

[0229] EXAMPLE 6: Effective pH Range in Non-Aqueous Storage Compositions

[0230] In this example, various pH ranges were evaluated for stabilization of RNA (Figure 12, 14, and 15) and DNA (Figure 13, Table 2, and Figure 16) in compositions comprised of propylene glycol (non-aqueous solvent), magnesium acetate tetrahydrate (pH buffer), a chelating agent (CDTA, HEDTA), and 5-sulfosalicylic acid dihydrate (an additive).

[0231] For the initial pH range evaluation (4.23-5.61), the composition was comprised of propylene glycol (non-aqueous solvent), magnesium acetate tetrahydrate (buffer; 500 mM), CDTA (chelating agent; 100 mM), 5-sulfosalicylic acid dihydrate (additive; 50 mM) and sodium dodecyl sulfate (SDS, surfactant; 229 mM). Briefly, 30 mL of urine was collected into an empty urine collection cup from four male and two female donors where two respective male urine samples were mixed to create two male pooled urine samples (MP1 and MP2) and two female urine samples were mixed to create a single female pooled urine sample (FP1). The pooled urine samples were mixed with compositions (see Table 11) at a 5:1 ratio (chemistry to urine by volume) to evaluate a functional pH range for nucleic acid (DNA and RNA) stability. Next, 100 pL of this mixture was transferred to a new deep well plate and spiked with 40 ng / pL Francisella philomiragia extracted RNA (extracted as described above) and 40 ng / pL of Escherichia coli gDNA (extracted as described above). 50 pL of each spiked sample was transferred to another deep well plate and placed at 37°C±2°C for 3 days.

[0232] A 50 pL aliquot of each spiked sample was extracted at baseline (TO) or after 3 days (T3) storage at 37°C±2°C using the SAR extraction method, as described above. Extracted samples were run on the Agilent Tapestation for DNA or RNA analysis at TO and T3 storage at 37°C±2°C timepoints (Figure 12 and Figure 13).

[0233] As shown in Table 11, a pH range from about 4.23-5.61 was effective at maintaining DNA stability in the tested non-aqueous storage compositions and a pH range from about 4.71-5.61 was effective at maintaining both RNA and DNA stability under stressed or accelerated conditions, i.e., storage for 3 days at 37°C±2°C.Table 11: Summary of compositions to establish an effective pH range for nucleic acid stabilization for RNA and DNA, where pH was adjusted using acetic acid.

[0234] Next, a pH range (4.33-7.84) was evaluated in compositions comprised of propylene glycol (non-aqueous solvent) with various concentrations of magnesium acetate tetrahydrate (buffer; 250-800 mM), HEDTA (chelating agent; 0-100 mM) and SDS (surfactant; 0-300 mM). Briefly, 30 mL of urine was collected into an empty urine collection cup from two male and two female donors and mixed with compositions (see Table 12) at a 5:1 and 10:1 ratio by volume. 500 pL of respective mixtures was removed and placed into aseparate deep well plate and spiked with 40 ng / pL Francisella philomiragia extracted RNA, and ~1 x 105CFU / mL of S. pyogenes.Table 12: Summary of compositions across various functional ranges or concentrations to establish an effective pH and functional composition range for the stabilization of RNA (Figure 14) and S. pyogenes DNA (Table 13) via gPCR when mixed at a 10:1 ratio and stored for 33 days (T33) at room temperature 23°C±3°C. pH was adjusted using acetic acid in formulation # 14 -18. PS = partial stability.

[0235] 100 pL of each spiked sample was extracted using the Quick-DNA / RNA™ Viral Magbead Kit (Zymo Research, Catalog No. R2141) at baseline (TO) for both 5:1 and 10:1 ratios, after 7 days (T7) of storage at room temperature (23°C±3°C) for the 10:1 ratio samples, and after 33 days (T33) of storage at room temperature for the 10:1 ratio samples. Extracted samples were run on the Agilent Tapestation™ for RNA analysis at TO (not shown), T7 (not shown) and T33 timepoints (Figure 16) and S. pyogenes qPCR was performed for DNA analysis (Table 13) following the CDC single plex assay for the SPY gene (Group A Streptococcus Lab Resources and Protocols | CDC) using the standard Promega PCR master mix conditions (Promega, Catalog No. M7502).

[0236] Using the non-aqueous storage composition (Figure 14), precipitation was observed upon mixing with a subset of urine samples. Without wishing to be bound by theory, this may be attributed to the presence of high concentrations of SDS in the storage compositions. This precipitation only occured in the presence of SDS chemistries, similar to what has also been observed with aqueous storage compositions (e.g., Aptima®). In general, RNA degradation is typically correlated with SDS precipitation as SDS can be an important component for RNA stability. However, as shown in Figure 14, SDS can be used successfully in embodiments of the non-aqueous storage compositions having low pH (for example, in the range of 4.33 - 4.48).

[0237] Importantly, the present application further provides alternative embodiments using other surfactants (e.g., 4-DBS, and / or Tween 20), as shown above, which can be used at higher pH without precipitation of surfactant mixtures (Figure 15). In some embodiments, the surfactant includes Tween 20, which has been shown to improve sample homogeneity under cold storage. Selection of the appropriate surfactant will depend, at least in part, on factors such as biological sample type, conditions of collection, transportation temperatures and downstream use, while also considering the effect of these surfactants under different conditions.Table 13: S. pyogenes DNA detection via qPCR (Group A Streptococcus Lab Resources and Protocols I CDC) at baseline (TO) and after storage at room temperature for 33 days (T33) and calculated delta Ct values (T33-T0) after mixing with either two male or two female urine samples (Ml, M2, Fl, F2) at a 10:1 ratio with compositions containing propylene glycol, 250- 800 mM magnesium acetate tetrahydrate, 0 - 100 mM HEDTA and 0-229 mM ofSDS with pH range from 4.33-7.84. pH was adjusted using acetic acid in formulation # 15 -19.

[0238] Finally, a pH range (4.39 - 5.2) was evaluated across the compositions comprised of propylene glycol (solvent) and various concentrations of magnesium acetate tetrahydrate (buffer; 350 - 802 mM), HEDTA (chelating agent; 39-119 mM) and 4-dodecylbenzene sulfonic acid (surfactant; 50-381 mM) (Table 14). Briefly, 30 mL of urine was collected into an empty urine collection cup from two male and two female donors and mixed with the present compositions (see Table 14) at a 5:1 ratio. 500 pL of respective mixtures were removed, placed into a separate deep well plate, and spiked with 40 ng / pL Francisella philomiragia extracted RNA and 30 ng / pL of Escherichia coli gDNA.

[0239] A 140 pL aliquot of the spiked samples were then extracted using the QIAamp® Viral RNA Mini Kit (Qiagen, Catalog No. 52906) at baseline (TO) and after 3 days (T3) storage at 37°C±2°C. Extracted samples were run on the Agilent Tapestation™ for RNA (results shown in Figure 15) and DNA (results shown in Figure 16) analysis at TO and T3 storage at 37°C±2°C timepoints.Table 14: Summary of present compositions across various components to establish an effective pH and functional composition range for the stabilization of RNA (Figure 15) and DNA (Figure 16) when mixed at a 5:1 ratio and stored for 3 days (T3) at 37°C±2°C.

[0240] As shown in Table 14, and in Figures 15 and 16, a pH range from about 4.23-5.61 was effective at maintaining RNA and DNA stability in the tested non-aqueous storage compositions under stressed, or accelerated, conditions, i.e., storage for 3 days at 37°C±2°C.

[0241] EXAMPLE 7: Effect of Chelators on Nucleic Acid Stability in Non-Aqueous Storage Compositions

[0242] In this example, various chelators were studied to evaluate their impact on nucleic acid stabilization.

[0243] In a first study (chelators each at 100 mM concentration; see Table 15) were evaluated for RNA (Figure 17) and DNA (Figure 18) stability in compositions comprising propylene glycol (solvent), magnesium acetate tetrahydrate (buffer; 500 mM), 5- sulfosalicylic acid dihydrate (CAS#5965-83-3) (additive; 50 mM)) and either trans-1, 2- diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate (CDTA; 100 mM, CAS# 125572- 95-4), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA; 100 mM, CAS#150-39-0) or a combination thereof (50 mM of CDTA and HEDTA each in combination). Additionally, the composition was evaluated for RNA (Figure 19) and DNA (Figure 20) stabilization without the presence of a chelating agent (i.e., only 500 mM magnesium acetate tetrahydrate and 229 mM of SDS; and pH adjusted to about 5.15 with acetic acid).

[0244] 30 mL of urine was collected into an empty urine collection cup from two males and two female donors and pooled to create one male pooled (MP) and female pooled (FP) sample. Pooled urine samples were mixed with the non-aqueous storage compositions as set out in Table 15 at a 5:1 ratio using 500 pL of urine and 100 pL of the storage composition in a deep well plate. Next a 100 pL aliquot was removed to a separate deep well plate and spiked with 45 ng / pL Francisella philomiragia extracted RNA (extracted as described above) and 40 ng / pL of Escherichia coli gDNA (extracted as described above). 50 pL of sample was transferred to another deep well plate and placed at 37°C±2°C for 3 days.

[0245] 50 pL of each sample was extracted at baseline (TO) and after 3 days (T3) storage at 37°C±2°C using a spike and recover (SAR) extraction method, as described above. Extracted samples were run on the Agilent Tapestation™ for DNA or RNA analysis at TO or T3 storage at 37°C±2°C (Figure 17 - 20).Table 15: Summary of chelators evaluated for nucleic acid stability (DNA / RNA) in urine samples held for 3 days storage at 37°C±2°C. PS = partial stability.

[0246] Compositions in the presence of chelating agents (CDTA and HEDTA) preserved nucleic acid in urine samples exposed to elevated temperature for 3 days. Interestingly, in the absence of chelating agent, high molecular weight ribosomal RNA (Figure 19) was intact, and DNA (Figure 20) was partially stabilized when stored at elevated temperature for a prolonged period.

[0247] In another study, compositions without the presence of a chelator (Table 16) and comprised of magnesium acetate tetrahydrate (buffer; 350 mM-623 mM) and 4-DBS (surfactant; 381 mM); with a pH of about 4.88-5.12 were evaluated for RNA (Figure 21) and DNA (Figure 22) stability.

[0248] 30 mL of urine was collected into an empty urine collection cup from two male and two female donors. Urine samples were mixed with the compositions without the presence of a chelator (Table 16) at a 5:1 ratio using 1 mL of each urine sample and 200 pL ofchemistry. Next, 300 pL of this mixture was transferred to a new deep well plate and spiked with 40 ng / pL Francisella philomiragia extracted RNA and 30 ng / pL of Escherichia coli gDNA.Table 16: Summary of compositions without the presence of a chelator for nucleic acid stability in urine samples held for 3 days at 37°C±2°C comprised of Propylene Glycol, Magnesium Acetate Tetrahydrate (buffer; 350 mM-632 mM), 4-Dodecylbenzene Sulfonic Acid (381 mM) and a pH range of about 4.88-5.12.

[0249] The results of this study demonstrate both RNA (Figure 21) and DNA (Figure 22) stabilization in urine mixed with a non-aqueous storage composition that does not contain chelating agent(s) and exposed to 3 days at 37°C. These results indicate that chelating agents are optional components of the present non-aqueous storage composition.

[0250] In a third study, compositions with propylene glycol, magnesium acetate tetrahydrate (buffer; 500 mM), SDS (surfactant; 229 mM) and HEDTA with a range from 0 mM (chelator removed) to 100 mM were evaluated (Table 17) for the detection of S. pyogenes DNA when mixed with two male and two female urine samples at a 10:1 ratio and held for 33 days at room temperature.

[0251] 30 mL of urine was collected into an empty urine collection cup from two male and two female donors and mixed with compositions from Table 17 at a 10:1 ratio (urine to composition). 500 pL of respective mixtures was removed and placed into a separate deep well plate and spiked with ~1 x 105CFU / mL of S. pyogenes. qPCR was performed following the CDC single plex assay for the SPY gene (https: / / www.cdc.gov / streplab / groupa- strep / resources.html) using the standard Promega PCR master mix conditions (Promega, Catalog No. M7502). Ct values were largely unchanged for samples mixed with the storage composition in the presence or absence of chelating agent, indicating that DNA waspreserved for at least 33 days at room temperature. Again, these results demonstrate effective nucleic acid stabilization even without the presence of a chelator in the nonaqueous storage composition.Table 17: Summary of compositions comprised of Propylene Glycol (solvent), SDS (surfactant; 229 mM); Magnesium Acetate Tetra hydrate (buffer; 500 mM) with varying concentrations of HEDTA (chelator; 0-100 mM) or with HEDTA removed from the formulation for detection ofS. pyogenes DNA via gPCR when mixed at a 10:1 ratio and stored for 33 days at room temperature (RT).

[0252] In a fourth study, non-aqueous solvent compositions with various chelators (Table 18) were evaluated for DNA stability (Figure 23). The compositions used in this study comprised various glycols, as defined herein (non-aqueous solvent), chelating agents (50 mM), with or without lithium acetate dihydrate (buffer; 750 mM), and with or without sodium dodecyl sulfate (surfactant; 6.6% (w / v)).

[0253] 30 mL of urine was collected into an empty urine collection cup from two males and two female donors and pooled to create one male pooled (MP) and female pooled (FP) sample. Pooled urine samples were mixed with the compositions (Table 18) at a 1:1 ratio using 100 pL of urine and 100 pL of the composition.

[0254] Next a 100 pL aliquot was removed to a 1.5 mL LoBind® Tube (Eppendorf, Cat No. 022431021) and spiked with 40 ng / pL of Escherichia coli gDNA. 50 pL of sample was transferred to a separate 1.5 mL LoBind® Tube and placed at 37°C for 3 days.

[0255] 50 pL of this sample was extracted at baseline (TO) and after 3 days (T3) storage at 37°C using a SAR extraction method, as described above. Extracted samples were run on the Agilent Tapestation™ for DNA at TO or T3 storage at 37°C±2°C.Table 18: Summary of compositions evaluated for DNA stability when mixed with one male and one female pooled urine sample at a 1:1 ratio and held at 37°Cfor 3 days.

[0256] Each of the non-aqueous storage compositions studied provided DNA stabilization.These results demonstrate that various chelators can be successfully used together with different non-aqueous solvents to provide a non-aqueous composition suitable for storage of nucleic acid.

[0257] EXAMPLE 8: Contribution of Non-Aqueous Solvent on Nucleic Acid Stabilization

[0258] In this example, various individual solvents (Table 19) were studied for their effect on DNA (Figure 24) and RNA (Figure 25) stabilization upon mixing with a pooled urine sample from two male and two female donors (n=4) at a 1:1 ratio (by volume). No chelating agents, buffering agents, or surfactants were included in these mixtures with urine, and the pH was not adjusted.

[0259] Briefly, 30 mL of urine was collected into an empty urine collection cup from 2 male and 2 female donors. Urine from the 4 donors was pooled to create a homogenous sample and then mixed with various solvents (Table 19) at a 1:1 ratio using 200 pL of respective solvent and 200 pL of the pooled urine sample. Next, 100 pL of the mixture was transferred to a new deep well plate and spiked with 25 ng / pL of Francisella philomiragia extracted RNA (extracted as described above) and a separate 100 pL was spiked with 25 ng / pL of Escherichia coli gDNA (extracted as described above). 50 pL was removed from the respective DNA or RNA-spiked aliquots for baseline (TO) extraction using the SAR extraction method. Extracted samples were run on the Agilent Tapestation™ for DNA (Figure 24) or RNA (Figure 25) analysis for the TO baseline timepoint and compared to the intact nucleic acid (DNA or RNA) spiked into the solvents in Table 19.Table 19: Summary of solvents evaluated for nucleic acid stabilization in a pooled urine sample. PS = partial stability.

[0260] As summarized in Table 19, none of the non-aqueous solvents tested were capable of protecting either high molecular weight DNA or RNA from degradation when the solvent was mixed with a pooled urine sample. The nucleic acid immediately degraded, partially or completely, upon spike in (Figure 24 and 25). Partially degraded DNA was observed in pooled urine samples mixed with tripropylene glycol ethyl ether, tetraethylene glycol, diethylene glycol butyl ether and dipropylene glycol monomethyl ether (Figure 24).

[0261] These results, combined with the results from the preceding examples, demonstrate that the nucleic acid stabilizing activity of the non-aqueous storage compositions of the present application is a result of the combination of detergent / surfactant, buffer (maintaining pH within an effective range) and, optionally, chelator present. However, notall non-aqueous solvents can be employed in such stabilizing compositions. For example, the present inventors have found that polar aprotic solvents (other than dimethylsulfoxide (DMSO)), esters, ketones and phenols do not sufficiently solubilize the stabilizing components. In contrast, glycols, as defined herein, glycerol and DMSO are able to solubilize components in nucleic acid storage compositions, including, for example, surfactants, buffers, chelators, salts, and additives.

[0262] EXAMPLE 9: Non-aqueous storage composition comprising 4-DBS and Tween 20 under freeze / thaw cycling or ambient storage at room temperature

[0263] In this example compositions (Table 20) containing propylene glycol (solvent, CAS# 57-55-6) were mixed with urine samples and evaluated for DNA (Figure 26, Figure 27) and RNA (Figure 28, Figure 29) stability and qPCR delta Ct values (Tx - TO) were evaluated under freeze / thaw cycling conditions (Figure 30, 31 & 32) and after 14 days storage at room temperature (23°C±3°C) (Figures 33 & 34).

[0264] Briefly, 30 mL of urine was collected into an empty urine collection cup from 3 male and 3 female donors. Urine from the 6 donors (3 males, 3 females) was mixed with compositions A and B (Table 20) at a 5:1 urine to chemistry ratio (composition A) or at both 5:1 and 10:1 ratios (composition B). Next, a 250 pL aliquot was removed and mixtures were spiked with F. Philomriagia RNA at 12.25 ng / pL, S. pyogenes cells at 1 x 105CFU / mL, and 4.28 ng / pL of Escherichia coli gDNA.

[0265] Next, 100 pL of sample was removed and nucleic acid were extracted from the mixtures at baseline (TO) using the Zymo Quick-DNA / RNA™ Magbead Kit (Zymo, Catalog No. R2141). 100 pL of the mixtures were removed post freeze / thaw cycling (F / T) from 40°C (24- hour storage) to -20°C (overnight storage) and nucleic acid extracted using the Zymo Quick- DNA / RNA™ Magbead Kit (Zymo, Catalog No. R2141). qPCR was performed for the P-globin target (DNA; Figure 30) and RT-qPCR performed for GAPDH (RNA; Figure 31). Next, exogenous microbial DNA qPCR was performed for S. pyogenes (https: / / www.cdc.gov / streplab / groupa-strep / resources.html) and delta Ct's calculated for the various qPCR targets under freeze / thaw conditions (F / T -TO) or room temperature storage (23°C±3°C) for 14 days (T14 - TO). For comparison with a commercially available preservative, Aptima® Urine Specimen Transport Tubes (Hologic; Catalog No. 105575) orCobas® PCR Media (Roche; Catalog No. 5170486190) were tested with the same urine samples.Table 20: Summary of compositions and ratios evaluated for DNA and RNA stability under freeze / thaw cycling conditions or after ambient room temperature at 23°C(±3°C) storage for 14 days.‘Ratio of urine:storage composition

[0266] The results provided in this example demonstrate successful use of non-aqueous storage compositions of present invention to maintain effective nucleic acid stabilization (summarized in Table 20) for both DNA and RNA under freeze / thaw cycling and after storage at room temperature for 14 days. Importantly, a mixture of surfactants including 4DBS and Tween 20 were effective at maintaining DNA and RNA stability under freeze thaw cycling conditions and storage for up to 14 days at room temperature.

[0267] EXAMPLE 10: Specific Ion Interaction in Non-aqueous Solvent Formulations

[0268] In this example, storage compositions of the present application were compared to equivalent aqueous solvent (water)-based formulations for the stabilization of nucleic acid within urine samples.

[0269] Briefly, compositions (Table 21) were prepared and mixed with two male pooled urine samples and one female pooled urine sample (n=2 individual urine samples in each pooled sample) at a 5:1 ratio. These mixtures were spiked with ~ 40 ng / pL of E.coli DNA and F. philomarigia RNA. Nucleic acid was extracted at baseline (TO) and following three days(T3) storage at 37°C±2°C using the SAR extraction method. Extracted nucleic acid was run on the TapeStation™ (Agilent) for respective DNA or RNA analysis (Figures 35 and 36).Table 21: Summary of non-aqueous solvent compositions evaluated for nucleic acid stability (DNA / RNA) in urine samples stored at 37°C±2°Cfor 3 days (T3).[Y]= Yes, [N]= No

[0270] As summarized in Table 21, only the non-aqueous solvent composition was found to be effective at stabilizing both DNA and RNA from urine samples after storage at 37°C±2°C for 3 days (T3). In contrast, the aqueous composition failed to maintain nucleic acid stabilization immediately upon spike in.

[0271] Next, non-aqueous solvent compositions (Table 22) containing either magnesium acetate tetrahydrate or lithium acetate dihydrate were compared to their equivalent aqueous solvent-based compositions for the stabilization of nucleic acid within a urine sample, after storage at 37°C for 3 days (T3). Briefly, compositions were mixed at a 5:1 ratio with two male pooled urine samples and one female pooled urine sample (n=2 in each pooled sample) at a 5:1 ratio and spiked with nucleic acid as described above and extracted using the SAR extraction method. Extracted nucleic acid was run on the TapeStation™ (Agilent) for respective DNA or RNA analysis (Figures 37 and 38).Table 22: Summary of compositions evaluated comparing non-aqueous solvent compositions to aqueous equivalent compositions for nucleic acid stability (DNA / RNA) in urine samples stored at 37°C±2°Cfor 3 days.Y]= Yes, [N]= No, [PS]= partial stability.

[0272] Surprisingly, a specific ion interaction was observed with the magnesium acetate component and propylene glycol as the non-aqueous solvent. It was determined that magnesium can be utilized in the non-aqueous solvent composition and still effectively maintain both DNA and RNA stability in a biological sample, after storage for 3 days (T3) at 37°C±2°C (Figures 37 and 38). In contrast, compositions containing lithium acetate can be utilized in an aqueous composition and effectively maintain nucleic acid, although the results were suboptimal compared to magnesium acetate. Surprisingly, the comparison between magnesium and lithium acetate within non-aqueous solvent compositions vs aqueous equivalent compositions demonstrate a specific ion interaction is occurring, providing effective nucleic acid stabilization with magnesium acetate and propylene glycol.

[0273] Finally, compositions (Table 23) containing magnesium acetate tetrahydrate in both aqueous and non-aqueous solvent systems were evaluated for compatibility with two commercially available extraction kits (Qiagen; QIAamp® Viral RNA Mini kit Cat No. 52904and Zymo Research; Quick-DNA / RNA™ Viral Magbead™ kit Cat No. R2130). Briefly, two male and two female urine samples were mixed with compositions from Table 23 at a 5:1 ratio and extracted at baseline (TO) and after storage at 37°C for 3 days (T3) as per the manufacturer's instructions for use. These compositions were compared to Aptima® urine transportation media (5:1 ratio) and unstabilized urine.Table 23: Summary of compositions evaluated comparing non-aqueous solvent compositions to aqueous equivalent compositions for nucleic acid stability (DNA / RNA) in urine samples stored at 37°C±2°Cfor 3 days.[Y]= Yes, [N]= No

[0274] Extracted DNA at both timepoints was amplified using qPCR for the human P-globin target and the delta Ct values (T3 - TO) were calculated (Figure 39). Extracted RNA was DNase treated and amplified using RT-qPCR for the GAPDH target via TaqMan® assay (Figure 40).

[0275] Once again, aqueous compositions containing magnesium acetate tetrahydrate failed to effectively maintain either DNA or RNA stabilization in a urine sample. In contrast, stability of DNA and RNA was maintained when stored in the non-aqueous solvent compositions containing propylene glycol. Importantly, non-aqueous solvent compositions were found to be effective at maintaining nucleic acid stabilization and were also compatible with two commercially available extraction kits.

[0276] EXAMPLE 11: Storage of Urine Samples Containing Bacterial Nucleic Acid

[0277] The present Example provides an evaluation of the impact of storing urine samples, co-spiked with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) bacteria and mixed with the present composition, at -80°C for 90 days on analyte (nucleic acid) detection, quantification, and long-term stability.

[0278] Transport and storage conditions to which collected biological samples are often subjected can greatly impact the integrity of analytes and / or biomolecules, such as nucleic acid, compromising the accurate detection and monitoring of disease and / or infection. In this example, long-term storage (-80°C for 90 days) of urine samples mixed with the present composition was investigated for its impact on the stability of nucleic acid and subsequent detection and quantification of DNA from microorganisms capable of causing sexually transmitted infection (STI), such as Chlamydia and Gonorrhea.

[0279] In this study with two cohorts (Male and Female), a minimum of 11 healthy adult donors (ages 18+) were recruited to collect first-void (FV) urine using a commercially available urine collection device, such as the neat Colli-Pee™ FV-5040 Device (no urine preservative; DNA Genotek, Inc., Canada). FV urine samples were pooled to create a final pool of urine per cohort. Each pool of urine was co-spiked with a "low" concentration of STI- causing micro-organisms, Chlamydia trachomatis (CT, 20 EB / mL for Males, 8 EB / mL for Females) and Neisseria gonorrhoeae (NG, 0.5 CFU / mL for Males and Females). The cospiked urine (3.5 mL per device) was added to Colli-Pee™ FV-004 Devices prefilled with 0.67 mL of the present composition per device. Alternately, co-spiked urine could be mixed with the present composition and stored in standard laboratory tubes or receptacles with appropriate caps, such as screwcaps. For this study, the non-aqueous storage composition comprised 100 mM HEDTA, 500 mM magnesium acetate tetrahydrate, 229 mM 4- dodecylbenzene sulfonic acid (4-DBS), and 50 mM Tween-20 in propylene glycol (pH 5.0).

[0280] A subset of urine samples mixed with the present composition, with or without a cospike of CT and NG, were processed immediately (baseline or TO), while the remaining samples were stored at -80°C for 90 days prior to processing and analysis. In a CLIA certified processing laboratory, each urine sample was analyzed using a representative (commercially-available), automated, nucleic acid diagnostic test which incorporates nucleicacid extraction and utilizes real-time polymerase chain reaction (rt-PCR), for the direct detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) DNA.

[0281] Storage of CT and NG co-spiked urine in the present composition at -80°C for up to 90 days did not negatively impact the ability to detect nucleic acid from Chlamydia trachomatis and Neisseria gonorrhoeae by rt-PCR, compared to baseline (Figure 41). For both CT and NG, DNA from these bacteria was readily detected and quantified in co-spiked urine samples from both female (Figure 41, Panel A) and male donors (Figure 41, panel B). By comparing baseline (TO) CT and NG co-spiked urine samples (never frozen) to CT and NG co-spiked urine samples stored at -80°C for 90 days, it was determined there was no statistical difference in the quantity of both CT and NG DNA targets in urine samples from both male and female donors. In this rt-PCR, the average delta Ct values ranged from -0.18 cycles to 0.22 cycles for CT and NG DNA, respectively, in male urine samples at baseline versus 90 days storage at -80°C. Similarly, the average delta Ct values ranged from 0.19 cycles to -0.10 cycles for CT and NG DNA, respectively, in female urine samples at baseline versus 90 days storage at -80°C. Hence, the present composition stabilizes bacterial nucleic acid in co-spiked urine samples, from female and male donors, for at least 90 days at -80°C and is compatible with and enables detection of CT and NG DNA over this period using a commercially-available, rt-PCR-based diagnostic test.

[0282] EXAMPLE 12: Storage of Nucleic Acid from Urine Samples Under Freeze / Thaw Cycle and Extreme Conditions

[0283] Nucleic acid in first-void urine samples were collected into a non-aqueous storage composition of the present application and co-spiked with CT and NG bacteria. As demonstrated in this example the CT and NG nucleic acid was stably stored even following repeated exposure of these urine samples to freeze / thaw cycles (-20°C to room temperature) or extreme temperature cycling profiles ( -10°C to +18°C or 22°C to 40°C), followed by an additional 33-days storage at 0°C or 32°C.

[0284] In this example, the suitability of the present composition for long-term stabilization of nucleic acid in collected urine samples spiked with sexually transmitted infection (STI)- causing microorganisms was investigated. These spiked urine samples were exposed to various transient temperature cycles to simulate the extreme conditions often encounteredduring shipping or transport from the point of collection (e.g. a home, clinic, or doctor's office) to the laboratory or during sample handling conditions within the laboratory, followed, in some cases, by long-term storage at extreme ambient temperatures (0°C or 32°C). Specifically, the post-collection stability of first -void (FV) urine samples co-spiked with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) bacteria and mixed with the present composition (5:1) was evaluated following: 1) exposure to 3 freeze / thaw cycles to simulate laboratory handling; 2) exposure to a simulated "Summer" transport profile (over 5 days) with an additional 33 days storage at 0°C or 32°C; and 3) exposure to a simulated "Winter" transport profile (over 5 days) with an additional 33 days storage at 0°C or 32°C.

[0285] In this study, 10 healthy adult male donors (ages 18+) were recruited to collect FV urine. While any commercially available collection device for FV urine can be used, the neat Colli-Pee™ FV-5040 Device (DNA Genotek, Inc., Canada) was utilized in this study to collect approximately 40 mL of FV urine from each donor. At baseline (TO), FV urine samples were pooled. For negative controls, aliquots (3.5 mL) of pooled urine were mixed with the present composition (0.67 mL) in a 5:1 ratio and then stored in neat Colli-Pee™ FV-004 Devices. Alternately, samples can be stored in standard laboratory tubes or receptacles with caps.

[0286] The remaining pool of FV urine was split into two fractions. One fraction was cospiked with a "low" concentration of STI-causing microorganisms, Chlamydia trachomatis (CT, 20 EB / mL) and Neisseria gonorrhoeae (NG, 0.5 CFU / mL), while the second fraction was co-spiked with a "medium" concentration of CT (50 EB / mL) and NG (2.5 CFU / mL). Low and medium co-spiked FV urine (3.5 mL per device) was dispensed into Colli-Pee™ FV-5004 Devices prefilled with 0.67 mL of the present composition and then subjected to the challenging temperature cycles described above. For this study, the non-aqueous storage composition comprised 100 mM HEDTA, 500 mM magnesium acetate tetrahydrate, 229 mM 4-dodecylbenzene sulfonic acid (4-DBS), and 50 mM Tween-20 in propylene glycol (pH 5.0). Alternately, co-spiked FV urine could also be mixed with the present composition and stored in standard laboratory tubes or receptacles with caps.

[0287] A subset of urine samples, with or without a co-spike of CT and NG, was processed immediately following urine collection (baseline, TO), while the remaining co-spiked urine samples were exposed to: 1) 3 freeze / thaw (F / T) cycles, where a cycle consisted of a minimum of 3 hours at -20°C, followed by a minimum of 3 hours at room temperature(20°C-26°C); 2) a simulated 5-day "Winter" (W) transport profile ( Table 24), followed by an additional 33 days storage at 0°C or 32°C; or 3) a simulated 5-day "Summer" (S) transport profile (Table 25), followed by an additional 33 days storage at 0°C or 32°C. Following these challenging conditions, all samples were shipped to a CLIA certified processing laboratory and analyzed using a representative (commercially available), automated, nucleic acid diagnostic test incorporating nucleic acid extraction and utilizing real-time polymerase chain reaction (rt-PCR) for the direct detection of CT and NG DNA.Table 24. "Winter" transport profile for CT and NG co-spiked FV urine samples stored in the present composition.Table 25. "Summer" transport profile for CT and NG co-spiked FV urine samples stored in the present composition.

[0288] In the freeze / thaw (F / T) study, 100% of CT and NG co-spiked FV urine samples (Negatives, Low Positive and Medium Positive) in the present composition resulted in the correct calls (Positive / Negative) relative to Baseline (TO), for both CT and NG targets, as measured by rt-PCR (Figure 42). Specifically, for Low CT Positive samples, the A Ct (Mean) was 0.17 cycles with a maximum A Ct of 1.56 cycles. For Medium CT Positive samples, the A Ct (Mean) was 0.14 cycles with a maximum A Ct of 0.49 cycles. For Low NG Positive samples,the A Ct (Mean) was 0.57 cycles with a maximum A Ct of 1.68 cycles. For Medium NG Positive samples, the A Ct (Mean) was 0.12 cycles with a maximum A Ct of 1.24 cycles. Hence, it was concluded that both CT and NG DNA targets were stable in the present composition mixed with FV urine, when subjected to freeze / thaw cycling conditions expected during routine laboratory processing.

[0289] In the simulated "Winter" and "Summer" transport studies, 100% of CT and NG cospiked FV urine samples (Negatives, Low Positive and Medium Positive) in the present composition resulted in the correct calls (Positive / Negative) for both CT and NG targets, as measured by rt-PCR (Figure 43).

[0290] Specifically, for Low CT Positive samples exposed to "Winter" transport conditions, the A Ct (Mean) was 0.45 cycles with a maximum A Ct of 1.39 cycles. For Medium CT Positive samples exposed to "Winter" transport conditions, the A Ct (Mean) was 0.56 cycles with a maximum A Ct of 1.25 cycles. For Low NG Positive samples exposed to "Winter" transport conditions, the A Ct (Mean) was 0.03 cycles with a maximum A Ct of 2.02 cycles. For Medium NG Positive samples exposed to "Winter" transport conditions, the A Ct (Mean) was 0.42 cycles with a maximum A Ct of 1.49 cycles.

[0291] Similarly, for Low CT Positive samples exposed to "Summer" transport conditions, the A Ct (Mean) was 0.09 cycles with a maximum A Ct of 1.25 cycles. For Medium CT Positive samples exposed to "Summer" transport conditions, the A Ct (Mean) was 0.23 cycles with a maximum A Ct of 0.66 cycles. For Low NG Positive samples exposed to "Summer" transport conditions, the A Ct (Mean) was 0.29 cycles with a maximum A Ct of 2.43 cycles. For Medium NG Positive samples exposed to "Summer" transport conditions, the A Ct (Mean) was 0.11 cycles with a maximum A Ct of 1.43 cycles.

[0292] Hence, it was concluded that both CT and NG DNA targets were stable in the present composition mixed with FV urine when subjected to "Winter" or "Summer" transport conditions.

[0293] Finally, when "Winter" and "Summer" transport conditions were followed by an additional 33 days at 0°C or 32°C, 100% of CT and NG co-spiked FV urine samples (Negatives, Low Positive and Medium Positive) in the present composition resulted in the correct calls (Positive / Negative) for both CT and NG targets, as measured by rt-PCR. Analysis of these rt-PCR results also indicated that both CT and NG targets were stable in the present composition mixed with FV urine and subjected to these extreme conditions (Figure 44).

[0294] Specifically, for Low CT Positive samples exposed to "Winter" transport conditions followed by a 33 day hold at 0°C, the A Ct (Mean) was 0.14 cycles with a maximum A Ct of 0.96 cycles. For Medium CT Positive samples, the A Ct (Mean) was -0.36 cycles with a maximum A Ct of -0.08 cycles. For Low CT Positive samples exposed to "Winter" transport conditions followed by a 33 day hold at 32°C, the A Ct (Mean) was -0.04 cycles with a maximum A Ct of 0.76 cycles. For Medium CT Positive samples, the A Ct (Mean) was -0.47 cycles with a maximum A Ct of 0.06 cycles.

[0295] Similarly, for Low NG Positive samples exposed to "Winter" transport conditions followed by a 33 day hold at 0°C, the A Ct (Mean) was 0.07 cycles with a maximum A Ct of 2.36 cycles. For Medium NG Positive samples, the A Ct (Mean) was 0.10 cycles with a maximum A Ct of 0.74 cycles. For Low NG Positive samples exposed to "Winter" transport conditions followed by a 33 day hold at 32°C, the A Ct (Mean) was 0.04 cycles with a maximum A Ct of 1.01 cycles. For Medium NG Positive samples, the A Ct (Mean) was 0.57 cycles with a maximum A Ct of 1.32 cycles.

[0296] For Low CT Positive samples exposed to "Summer" transport conditions followed by a 33 day hold at 0°C, the A Ct (Mean) was 0.09 cycles with a maximum A Ct of 0.90 cycles. For Medium CT Positive samples, the A Ct (Mean) was 0.56 cycles with a maximum A Ct of 0.79 cycles. Low CT Positive samples exposed to "Summer" transport conditions followed by a 33 day hold at 32°C, the A Ct (Mean) was 0.19 cycles with a maximum A Ct of 0.93 cycles. For Medium CT Positive samples, the A Ct (Mean) was 0.65 cycles with a maximum A Ct of 1.29 cycles.

[0297] Low NG Positive samples exposed to "Summer" transport conditions followed by a 33 day hold at 0°C, the A Ct (Mean) was -0.24 cycles with a maximum A Ct of 0.83 cycles. For Medium NG Positive samples, the A Ct (Mean) was 0.19 cycles with a maximum A Ct of 1.78 cycles. Low NG Positive samples exposed to "Summer" transport conditions followed by a 33 day hold at 32°C, the A Ct (Mean) was -0.04 cycles with a maximum A Ct of 1.10 cycles. For Medium NG Positive samples, the A Ct (Mean) was 0.50 cycles with a maximum A Ct of 1.09 cycles.

[0298] In summary, the results presented in this Example demonstrate stability CT and NG nucleic acid targets in the non-aqueous composition of the present application mixed with FV urine, even when subjected to extreme conditions.

[0299] EXAMPLE 13: Concentration of Nucleic Acid from Urine Samples

[0300] This Example demonstrates use of the non-aqueous storage composition of this application in separation and concentration of nucleic acid from urine samples following exposure to cold temperatures.

[0301] In this study, two cohorts (Male and Female) with 9 healthy adult donors (ages 18+) each were recruited to collect first-void (FV) urine using a commercially available device, such as the Colli-Pee™ FV-5040 Device (no urine preservative; DNA Genotek, Inc., Canada). FV urine samples (40 mL / donor) were pooled to create a male pooled (MP) and female pooled (FP) urine sample for each respective male or female cohort.

[0302] Commercially sourced, purified nucleic acid (NA) from three different microorganisms was spiked into the respective MP and FP urine samples, or nuclease-free (NF) water (Invitrogen, Catalog No. 10-977-023) as a control. The microorganisms used in the study were Trichomonas vaginalis (TV, 0.06 ng / pL; ATCC Catalog No. 30001D), Chlamydia trachomatis (CT, 0.007 ng / pL; ATCC Catalog No. VR-885D Serovar D, strain UX- 31Cx) and Neisseria gonorrhoeae (NG, 0.33 pg / pL (spiked into urine samples), 3.34 pg / pL (spiked into water samples); ATCC Catalog No. 49226). Next, CT, NG, and TV NA spiked urine or water samples were thoroughly mixed at a 5:1 ratio (1 mL of sample to 200 pL of present composition) with a non-aqueous storage composition containing 100 mM of HEDTA, 500 mM of Magnesium Acetate tetrahydrate, 229 mM of 4-DBS in propylene glycol (pH 4.81), or a surfactant-free non-aqueous composition containing 100 mM of HEDTA, 500 mM of Magnesium Acetate tetrahydrate in propylene glycol (pH 4.81). Urine and water samples spiked with CT, NG, and TV nucleic acid were subsequently divided into 1 mL aliquots and held briefly at room temperature (23°C±3°C) or frozen at -20°C for 7 days. Also, a positive spike-in control (C) was prepared in NF water with Trichomonas vaginalis (TV, 0.06 ng / pL), Chlamydia trachomatis (CT, 0.007 ng / pL) and Neisseria gonorrhoeae (NG, 3.34 pg / pL) and diluted at a 5:1 ratio.

[0303] Upon mixing water and urine samples at a 5:1 ratio with these compositions at room temperature, a distinct cloud formation or turbidity was observed immediately upon mixing water and urine samples with the composition containing the surfactant 4-DBS, whereas no cloud formation occurred when the water and urine samples were mixed with the composition containing no surfactant (Table 26).Table 26. Visual assessment of cloud formation in compositions with a surfactant (4-DBS) and without a surfactant when mixed with FV urine or water at room temperature (23°C±3°C).

[0304] As used herein, "cloud formation" refers to formation of a binary or multicomponent mixture, that becomes turbid due to phase separation. Cloud formation can occur when a homogeneous aqueous solution containing a surfactant is heated above a specific temperature and suddenly becomes visibly cloudy or turbid as a direct result of micelle rearrangement [1], This phenomenon results in the formation of two distinct phases, i.e., liquid-liquid or liquid-solid (a precipitate). When two distinct liquid phases are formed, one phase is enriched with surfactant, while the second aqueous phase contains surfactant at a concentration lower than the critical micellar concentration [1],

[0305] Next, six aliquots from each sample mixture (MP / FP urine and water) were collected. Three aliquots were processed immediately (baseline samples) while the remaining three aliquots were stored at -20°C for 7 days. At each time point (baseline and T7), the samples were processed as follows: the first aliquot was set aside and vortexed right before isolation and designated as a "Mixed" sample (M). The remaining two aliquots were spun down at 8,000 RPM for 1 minute. The smaller volume lower phase and / or precipitate in the sample was isolated and designated as the "Lower" phase (L). The supernatant was removed, designated as "Upper" phase (U), and was transferred to a separate tube after centrifugation. Surprisingly, in baseline centrifuged "L" aliquots, a distinct phase separation was observed in the water sample, while a white precipitate was observed in MP and FPurine samples. Interestingly, upon thawing the T7 samples at room temperature, a distinct visible brownish-yellow pellet or precipitate was observed in the urine samples mixed with the composition containing 100 mM of HEDTA, 500 mM of Magnesium Acetate tetrahydrate, 229 mM of 4-DBS in propylene glycol, while a visible phase separation occurred in water prior to centrifugation.

[0306] Nucleic acid was extracted from the respective Lower, Upper and Mixed aliquots using the Quick-DNA / RNA Magbead™ kit (Zymo, Catalog No. R2141). Specifically, 50 pL of each aliquot (L, M, U) was mixed with 150 pL of DNA / RNA Shield™ and then purified, as per the manufacturer's instructions (Zymo, Catalog No. R2141). Extracted nucleic acid was amplified using qPCR with a Bio-Rad C1000 Touch Thermal Cycler™ with cycling conditions consisting of 95°C for 20 seconds, 40 cycles of 95°C for 3 seconds to 60°C for 30 seconds using primer and probe sequences outlined in Table 27Error! Reference source not found, for CT, NG and TV.Table 27. Primer and probe sequences for CT, NG, TV qPCR assay (Integrated DNA Technologies).

[0307] Table 28Ct values for CT, NG, and TV (Table 28) obtained after qPCR of the material recovered from the L fractions from urine and water samples at baseline (i.e., prior to storage) were about 3.29 to 5.15 higher than the Ct values obtained using M or U fractions or a positive spike-in control (C). Surprisingly, indicating over 10-fold difference in analyte concentration in the U and M fraction for 7 of 9 samples, while the L fraction was significantly depleted of nucleic acid at baseline, prior to cold storage.Table 28 Ct Values (CT, NG, TV) for exogenous spiked nucleic acid from the "Lower" (L), "Upper" (U) aqueous phase and a completely "Mixed" (M) aliquot from respective FV urine samples and water samples at room temperature (23°C±3°C) at baseline, compared to a positive spike-in control (C).

[0308] The results of the qPCR for CT, NG and TV and Ct values are reported (Table 29) after storing samples for 7 days at -20°C. Remarkably, the respective Ct values for the L fractions from MP / FP urine samples were indicative of a marked concentration effect (~10-fold increase), corresponding to lower Ct values of about 2.45 to 3.90, when compared to M or U fractions. Compared to baseline, this resulted in a flip of the nucleic acid concentrated fraction after storage, with the exception of the water only samples.Table 29. Ct Values (CT, NG, TV) for exogenous spiked nucleic acid from the "Lower" (L), "Upper" (U) aqueous phase and a completely "Mixed" (M) sample from respective FV urine samples and water samples after storage of the mixtures at -20°Cfor 7 days, compared to a positive spike-in control (C).

[0309] These results demonstrate a remarkably simple method to concentrate nucleic acid from relatively large volumes of urine samples, whereby upon mixing the urine samples with the non-aqueous storage composition containing a surfactant (e.g., 4-DBS), followed by storage and centrifugation, the nucleic acid in the sample can be successfully partitioned in the smaller volume, lower phase. This mechanism appears to be in part mediated by cloud formation, with surfactant accumulating in the smaller volume, lower phase. This concentration effect is enhanced when the samples were stored under cold temperatures, but a similar result can be obtained during storage at ambient temperatures, per this Example.

[0310] Nucleic acid was not concentrated in L fractions in the water only samples, as Ct values for the L fraction were comparable to C and M fractions. This demonstrates that cloud formation is facilitated by use of the non-aqueous storage composition and may require the presence of biological material or salts to contribute to the process of phase separation.

[0311] One skilled in the art will recognize use of the non-aqueous storage composition for concentrating nucleic acid, and related method, can be practiced with other biological sample types and combined with other extraction techniques.

[0312] References[1] Halko R, Hagarova I, and Andruch V (2023) Innovative approaches in cloud-point extraction. Journal of Chromatography A. Volume 1701, 464053.[2] Venter JME, Mahlangu PM, Muller EE, Lewis DA, Rebe K, Struthers H, McIntyre J, Kularatne RS. Comparison of an in-house real-time duplex PCR assay with commercial HOLOGIC® APTIMA assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urine and extra-genital specimens. BMC Infect Dis. 2019 Jan 3;19(1):6. doi: 10.1186 / sl2879-018-3629-0. PMID: 30606127; PMCID: PMC6318993.[3] Caliendo AM, Jordan JA, Green AM, Ingersoll J, Diclemente RJ, Wingood GM. Real-time PCR improves detection of Trichomonas vaginalis infection compared with culture using self-collected vaginal swabs. Infect Dis Obstet Gynecol. 2005 Sep;13(3):145-50. doi: 10.1080 / 10647440500068248. PMID: 16126499; PMCID: PMC1784568.[4] Centers for Disease Control and Prevention. "Multiplex Assay for Detection of SARS-CoV-2." CDC, 2023, https: / / www.cdc.gov / coronavirus / 2019- ncov / lab / mu ltiplex.html.

[0313] EXAMPLE 14: Stabilization of Nucleic acid from Vaginal Swab Samples

[0314] The present Example provides a study of the use of the non-aqueous storage composition to stabilize nucleic acid in vaginal swab samples for at least 6 days at room temperature.

[0315] Vaginal swab samples are commonly used in healthcare for diagnostic testing, including the detection of sexually transmitted infections (STIs) and bacterial vaginosis (BV). Mucins are a major component of vaginal secretions, including cervical mucus. The mucinous nature of certain biological samples (e.g., saliva, throat and oral cavity samples, sputum, vaginal secretions) can contribute to difficulties in sample handling at the laboratory and have negative impacts on nucleic acid stability and extraction.

[0316] Six healthy donors self-collected vaginal swab samples with Purflock™ Ultra swabs (Puritan Medical Products Co., Catalog No. 3306-U), which were then placed into into tubes containing a non-aqueous storage composition according to one embodiment of the present application. For this study, the non-aqueous storage composition (100 mM HEDTA, 500 mM magnesium acetate tetrahydrate, 229 mM 4-dodecylbenzene sulfonic acid (4-DBS), and 50 mM Tween-20 in propylene glycol) was diluted 1 / 5 with nuclease-free water, prior to dispensing 1 mL of the mixture into each tube. Tubes containing swabs with collected vaginal samples were vortexed for 10 seconds and then stored at room temperature for 6 days. Interestingly, in the absence of a heating step and / or enzymatic treatment, the viscosity of each vaginal sample was clearly reduced upon mixture with the present composition, and this coincided with an increase in turbidity of the mixture, suggesting the rapid release of vaginal samples from the flocked swabs.

[0317] After 6 days storage at room temperature, total nucleic acid was extracted from a 200 pL aliquot of each vaginal sample using an OMNIgene™»XTRACT ULTRA extraction kit (DNA Genotek, Inc., Catalog No. OM-XTU-50) and following manufacturer's instructions. Extracted samples were run on the Agilent TapeStation™ for visualization of DNA (Figure 45) and RNA (Figure 46).

[0318] Interestingly, the present embodiment of the composition appears to rapidly release vaginal samples from flocked swabs, without the need for a heating step and / or enzymatic treatment. This released material contains nucleic acid (both DNA and RNA) which is stabilized by the present composition for at least 6 days at room temperature. As demonstrated in Figures 45 and 46, the DNA and RNA, respectively, extracted from these mucoid vaginal swab samples from most donors are high molecular weight and / or largely intact. The faint bands observed in Figures 45 and 46 for donor 3 can be explained by the relatively low yields of nucleic acid in this particular self-collected sample. With selfcollected biological sampling, including vaginal sampling, one can expect to see donor to donor variability, resulting in a wider range of nucleic acid concentrations recovered per sample type.

[0319] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent applications was specifically and individually indicated to be incorporated by reference.

[0320] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising:(a) a non-aqueous solvent that comprises dimethylsulfoxide, a glycol, glycerol or a mixture of two or more thereof;(b) one or more surfactant;(c) a buffer to provide a pH in the range of from about 4 to about 8, or from about4.6 to about 5.7; and(d) optionally, a chelator.

2. The composition according to claim, wherein the glycol is a compound having the structure of Formula I:I where:R1is absent or R1is a bivalent straight-chain, branched or cyclic hydrocarbon with 1 to 20, preferably 1 to 10, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'- -CR'=CR"-, or - CY'=CY"- in such a manner that O and / or S atoms are not linked directly to one another, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L; where each R2is independently H , F , Cl , CN , or straight-chain, branched or cyclic alkyl with 1 to 20, preferably 1 to 10, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'- -CR'=CR"-, or -CY'=CY"- in such a manner that O and / or S atoms are not linked directly to oneanother, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;L is F, Cl, -CN, -NC, -NCO, -NCS, -OCN, -SCN, -R', -OR', -SR', -C(=O)R'-, -C(=O)- OR'-, -NH2, -NHR', -NR'R", -SO3R', -SO2R', -OH, -NO2, -CF3, or optionally substituted silyl, carbyl or hydrocarbyl with 1 to 30 C atoms that is optionally substituted and optionally comprises one or more hetero atoms;R' and R" are each independently H or a straight-chain or branched alkyl with 1 to 6 C atoms; andY' and Y" are each independently H, F, Cl or CN.

3. The composition according to claim 2, wherein the glycol is propylene glycol, diethylene glycol, triethylene glycol, 1,2-butanediol, N-methyl diethanolamine, 2,2- thiodiethanol or a combination thereof.

4. The composition according to any one of claims 1 to 3, wherein the one or more surfactant comprises a surfactant compound of Formula IIII where:R3is CH, — O— , aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;each R4is independently H or a straight-chain, branched or cyclic alkyl with 1 to 20, preferably 10 to 18, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR'-, -SiR'R"-, -CR'=CR"-, -CY'=CY"- or -C=C- in such a manner that O and / or S atoms are not linked directly to one another , and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by a cationic or anionic group, or aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L, and wherein at least one R4is not H; each R', R", Y', Y", and L is as defined in claim 2;M is an ammonium compound, H+, Li+, Na+, K+, Ca2+, or Mg2+, preferably Li+, Na+, or Mg2+; and n is an integer between 1 and 2, and n is equal to the oxidation state of M.

5. The composition according to claim 4, wherein the one or more surfactant comprises sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, alkylbenzene sulfonic acid (e.g., dodecylbenzene sulfonic acid) or a combination of SDS with another surfactant, such as docusate sodium sulfate, or Tween 20 or a combination of alkylbenzene sulfonic acid with another surfactant, such as Tween 20.

6. The composition according to any one of claims 1 to 5, wherein the buffer is carboxylate of Formula III or a salt thereof,III where:R5is a straight-chain, branched or cyclic alkyl with 1 to 8, preferably 1 to 6, C atoms, in which one or more CH2 groups are optionally replaced by -O-, -S-, — C(=O)— , - C(=O)-O-, -O-C(=O)-, -C(=O)-OH, -C(=O)-OM, -NR6-, -NR'-, -CR'=CR"-, -CY'=CY"-or — C=C— in such a manner that O and / or S atoms are not linked directly to one another, and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I, C(=O)-OH, C(=O)-O_, or CN, and in which one or more CH2, or CH3 groups are optionally replaced by an aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein each of the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;R6is a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof; and each R', R", Y', Y", and L is as defined in claim 2.

7. The composition of claim 6, wherein the buffer comprises an acetate buffer, such as lithium acetate, magnesium acetate, tetraethyl ammonium acetate, sodium acetate, ammonium acetate, or a combination thereof, preferably, lithium acetate or magnesium acetate, or a combination thereof.

8. The composition according to any one of claims 1 to 7, wherein the chelator is present and comprises a compound of Formula IVFormula IV where: each R7is independently a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof; andR8is a bivalent straight-chain, branched or cyclic hydrocarbon with 1 to 12, preferably 1 to 8, C atoms, in which one or more CH2 groups are optionally replaced by -CR'R"-, -O-, -S-, -C(=O)- , -C(=O)-O-, -O-C(=O)-, -NR9-, -NR'-, -CR =CR"-, - CY'=CY"- or -C=C- in such a manner that O and / or S atoms are not linked directly to one another , and in which one or more H atoms are optionally replaced by OH, F , Cl , Br , I or CN, and in which one or more CH2, or CH3 groups are optionally replaced by an aryl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy or heteroaryloxy, wherein eachof the aforementioned cyclic groups has 5 to 20 ring atoms, is mono- or polycyclic, does optionally contain fused rings, and is unsubstituted or substituted by one or more identical or different groups L;R9is a straight-chain or branched alkyl with 1 to 6, preferably 1 to 4, C atoms, which terminates with a -C(=O)-OH, -OH, -C(=O)-O_or a salt thereof; and each R', R", Y', Y", and L is as defined in claim 2.

9. The composition according to claim 8, wherein the compound of Formula IV is a compound of Formula IVawhere: where R10and R11, together with the carbon atoms to which they are attached, form a cycloalkyl, such as a cyclohexyl.

10. The composition according to claim 8, wherein the chelator comprises ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetra acetic acid (EGTA), cyclohexanediaminetetraacetic acid (CDTA), 2,2',2",2’"-(l,2- propanediyldinitrilo)tetraacetic acid (PDTA), l,3-diamino-2-hydroxypropane- N,N,N',N'-tetraacetic acid or hydroxyethylethylenediaminetriacetic acid (HEDTA).

11. The composition according to any one of claims 1 to 10, further comprising one or more additive, such as an antioxidant, denaturing agent, antimicrobial agent, reducing agent, colorant, bittering agent, enzyme inhibitor (such as a protease, RNase inhibitor, or DNase inhibitor), emulsifying agent, defoamer or antifoaming agent, viscosity modifier, or coating additive.

12. The composition according to claim 11, wherein the one or more additive comprises one or more denaturing agent, such as, 5-su Ifosal icy lie acid, urea, or a combination thereof.

13. The composition according to any one of claims 1 to 12, wherein the composition comprises or consists of a liquid, gel, solid, semi-solid, slurry, film (coating), emulsion, powder, lyophilizate, cream or suspension.

14. The composition according to any one of claims 1 to 13 for use in stabilizing nucleic acid in or from a biological sample.

15. The composition according to any one of claims 1 to 13 for use in concentrating nucleic acid from the biological sample, wherein the biological sample is a liquid sample.

16. A kit for stabilizing nucleic acid contained in a biological sample at ambient temperature, the kit comprising:(a) a sample container having a resealable closure for receiving the biological sample;(b) the composition of any one of claims 1 to 13; and(c) instructions for use.

17. The kit according to claim 16, further comprising:(i) a means to transfer the biological sample, or a portion thereof, into the sample container; and / or(ii) a mixing or homogenization means, optionally contained within the sample container.

18. The kit of claim 16 or 17, wherein the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

19. The kit according to any one of claims 16 to 18, wherein the biological sample is a saliva sample, a buccal sample, a sputum sample, a blood sample, a plasma sample, a serum sample, a urine sample, a fecal sample, a rectal swab sample, a throat swab sample, a tongue swab or scrapper sample, a vaginal sample, a swab containing a biological material, a tissue sample, a skin sample, an environmental sample, a surface sample, a forensic sample, a soil sample, a sewage sample, a wastewater sample, or a water sample.

20. The kit according to any one of claims 16 to 19, wherein the nucleic acid is from a human, an animal, a bacterium, a virus, a parasite or plant.

21. The kit according to any one of claims 16 to 20, wherein the kit is additionally for concentrating the nucleic acid from the biological sample.

22. A method of stabilizing nucleic acid contained in a biological sample at ambient temperature comprising the steps of:(a) obtaining a biological sample comprising nucleic acid;(b) mixing the biological sample with the composition according to any one of claims 1 to 13; and(c) storing the mixture from step (b) at ambient temperature.

23. The method according to claim 22, wherein the biological sample is a saliva sample, a buccal sample, a sputum sample, a blood sample, a plasma sample, a serum sample, a urine sample, a fecal sample, a rectal swab sample, a throat swab sample, a tongue swab or scrapping sample, a vaginal sample, a swab containing a biological material, a tissue sample, a skin sample, an environmental sample, a surface sample, a forensic sample, a soil sample, a sewage sample, a wastewater sample, or a water sample, and optionally wherein, the nucleic acid are from a human, an animal, a bacterium, a virus, a parasite or plant.

24. The method according to claim 22 or 23, wherein in step (c) storage is for a time sufficient to permit phase separation of the mixture into an upper and a lower phase, and wherein the method additionally comprises collecting the phase containing concentrated nucleic acid.

25. A method of concentrating nucleic acid or other analyte of interest contained in a liquid biological sample comprising the steps of:(a) obtaining the liquid biological sample comprising nucleic acid;(b) mixing the biological sample with the composition according to any one of claims 1 to 13;(c) incubating the mixture from step (b) at an incubation temperature for an incubation time sufficient to permit phase separation of the mixture into an upper and a lower phase; and(d) collecting the phase containing the concentrated nucleic acid or other analyte(s) of interest.

26. The method according to claim 25, wherein the incubation temperature is from about 35°C to about -80°C, from about 20°C to about -20°C, or from about 0°C to about -20°C, or about -20°C.

27. The method according to claim 25 or 26, wherein in step (c) the mixture is incubated for an incubation time of from about 1 minute to about 1 week, or at least one minute, at least 5 minutes, at least 15 minutes, at least 1 hour, at least 4 hours, at least 8 hours, at least 1 day, at least 2 days, at least 5 days or at least a week before step (d).

28. The method according to any one of claims 25 to 27, wherein the nucleic acid or other analyte(s) of interest is concentrated by at least 2 fold, at least 5 fold, at least 10 fold or greater than 10 fold over the concentration of the nucleic acid or other analyte(s) of interest in the biological sample.

29. The method according to any one of claims 25 to 28, wherein the method additionally comprises:(i) storing the collected phase containing the concentrating nucleic acid or other analyte(s) of interest;(ii) extracting the nucleic acid from the collected phase; or(iii) analyzing the nucleic acid in the collected phase.

30. The method according to any one of claims 25 to 29, wherein: the biological sample is a plasma sample, a urine sample, a sewage sample, a wastewater sample, a water sample or an liquid sample obtained from extraction of a swab, such as a surface swab, a buccal swab, a tongue swab or scrapping, a throat swab, a nasopharyngeal swab, a rectal swab, a vaginal swab, or a skin swab; and optionally wherein, the nucleic acid is from a human, an animal, a bacterium, a virus, a parasite or plant.

31. The method according to any one of claims 25 to 30, wherein the composition comprises an anionic surfactant, such as a linear sulfonic acid (e.g., SDS) or an alkylbenzene sulfonic acid (e.g. 4-DBS).

Images