Compositions and methods for storing biological samples - Patents.com

Abstract

The present invention relates to an aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising a first component comprising: i) an anionic surfactant in an amount of about 1%-20% (w / v); ii) a Group I metal hydroxide in an amount of about 1%-5% (w / v); iii) a chelating agent in an amount of about 0.5%-5% (w / v); and iv) a first buffer, wherein the composition further comprises a diluent component selected from the group consisting of water, a second buffer, a transport fluid, sodium chloride (aq), and combinations thereof, wherein the first component has been diluted with the diluent component to a maximum of about 50% (v / v).

Description

[Technical field]

[0001] The present invention relates to an aqueous composition for storing biological samples for subsequent nucleic acid testing, comprising an anionic surfactant, a chelating agent, a Group I metal hydroxide, and a buffering agent. The composition of the present invention provides for a) obtaining the sample, b) lysis of viruses, cells or tissues to render the nucleic acid free of cellular debris and extraneous biomolecules, resulting in c) inactivation of viruses, bacteria, fungi, parasites and other microorganisms present in the sample, d) protection of the nucleic acid from degradation by nuclease activity, and preservation of the nucleic acid for subsequent isolation, detection, amplification, and / or molecular analysis. The five functions can be accomplished using a single composition and in a single reaction vessel, and the resulting sample can be stored at room temperature for extended periods of time without significant degradation of the polynucleotides contained within the sample. [Background technology]

[0002] The current COVID-19 pandemic has created an urgent need for rapid and reliable protocols for the diagnosis of viral pathogens in a large number of individuals. COVID-19 is caused by SARS-CoV-2, a positive-sense single-stranded RNA virus. Diagnosis of this disease and other common diseases caused by viruses and other pathogens is based on the analysis of the pathogen's nucleic acid. The development of nucleic acid-based detection platforms such as real-time PCR, transcription-mediated amplification (TMA), ligase chain reaction (LCR), microarrays, next-generation sequencing (NSG), and pathogen gene chips has significantly transformed the field of clinical molecular diagnostics. However, nucleic acids in biological samples degrade and / or denature rapidly at room temperature, and if diagnostic analysis is to be performed, the ability to keep said nucleic acids stable often determines whether the nucleic acid can be successfully analyzed. This is even more important when the nucleic acid desired for downstream analysis comprises, for example, ribonucleic acid (RNA), which is particularly susceptible to degradation by endonuclease and exonuclease activity.

[0003] Another significant concern when using biological samples is the potential inoculation, release, or seeding of live infectious agents or biological materials from the sample to the environment. If the sample is kept viable and / or biologically intact to preserve its integrity for testing, individuals involved in the collection, transportation, and testing process are potentially exposed to highly dangerous infections. As a result, the required safety measures typically increase the cost and effort required to transport such samples from one location to another and complete their analysis.

[0004] Thus, there is a need for safe collection, transport and storage compositions that preserve the integrity of nucleic acids even for hazardous biological samples, typically for further molecular analysis or diagnostic testing, without exposing personnel to risk.

[0005] Current protocols for handling biological samples containing nucleic acids from individuals suspected of having a viral or bacterial infection include the collection of the sample and the use of a collection medium that allows for the storage of the nucleic acid until diagnostic or molecular biological testing is performed.

[0006] For detection of SARS-CoV-2 and other respiratory viruses, collection of biological samples is usually performed by using a swab, which is then introduced into a test tube containing a collection medium, often referred to as viral transport fluid (VTM). VTM typically contains buffering proteins (serum, albumin or gelatin) and antibiotics to inhibit the growth of contaminating bacteria and fungi. For example, the CDC has recommended a viral transport fluid (VTM) for SARS-CoV-2 containing Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), gentamicin and amphotericin B (US Centers for Disease Control and Prevention SOP#: DSR-052-05).

[0007] Commercially produced VTM is available in screw-cap plastic test tubes. Nasal, nasopharyngeal or oropharyngeal swabs are directly introduced into the test tube containing the VTM solution until molecular testing is performed. The tube is then vortexed and the sample is transferred to a second tube containing an extraction buffer. The extraction buffer generally contains the reagents necessary to inactivate the virus, lyse the cells so that the nucleic acid is free of cellular debris and other biomolecules, and protect the nucleic acid from degradation by endonuclease activity. The transfer must be performed under a safety cabinet to avoid the spread of the virus, which increases the steps before molecular testing and delays the preparation of the sample. Thus, currently available protocols for collecting and processing samples have proven inadequate to meet the needs of pandemic situations, as well as other situations in which large numbers of samples must be collected and processed quickly.

[0008] Moreover, extraction buffers often contain harmful chaotropic agents such as guanidine thiocyanate, which acts as a denaturant for macromolecules and inactivates viruses.Guanidine thiocyanate can reduce the degradation of RNA molecules in samples by inactivating RNase, thus preserving the integrity of nucleic acids, which is fundamental for applications in molecular diagnostic methodologies where RNA integrity is essential.However, said chaotropic agents are harmful when in contact with skin, and may further interact with other reagents frequently used in test devices, such as sodium hypochlorite, to produce toxic gases. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, when it is necessary to rapidly collect, store and analyze a large number of samples suspected of containing pathogens, it is critical to reduce the number of protocol steps while reducing the risk of spreading the pathogen. [Means for solving the problem]

[0010] The inventors of the present invention have developed a novel composition that surprisingly inactivates any pathogens present in a sample while preserving the nucleic acid for further molecular testing. In addition, since the composition itself can lyse viruses and cells, and also inactivate endonucleases and exonucleases for preserving the nucleic acid, the composition can be used directly to collect the sample without the need for any transfer to a second composition or buffer. In addition, the composition is compatible with nucleic acid-based detection platforms, especially with commonly used sterilants such as sodium hypochlorite, and reduces the health risks associated with sample manipulation.

[0011] The invention also encompasses methods utilizing the compositions that can advantageously improve conventional harvesting, lysis, inactivation, storage and preservation methods for the preparation of nucleic acids from one or more biological sources.

[0012] overview The present invention relates to an aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising a first component comprising: i) an anionic surfactant in an amount of about 1%-20% (w / v); ii) a Group I metal hydroxide in an amount of about 1%-5% (w / v); iii) a chelating agent in an amount of about 0.5%-5% (w / v); and iv) a first buffer, the composition further comprising a diluent component selected from the group consisting of water, a second buffer, a transport medium, sodium chloride (aq), and combinations thereof, wherein the first component has been diluted with the diluent component to a maximum of about 50% (v / v).

[0013] In some embodiments, the present invention relates to an aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising an anionic surfactant in an amount of about 1%-20% (w / v), a Group I metal hydroxide in an amount of about 1%-5% (w / v), a chelating agent in an amount of about 0.5%-5% (w / v), and a first buffering agent, the composition being diluted up to about 50% (v / v) with a diluent component selected from the group consisting of water, a second buffering agent, a transport fluid, and sodium chloride (aq), or any combination thereof.

[0014] In some embodiments, the diluent component is sodium chloride (aq). In some preferred embodiments, the sodium chloride (aq) is 0.9% (w / v) sodium chloride.

[0015] In some embodiments, the anionic surfactant is a C 20 Alkyl sulfate anion, C5-C 20 Alkenyl sulfate anion, C5-C 20 alkynyl sulfate anion, or any combination thereof. In some preferred embodiments, the surfactant comprises lauryl sulfate anion.

[0016] In some embodiments, the first buffering agent is selected from Tris, MES, Bis-Tris, HEPES, MOPS, citrate, sodium bicarbonate, sodium phosphate, and combinations thereof. In some preferred embodiments, the first buffering agent comprises HEPES. In some embodiments, the first buffering agent is present in an amount of about 5%-30% (w / v). In some embodiments, the buffering agent is present in an amount sufficient to maintain a pH of 5-10.

[0017] In some embodiments, the Group I metal hydroxide is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, and combinations thereof, hi some embodiments, the Group I metal hydroxide comprises lithium hydroxide.

[0018] In some embodiments, the chelating agent is selected from EGTA, HEDTA, DTPA, NTA, EDTA, succinic acid, anhydrous citrate, sodium citrate, calcium citrate, ammonium citrate, ammonium dicitrate, citric acid, diammonium citrate, potassium citrate, magnesium citrate, ferric ammonium citrate, lithium citrate, or combinations thereof. In some embodiments, the chelating agent is selected from succinic acid, EGTA, EDTA, and combinations thereof.

[0019] In some embodiments of the aqueous compositions of the present invention, the first buffer is present in an amount of about 10%-25% (w / v), the surfactant is present in an amount of about 2%-15% (w / v), the chelating agent is present in an amount of about 1%-4% (w / v), and the hydroxide is present in an amount of about 1%-4% (w / v).

[0020] In some embodiments, the pH of the aqueous composition is from 5 to 10. In some embodiments, the pH of the aqueous composition is from 6 to 9.

[0021] In some embodiments, the aqueous compositions of the present invention further comprise an antifoaming agent in an amount of about 50 μL / L to 750 μL / L. In some preferred embodiments, the antifoaming agent comprises a silicone polymer, a polysorbate, an organic polyether dispersion, or a combination thereof.

[0022] In some embodiments, the aqueous compositions of the present invention further comprise a population of polynucleotides comprising RNA, DNA, or any combination thereof.

[0023] In some embodiments, the RNA comprises an in vitro synthesized transcript.

[0024] In another aspect, the present invention relates to an aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising: i) an anionic surfactant in an amount of about 1%-20% (w / v); ii) a Group I metal hydroxide in an amount of about 1%-5% (w / v); iii) a chelating agent in an amount of about 0.5%-5% (w / v); and iv) a first buffering agent, wherein the composition comprises an antifoaming agent in an amount of about 50 μL / L-750 μL / L.

[0025] In some embodiments, the antifoaming agent comprises a silicone polymer, a polysorbate, an organic polyether dispersion, or any combination thereof.

[0026] In some embodiments, the anionic surfactant comprises a C5-C20 alkyl sulfate anion, a C5-C20 alkenyl sulfate anion, a C5-C20 alkynyl sulfate anion, or any combination thereof. In some preferred embodiments, the surfactant comprises a lauryl sulfate anion.

[0027] In some embodiments, the first buffer is selected from Tris, MES, Bis-Tris, HEPES, MOPS, citrate, sodium bicarbonate, sodium phosphate, and combinations thereof, hi some preferred embodiments, the first buffer comprises HEPES.

[0028] In some embodiments, the first buffering agent is present in an amount of about 5% to 30% (w / v).

[0029] In some embodiments, the first buffering agent is present in an amount sufficient to maintain a pH of 5-10.

[0030] In some embodiments, the Group I metal hydroxide is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, and combinations thereof, hi some preferred embodiments, the Group I metal hydroxide comprises lithium hydroxide.

[0031] In some embodiments, the chelating agent is selected from EGTA, HEDTA, DTPA, NTA, EDTA, succinic acid, anhydrous citrate, sodium citrate, calcium citrate, ammonium citrate, ammonium dicitrate, citric acid, diammonium citrate, potassium citrate, magnesium citrate, ferric ammonium citrate, lithium citrate, and combinations thereof. In some preferred embodiments, the chelating agent is selected from succinic acid, EGTA, EDTA, and combinations thereof.

[0032] In some embodiments, the first buffering agent is present in an amount of about 10%-25% (w / v), the anionic surfactant is present in an amount of about 2%-15% (w / v), the chelating agent is present in an amount of about 1%-4% (w / v), and the Group I metal hydroxide is present in an amount of about 1%-4% (w / v).

[0033] In some embodiments, the pH of the aqueous composition is from 5 to 10. In some preferred embodiments, the pH of the aqueous composition is from 6 to 9.

[0034] In some embodiments, the composition is diluted up to about 50% (v / v) with a component selected from the group consisting of water, a second buffer, a transport fluid, sodium chloride (aq), and combinations thereof. In some preferred embodiments, the composition is diluted with sodium chloride (aq).

[0035] In some embodiments, the compositions of the invention further comprise a population of polynucleotides comprising RNA, DNA, or any combination thereof, hi some preferred embodiments, the RNA comprises an in vitro synthetic transcript.

[0036] The present invention also relates to a method for obtaining a population of polynucleotides from a sample suspected of containing nucleic acids, comprising the step of contacting the sample with an aqueous composition of the present invention at a temperature in the range of 2°C to 40°C.

[0037] In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the nucleic acid is RNA and / or DNA. In some preferred embodiments, the RNA comprises an in vitro synthetic transcript. In some embodiments, the nucleic acid is derived from at least one pathogen. In some preferred embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In preferred embodiments, the at least one pathogen is a fungus. In preferred embodiments, the at least one pathogen is a bacterium. In some preferred embodiments, the at least one pathogen is a parasite. In preferred embodiments, the at least one pathogen is a virus. In preferred embodiments, the virus is SARS-CoV-2.

[0038] In some embodiments, the method for obtaining a population of polynucleotides from a sample suspected of containing nucleic acids further comprises an amplification reaction for amplifying at least a target nucleic acid and detecting at least one target in the resulting amplification products.

[0039] The present invention also relates to a method for preserving the integrity of a population of polynucleotides in a sample comprising contacting the sample with an aqueous composition according to the present invention at a temperature ranging from 4° C. to 40° C. In some preferred embodiments, the sample after contacting with the aqueous composition of the present invention is stored at a temperature ranging from −25° C. to 40° C. for at least 1 hour up to 6 months, up to 9 months, up to 12 months, or up to 26 months. In some preferred embodiments, the sample after contacting with the aqueous composition is stored at −20° C., 4° C., RT, or 30° C.

[0040] In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the population of polynucleotides is a population of RNA and / or DNA. In some preferred embodiments, the RNA comprises in vitro synthetic transcripts. In some embodiments, the population of polynucleotides is derived from at least one pathogen. In some preferred embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In preferred embodiments, the at least one pathogen is a fungus. In preferred embodiments, the at least one pathogen is a bacterium. In some preferred embodiments, the at least one pathogen is a parasite. In preferred embodiments, the at least one pathogen is a virus. In preferred embodiments, the virus is SARS-CoV-2.

[0041] The present invention also relates to a method for inactivating a sample containing at least one pathogen, comprising contacting the sample with an aqueous composition according to the present invention, the aqueous composition representing at least 30% (v / v) of the resulting mixture. In some embodiments, the sample is contacted with the aqueous composition at 4°C, RT or 30°C. In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the at least one pathogen is a fungus, a bacterium, a parasite or a virus. In preferred embodiments, the at least one pathogen is a fungus. In preferred embodiments, the at least one pathogen is a bacterium. In some preferred embodiments, the at least one pathogen is a parasite. In preferred embodiments, the at least one pathogen is a virus. In preferred embodiments, the virus is SARS-CoV-2.

[0042] The present invention also relates to a method for obtaining a sample comprising contacting the sample with an aqueous composition according to the present invention, the aqueous composition representing at least 30% (v / v) of the resulting mixture, in some embodiments, the sample is a biological sample or an environmental sample.

[0043] In some embodiments, the sample is introduced directly into a collection device or collection vessel containing an aqueous composition according to the present invention.

[0044] In other embodiments, the sample is collected using a swab and introducing it into a collection device or collection vessel containing an aqueous composition according to the invention, hi some preferred embodiments, the swab is discarded after being rubbed against the collection device or collection vessel.

[0045] In some embodiments, the sample comprises at least one pathogen. In some embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In preferred embodiments, the at least one pathogen is a fungus. In preferred embodiments, the at least one pathogen is a bacterium. In some preferred embodiments, the at least one pathogen is a parasite. In preferred embodiments, the at least one pathogen is a virus. In preferred embodiments, the virus is SARS-CoV-2.

[0046] The present invention also relates to a collection device or container comprising an aqueous composition according to the present invention.

[0047] The present invention also relates to a sample collection kit comprising a collection device or collection vessel and an aqueous composition according to the present invention. In some embodiments, the sample collection kit further comprises a swab, curette, or culture loop.

[0048] The present invention also relates to the use of an aqueous composition according to the present invention for obtaining a sample suspected of containing a target nucleic acid. In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the sample comprises at least one pathogen. In some embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In a preferred embodiment, the at least one pathogen is a fungus. In a preferred embodiment, the at least one pathogen is a bacterium. In some preferred embodiments, the at least one pathogen is a parasite. In a preferred embodiment, the at least one pathogen is a virus. In a preferred embodiment, the virus is SARS-CoV-2. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 shows a photograph of a 96-well plate containing SARS-CoV-2 infected and uninfected Vero cells stained with crystal violet. [Diagram 2] FIG. 2 shows the stability of positive SeraCare and BEI samples stored at 4° C. or 30° C. for up to 6 months. FIG. 2 shows the % reactivity. [Diagram 3] Figure 3 shows the stability of positive SeraCare and BEI samples stored at 4° C. or 30° C. for up to 6 months. Figure 3 shows analyte RLU values ​​versus time. [Figure 4] Figure 4 shows the stability of positive SeraCare and BEI samples stored at 4° C. or 30° C. for up to 6 months. Figure 4 shows the internal control (IC) RLU values ​​over time. [Diagram 5] Figure 5 shows the stability of positive SeraCare and BEI samples stored at 4° C. or 30° C. for up to 6 months. Figure 5 shows Analyte-S / CO RLU values ​​versus time. [Figure 6] FIG. 1 shows the stability of Babesia IVT samples (500c / mL) stored at 5° C. for up to 12 months. [Figure 7-1]FIG. 1 shows the stability of Babesia IVT samples (100, 30 and 500 c / mL) stored at −20° C. for up to 26 months. [Figure 7-2] FIG. 1 shows the stability of Babesia IVT samples (100, 30 and 500 c / mL) stored at −20° C. for up to 26 months. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] The words "comprise" and "having", when used herein with reference to the present invention, are used to specify the presence of stated features, integers, steps or components, but do not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0051] It should be understood by those skilled in the art that the specific embodiments disclosed herein should not be read in isolation, and that the specification intends that the disclosed embodiments be read in combination with each other as opposed to individually, and thus each embodiment can serve as a basis for modifying or limiting other embodiments disclosed herein.

[0052] Concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a range. Such range formats are used merely for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values ​​specified as the limits of the range, but also all individual numerical values ​​and subranges subsumed within the range as if each numerical value or subrange were specified. By way of example, a numerical range of "10-100" should be interpreted not only to include the specified value of 10-100, but also to include the individual values ​​and subranges within the range indicated. Thus, individual values ​​such as 10, 11, 12, 13...97, 98, 99, 100, etc., as well as subranges such as 10-40, 25-40, and 50-60, etc., are included in this numerical range. This same principle applies to ranges in which only one numerical value is recited, such as "at least 10". Moreover, such an interpretation should apply regardless of the breadth of the range or feature being recited.

[0053] Compositions of the Invention In a first aspect, the present invention relates to an aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising a first component comprising: i) an anionic surfactant in an amount of about 1%-20% (w / v); ii) a Group I metal hydroxide in an amount of about 1%-5% (w / v); iii) a chelating agent in an amount of about 0.5%-5% (w / v); and iv) a first buffer, wherein the composition further comprises a diluent component selected from the group consisting of water, a second buffer, a transport fluid, sodium chloride (aq), and combinations thereof, and wherein the first component has been diluted with the diluent component to a maximum of about 50% (v / v).

[0054] In a second aspect, the present invention relates to an aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising: i) an anionic surfactant in an amount of about 1%-20% (w / v); ii) a Group I metal hydroxide in an amount of about 1%-5% (w / v); iii) a chelating agent in an amount of about 0.5%-5% (w / v); and iv) a first buffering agent, wherein the composition comprises an antifoaming agent in an amount of about 50 μL / L-750 μL / L.

[0055] In another aspect, the present invention relates to an aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising an anionic surfactant in an amount of about 1%-20% (w / v), a Group I metal hydroxide in an amount of about 1%-5% (w / v), a chelating agent in an amount of about 0.5%-5% (w / v), and a first buffering agent, the composition being diluted up to about 50% (v / v) with a diluent component selected from the group consisting of water, a second buffering agent, a transport fluid, and sodium chloride (aq), or any combination thereof.

[0056] In some preferred embodiments, the sodium chloride (aq) is 0.9% (w / v) sodium chloride.

[0057] In some embodiments, the anionic surfactant is a C 20 Alkyl sulfate anion, C5-C 20 Alkenyl sulfate anion, C5-C 20 In some embodiments, the surfactant comprises a lauryl sulfate anion. In some embodiments, the surfactant comprises lithium lauryl sulfate (LLS) or sodium dodecyl sulfate (SDS). In some embodiments, the surfactant comprises an LLS.

[0058] In some embodiments, the amount of anionic surfactant in the aqueous composition of the present invention is about 1% to 20% (w / v), about 2% to 20% (w / v), about 3% to 20% (w / v), about 5% to 20% (w / v), about 7% to 20% (w / v), about 9% to 20% (w / v), about 10% to 20% (w / v), about 15% to 20% (w / v), about 1% to 15% (w / v), about 1% to 12% (w / v), about 1% to 10% (w / v), about 1% to 9% (w / v), about 1% to 7% (w / v), or about 1% to 5% (w / v). In some embodiments, the amount of anionic surfactant in the compositions of the present invention is about 2%-15% (w / v), about 3%-12% (w / v), about 5%-10% (w / v), or about 7%-9% (w / v).

[0059] The term "buffering agent" as used herein means a weak acid or base used to maintain the pH of a solution. In some embodiments, the buffering agent is selected from tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 1,3-bis(tris(hydroxymethyl)methylamino)propane (Bis-Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), citrate, sodium bicarbonate, sodium phosphate, and combinations thereof. In a preferred embodiment, the buffering agent comprises HEPES.

[0060] In some embodiments, the amount of buffer in the aqueous composition of the present invention is 5%-30% (w / v), 5%-25% (w / v), 5%-20% (w / v), 5%-15% (w / v), about 5%-10% (w / v), about 10%-30% (w / v), about 15%-30% (w / v), or about 20%-30% (w / v). In some embodiments, the amount of buffer in the composition of the present invention is about 10%-25% (w / v), about 10%-22% (w / v), about 12%-20% (w / v), or about 15%-18% (w / v).

[0061] In some embodiments, the buffering agent is present in the aqueous composition of the present invention in an amount sufficient to maintain a pH of 5 to 10. More preferably, the pH is 6 to 9, 6.5 to 8.5, 7 to 8, or 7.2 to 7.8. In other embodiments, the pH is 6 to 10, 7 to 10, or 7.5 to 10. In other embodiments, the pH is 5 to 9, 5 to 8, 5 to 7.8, 5 to 7.5, or 5 to 7.2.

[0062] In some embodiments, the Group I metal hydroxide is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, and combinations thereof, hi more preferred embodiments, the Group I metal hydroxide is lithium hydroxide.

[0063] In some embodiments, the amount of Group I metal hydroxide in the composition of the invention is about 1%-5% (w / v), about 1%-4.5% (w / v), about 1%-4% (w / v), about 1%-3.5% (w / v), about 1%-3% (w / v), about 1%-2.5% (w / v), about 1.5%-5% (w / v), about 2%-5% (w / v), about 2.5%-5% (w / v), about 3%-5% (w / v), or about 3.5%-5% (w / v). In some embodiments, the amount of base in the composition of the invention is about 1.5%-4.5% (w / v), about 2%-4% (w / v), about 2.5%-3.5% (w / v), or about 2.5%-3% (w / v).

[0064] The term "chelating agent" as used herein means a compound that reacts with a metal ion to form a complex ring structure called a chelate. In some embodiments, the chelating agent is selected from ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (NTA), ethylenediaminetetraacetic acid (EDTA), succinic acid, anhydrous citrate, sodium citrate, calcium citrate, ammonium citrate, ammonium dicitrate, citric acid, diammonium citrate, potassium citrate, magnesium citrate, ferric ammonium citrate, lithium citrate, and combinations thereof. In preferred embodiments, the chelating agent is selected from succinic acid, EGTA, EDTA, and combinations thereof. In some preferred embodiments, the chelating agent is succinic acid.

[0065] In some embodiments, the amount of chelating agent in the composition of the present invention is about 0.5%-5% (w / v), about 0.5%-4.5% (w / v), about 0.5%-4% (w / v), about 0.5%-3.5% (w / v), about 0.5%-3% (w / v), about 0.5%-2.5% (w / v), about 0.5%-2% (w / v), about 0.5%-1.5% (w / v), about 1%-5% (w / v), about 1.5%-5% (w / v), about 2%-5% (w / v), about 2.5%-5% (w / v), about 3%-5% (w / v), about 3.5%-5% (w / v), or about 4%-5% (w / v). In some embodiments, the amount of chelating agent in the compositions of the present invention is about 1%-4% (w / v), about 1.5%-4% (w / v), about 2%-3% (w / v), or about 2.5%-3% (w / v).

[0066] In some embodiments of the aqueous compositions of the present invention, the buffer is present in an amount of about 10%-25% (w / v), the anionic surfactant is present in an amount of about 2%-15% (w / v), the chelating agent is present in an amount of about 1%-4% (w / v), and the Group I metal hydroxide is present in an amount of about 1%-4% (w / v).

[0067] In some embodiments of the aqueous compositions of the present invention, the buffer is present in an amount of about 15%-20% (w / v), the anionic surfactant is present in an amount of about 5%-10% (w / v), the chelating agent is present in an amount of about 2%-3% (w / v), and the Group I metal hydroxide is present in an amount of about 2%-3% (w / v).

[0068] In some embodiments, the aqueous composition of the present invention has a pH of 5 to 10. More preferably, the pH of the aqueous composition of the present invention is 6 to 9, 6.5 to 8.5, 7 to 8, or 7.2 to 7.8. In other embodiments, the pH is 6 to 10, 7 to 10, or 7.5 to 10. In other embodiments, the pH is 5 to 9, 5 to 8, 5 to 7.8, 5 to 7.5, or 5 to 7.2.

[0069] The term "antifoaming agent" or "antifoaming agent" as used herein means a chemical additive that reduces and prevents the formation of foam in a liquid composition. In the context of the present invention, said antifoaming agent prevents the formation of air bubbles that typically occur due to the presence of surfactants in the formulation and / or facilitates pipetting and handling of the disclosed compositions. In some embodiments, the compositions of the present invention further comprise an antifoaming agent. In some embodiments, the amount of antifoaming agent is between 50 μL / L and 750 μL / L, between 50 μL / L and 600 μL / L, between 50 μL / L and 500 μL / L, between 50 μL / L and 400 μL / L, between 50 μL / L and 300 μL / L, between 50 μL / L and 250 μL / L, between 50 μL / L and 200 μL / L, between 50 μL / L and 150 μL / L, between 75 μL / L and 750 μL / L, between 100 μL / L and 750 μL / L, between 150 μL / L and 750 μL / L, between 200 μL / L and 750 μL / L, between 250 μL / L and 750 μL / L, between 300 μL / L and 750 μL / L, or between 500 μL / L and 750 μL / L. In some preferred embodiments, the amount of antifoaming agent is 75 μL / L to 600 μL / L, 100 μL / L to 500 μL / L, 150 μL / L to 400 μL / L, or 200 μL / L to 300 μL / L. Exemplary antifoaming agents for the present invention include, but are not limited to, cocoamidopropyl hydroxysultaine, alkylaminopropionic acid, imidazoline carboxylate, sulfobetaine, sultaine, alkylphenol ethoxylate, alcohol ethoxylate, polyoxyethylenated polyoxypropylene glycol, polyoxyethylenated mercaptan, long chain carboxylic acid ester, alkanolamide, tertiary acetylene glycol, polyoxyethylenated silicone, N-alkylpyrrolidone, alkylpolyglycosidase, silicone polymer, polysorbate, organic polyether dispersion, or combinations thereof. In some preferred embodiments, the antifoaming agent comprises a silicone polymer, a polysorbate, an organic polyether dispersion, or a combination thereof. In more preferred embodiments, the antifoaming agent comprises a silicone polymer. In even more preferred embodiments, the silicone polymer is a three-dimensional siloxane.In an even more preferred embodiment, the antifoam agent comprises Foam Ban® MS-575.

[0070] For example, an antifoam agent can be included in the aqueous composition when the sample is to be analyzed in a platform that is prone to error when air bubbles are present in the sample.

[0071] The compositions of the present invention are diluted up to about 50% (v / v) with a component selected from the group consisting of water, a buffer, a transport fluid, and sodium chloride (aq), or any combination thereof.

[0072] In some embodiments, the aqueous composition is diluted with water up to about 50% (v / v), hi some embodiments, the aqueous composition is diluted up to about 30% (v / v), up to about 40% (v / v), up to about 45% (v / v), up to about 55% (v / v), up to about 60% (v / v), up to about 75% (v / v), up to about 80% (v / v), or up to about 80% (v / v).

[0073] The term "transport fluid" as used herein refers to any solution containing protective proteins and antimicrobial agents to avoid microbial growth for transport of biological samples. In some embodiments, the aqueous composition is diluted with the transport fluid up to about 50% (v / v). In some embodiments, the aqueous composition is diluted up to about 30% (v / v), up to about 40% (v / v), up to about 45% (v / v), up to about 55% (v / v), up to about 60% (v / v), up to about 75% (v / v), up to about 80% (v / v), or up to about 80% (v / v).

[0074] In some embodiments, the aqueous composition is diluted with sodium chloride (aq) up to about 50% (v / v). In some preferred embodiments, the sodium chloride (aq) is 0.9% (w / v) sodium chloride. In some embodiments, the aqueous composition is diluted up to about 30% (v / v), up to about 40% (v / v), up to about 45% (v / v), up to about 55% (v / v), up to about 60% (v / v), up to about 75% (v / v), up to about 80% (v / v), or up to about 80% (v / v).

[0075] In some embodiments, the aqueous composition is diluted with any combination of water, buffer, transport fluid, and 0.9% (w / v) sodium chloride up to about 50% (v / v). In some embodiments, the aqueous composition is diluted with any combination of water, buffer, transport fluid, and 0.9% (w / v) sodium chloride up to about 30% (v / v), up to about 40% (v / v), up to about 45% (v / v), up to about 55% (v / v), up to about 60% (v / v), up to about 75% (v / v), up to about 80% (v / v), or up to about 80% (v / v).

[0076] In some embodiments, the aqueous composition of the present invention further comprises a population of polynucleotides comprising RNA, DNA, or a combination thereof. In some embodiments, the RNA comprises an in vitro synthesized transcription product. In some embodiments, the population of polynucleotides can comprise primers, amplification oligomers, detection oligomers, probe protection oligomers, capture probe oligomers, or any other polynucleotides required to perform an amplification reaction to amplify a target nucleic acid and / or to detect the target nucleic acid. In other embodiments, the population of polynucleotides is a target nucleic acid.

[0077] The term "nucleic acid" refers to a multimeric compound containing two or more covalently linked nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases, or base analogs, where the nucleosides are linked together by phosphodiester bonds or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and analogs thereof.

[0078] By "isolated" it is meant that the sample containing the target nucleic acid is removed from its natural environment, although the term does not imply any degree of purification.

[0079] Methods of the Invention In a further aspect, the present invention relates to a method for obtaining a population of polynucleotides from a sample suspected of containing nucleic acids, comprising contacting the sample with an aqueous composition according to the present invention at a temperature ranging from 2° C. to 40° C. In some embodiments, the contacting is performed at a temperature ranging from 2° C. to 40° C., more preferably at a temperature ranging from 4° C. to 35° C., more preferably at 4° C. to 30° C. In some embodiments, the contacting is performed at room temperature. The term "room temperature" (RT) as used herein generally refers to a range of temperatures from 15° C. to 30° C.

[0080] In some embodiments, the sample is preferably contacted with the aqueous composition for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 10 minutes, or at least 15 minutes.

[0081] In some embodiments, the sample is contacted with an aqueous composition according to the present invention at a temperature in the range of 2° C. to 40° C. for at least 1 minute.

[0082] A sample can be an isolated sample, including "biological samples," "environmental samples," and sample collection devices (e.g., swabs) that have been contacted with a biological or environmental sample or any other sample suspected of containing pathogen nucleic acid or components thereof.

[0083] A "biological sample" includes bodily fluids such as urine, blood, plasma, serum, peripheral blood, red blood cells, lymph nodes, gastrointestinal tissue, feces, cerebrospinal fluid (CSF), semen, sputum, saliva, or other bodily fluids or materials, as well as solid tissues. Biological samples also include cells (such as cell lines, cells isolated from tissues, whether or not the isolated cells are cultured after isolation from the tissue, fixed cells, such as cells fixed for histology and / or immunohistochemistry analysis), tissues (such as biopsies), or fluids obtained from a mammal, including from upper respiratory tract tissues (such as nasopharyngeal washes, nasopharyngeal aspirates, nasopharyngeal swabs, and oropharyngeal swabs), from lower respiratory tract tissues (such as bronchial lavages, tracheal aspirates, pleural punctures, sputum, etc.), and tissues from any organ, such as, but not limited to, the lungs, heart, spleen, liver, brain, kidneys, and adrenal glands. Also included are nucleic acids (e.g., DNA and RNA) isolated from cells and / or tissues. In some preferred embodiments, biological samples include anterior and middle turbinate nasal swabs, nasopharyngeal (NP) and oropharyngeal (OP) swabs, nasopharyngeal washes / aspirates or nasal aspirates, and bronchoalveolar lavage (BAL) samples. In more preferred embodiments, the samples are obtained from individuals suspected of having COVID-19.

[0084] "Environmental samples" include environmental materials such as surface matter, soil, water, and industrial materials, as well as materials obtained from food and dairy processing equipment, devices, facilities, disposable and non-disposable items.

[0085] In some embodiments of the method for obtaining a population of polynucleotides from a sample suspected of containing nucleic acid, the sample is a biological sample. In other embodiments, the sample is an environmental sample. In some embodiments, the nucleic acid is RNA and / or DNA. In some embodiments, the RNA comprises an in vitro synthetic transcript.

[0086] The term "sample" includes any specimen that may contain or is suspected of containing at least one pathogen or a component thereof, such as a nucleic acid, or a fragment of a pathogen nucleic acid. In some embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In some embodiments, the at least one pathogen is a virus. In other embodiments, the virus is a respiratory virus. In some embodiments, the respiratory virus is from the family Coronaviridae. In more preferred embodiments, the virus is SARS-CoV-2. Other non-respiratory viruses, such as HIV, HCV, HBV, HAV, HEV, parvovirus, West Nile virus, Ustu virus, Chikungunya virus, Dengue virus, or Zika virus, among others, are also contemplated. In some embodiments, the at least one pathogen is a bacterium. In some embodiments, the at least one pathogen is a fungus. In some embodiments, the at least one pathogen is a parasite. Thus, other pathogens, such as Babesia or Plasmodium, are also contemplated within the scope of the present invention.

[0087] In some embodiments, the method for obtaining a population of polynucleotides from a sample suspected of containing a nucleic acid further comprises an amplification reaction for amplifying at least a target nucleic acid. In some embodiments, the method further comprises detecting at least the target from the resulting amplification products. In some embodiments, the amplification reaction is an isothermal amplification reaction. In a more preferred embodiment, the amplification reaction is a transcription-mediated amplification.

[0088] The term "nucleic acid-based detection" or "nucleic acid test" as used herein refers to an assay for the detection of a target sequence. In certain embodiments, the nucleic acid-based detection assay includes the detection of a target sequence by distinguishing it from other sequences, for example by sequencing or by utilizing one or more oligonucleotides that specifically hybridize to the target sequence. In certain embodiments, the nucleic acid-based detection assay is an "amplification-based assay", i.e., an assay that utilizes one or more steps to amplify a nucleic acid target sequence. For clarity, an amplification-based assay can include one or more steps that do not amplify the target sequence, such as, for example, steps used in non-amplification-based assay methods (e.g., hybridization assays or cleavage-based assays). Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), quantitative PCR (qPCT), real-time PCR (rt-PCT), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated or transcription-associated amplification (TMA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and polymerase spiral reaction (PSR). Such amplification methods are well known in the art and can be easily used according to the methods disclosed herein. Other non-targeted amplification techniques known by those skilled in the art, such as strand displacement amplification, also referred to as whole genome amplification (WGA), are also included within the spirit of the present invention. In other embodiments, the nucleic acid-based detection assay is a "non-amplification-based assay", i.e., an assay that does not rely on any step to amplify a nucleic acid target sequence.

[0089] "Transcription-associated amplification," also referred to herein as "transcription-mediated amplification" (TMA), is an isothermal amplification reaction that refers to nucleic acid amplification using an RNA polymerase to generate multiple RNA transcripts from a nucleic acid template. TMA generally utilizes an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a template-complementary oligonucleotide that includes a promoter sequence and can optionally include one or more other oligonucleotides.

[0090] In a further aspect, the present invention relates to a method for preserving the integrity of a population of polynucleotides in a sample comprising the step of contacting the sample with an aqueous composition according to the present invention at a temperature in the range of 4°C to 40°C.

[0091] In some preferred embodiments, the sample is contacted with the aqueous composition for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, or at least 10 minutes. In some embodiments, the sample is contacted with the aqueous composition for at least 1 day, at least 2 days, at least 5 days, at least 7 days, at least 15 days, at least 30 days, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 24 months, or at least 26 months.

[0092] In some embodiments, the contacting is performed at a temperature ranging from 4° C. to 40° C., more preferably at a temperature ranging from 4° C. to 35° C., more preferably at 4° C. to 30° C. In some embodiments, the contacting is performed at room temperature. The term "room temperature" as used herein generally refers to a temperature range of from 15° C. to 30° C. In some embodiments, the sample is contacted with an aqueous composition according to the present invention at a temperature ranging from 4° C. to 30° C. for at least 2 minutes.

[0093] In some embodiments of the method for preserving the integrity of a population of polynucleotides in a sample, the sample after contacting with the aqueous composition is stored at a temperature ranging from -25°C to 40°C for at least 1 hour and up to 6 months, up to 9 months, up to 12 months, or up to 26 months. In more preferred embodiments, the sample after contacting with the aqueous composition is stored at -20°C, 4°C, RT, or 30°C for at least 1 hour and up to 6 months, up to 12 months, up to 24 months, or up to 26 months. In some embodiments, the sample after contacting with the aqueous composition is stored at a temperature ranging from 2°C to 10°C for at least 1 hour and up to 9 months. In some embodiments, the sample after contacting with the aqueous composition is stored at a temperature ranging from -25°C to -5°C for at least 1 hour and up to 26 months.

[0094] In some embodiments of the method for preserving the integrity of a population of polynucleotides in a sample, the sample is a biological sample as defined herein. In some preferred embodiments, the biological sample includes anterior and middle turbinate nasal swabs, nasopharyngeal (NP) and oropharyngeal (OP) swabs, nasopharyngeal wash / aspirate or nasal aspirate, and bronchoalveolar lavage (BAL) samples. In more preferred embodiments, the sample is obtained from an individual suspected of COVID-19. In other embodiments, the sample is an environmental sample as defined herein.

[0095] In some embodiments, the population of polynucleotides is a population of RNA and / or DNA. In some embodiments, the population of polynucleotides is derived from at least one pathogen. In some embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In some embodiments, the at least one pathogen is a virus. In other embodiments, the virus is a respiratory virus. In some embodiments, the respiratory virus is from the Coronaviridae family. In more preferred embodiments, the virus is SARS-CoV-2. Other non-respiratory viruses, such as HIV, HCV, HBV, HAV, HEV, parvovirus, West Nile virus, Ustu virus, Chikungunya virus, Dengue virus, or Zika virus, among others, are also contemplated. In some embodiments, the at least one pathogen is a bacterium. In some embodiments, the at least one pathogen is a fungus. In some embodiments, the at least one pathogen is a parasite. Thus, other pathogens, such as Babesia or Plasmodium, are also contemplated within the scope of the present invention.

[0096] In a further aspect, the present invention relates to a method for inactivating a sample containing at least one pathogen, comprising the step of contacting the sample with an aqueous composition according to the present invention, wherein the aqueous composition represents at least 30% (v / v) of the resulting mixture.

[0097] In some embodiments, the aqueous composition represents at least 2% (v / v) of the resulting mixture, more preferably at least 5% (v / v) of the resulting mixture, more preferably at least 10% (v / v) of the resulting mixture, more preferably at least 20% (v / v) of the resulting mixture, more preferably at least 30% (v / v) of the resulting mixture, more preferably at least 35% (v / v) of the resulting mixture, more preferably at least 40% (v / v) of the resulting mixture, more preferably at least 50% (v / v) of the resulting mixture, more preferably at least 60% (v / v) of the resulting mixture, more preferably at least 70% (v / v) of the resulting mixture, more preferably at least 80% (v / v) of the resulting mixture, more preferably at least 85% (v / v) of the resulting mixture, more preferably at least 90% (v / v) of the resulting mixture, more preferably at least 95% (v / v) of the resulting mixture, more preferably at least 98% (v / v) of the resulting mixture, more preferably at least 99% (v / v) of the resulting mixture.

[0098] In some embodiments, the sample is contacted with the aqueous composition for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, or at least 30 minutes. In some embodiments, the contacting is performed at a temperature ranging from 2° C. to 40° C., more preferably at a temperature ranging from 4° C. to 35° C., more preferably at 4° C. to 30° C. In some embodiments, the contacting is performed at room temperature. The term "room temperature" as used herein generally refers to a range of temperatures from 15° C. to 30° C. In some preferred embodiments, the sample is contacted with the aqueous composition at 4° C., RT, or 30° C.

[0099] In some embodiments, the sample is contacted with an aqueous composition according to the present invention at a temperature in the range of 4° C. to 30° C. for at least 1 minute.

[0100] In some embodiments of the method for inactivating a sample containing one or more pathogens of the present invention, the sample is as defined herein. In some preferred embodiments, the sample is a biological sample as defined herein. In some preferred embodiments, the biological sample includes anterior and middle turbinate nasal swabs, nasopharyngeal (NP) and oropharyngeal (OP) swabs, nasopharyngeal wash / aspirate or nasal aspirate, and bronchoalveolar lavage (BAL) samples. In more preferred embodiments, the sample is obtained from an individual suspected of COVID-19. In other embodiments, the sample is an environmental sample as defined herein.

[0101] In some embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In some embodiments, the at least one pathogen is a virus. In other embodiments, the virus is a respiratory virus. In some embodiments, the respiratory virus is from the Coronaviridae family. In more preferred embodiments, the virus is SARS-CoV-2. Other non-respiratory viruses, such as HIV, HCV, HBV, HAV, HEV, parvovirus, West Nile virus, Ustu virus, Chikungunya virus, Dengue virus, or Zika virus, among others, are also contemplated. In some embodiments, the at least one pathogen is a bacterium. In some embodiments, the at least one pathogen is a fungus. In some embodiments, the at least one pathogen is a parasite. Thus, other pathogens, such as Babesia or Plasmodium, are also contemplated within the scope of the present invention.

[0102] In a further aspect, the present invention relates to a method for obtaining a sample comprising contacting the sample with an aqueous composition according to the present invention, wherein the aqueous composition represents at least 30% (v / v) of the resulting mixture. In some embodiments, the aqueous composition represents at least 30% (v / v) of the mixture resulting from contacting the sample with the aqueous composition. In some embodiments, the aqueous composition represents at least 2% (v / v) of the resulting mixture, or at least 5% (v / v) of the resulting mixture, or at least 10% (v / v) of the resulting mixture, or at least 20% (v / v) of the resulting mixture, or at least 30% (v / v) of the resulting mixture, or at least 35% (v / v) of the resulting mixture, or at least 40% (v / v) of the resulting mixture, or at least 50% (v / v) of the resulting mixture, or at least 60% (v / v) of the resulting mixture, or at least 70% (v / v) of the resulting mixture, or at least 80% (v / v) of the resulting mixture, or at least 85% (v / v) of the resulting mixture, or at least 90% (v / v) of the resulting mixture, or at least 95% (v / v) of the resulting mixture, or at least 98% (v / v) of the resulting mixture, or at least 99% (v / v) of the resulting mixture.

[0103] Contact of the sample with the aqueous composition can be carried out at a temperature ranging from 2°C to 40°C, more preferably from 4°C to 40°C, and for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 15 minutes.

[0104] In some embodiments of the method for collecting a sample of the present invention, the sample is a sample as defined herein. In some preferred embodiments, the sample is a biological sample as defined herein. In some preferred embodiments, the biological sample includes anterior and middle turbinate nasal swabs, nasopharyngeal (NP) and oropharyngeal (OP) swabs, nasopharyngeal wash / aspirate or nasal aspirate, and bronchoalveolar lavage (BAL) samples. In more preferred embodiments, the sample is obtained from an individual suspected of COVID-19. In other embodiments, the sample is an environmental sample as defined herein.

[0105] In some preferred embodiments, the sample is introduced directly into a collection device or collection vessel containing a composition according to the invention.

[0106] In other preferred embodiments, the sample is collected using a swab and introducing it into a collection device or collection container containing a composition according to the invention. In a more preferred embodiment, the swab is discarded after being rubbed against the collection device or collection container.

[0107] In some embodiments, the sample contains at least one pathogen. In some embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In some embodiments, the at least one pathogen is a virus. In other embodiments, the virus is a respiratory virus. In some embodiments, the respiratory virus is from the Coronaviridae family. In more preferred embodiments, the virus is SARS-CoV-2. Other non-respiratory viruses, such as HIV, HCV, HBV, HAV, HEV, parvovirus, West Nile virus, Ustu virus, Chikungunya virus, Dengue virus, or Zika virus, among others, are also contemplated. In some embodiments, the at least one pathogen is a bacterium. In some embodiments, the at least one pathogen is a fungus. In some embodiments, the at least one pathogen is a parasite. Thus, other pathogens, such as Babesia or Plasmodium, are also contemplated within the scope of the present invention.

[0108] In a further aspect, the present invention also relates to a collection device or container comprising an aqueous composition as described herein. The present invention also relates to a sample collection kit comprising a collection device or container and an aqueous composition as described herein. In some embodiments, the sample collection kit further comprises a swab, curette, or culture loop. In some preferred embodiments, the sample collection kit comprises a collection device or container, an aqueous composition as described herein, and a swab.

[0109] In a further aspect, the present invention relates to the use of a composition as described herein for obtaining a sample suspected of containing a target nucleic acid. In a preferred embodiment, the sample is as defined herein. In some preferred embodiments, the sample is a biological sample as defined herein. In some preferred embodiments, the biological sample includes anterior and middle turbinate nasal swabs, nasopharyngeal (NP) and oropharyngeal (OP) swabs, nasopharyngeal washes / aspirates or nasal aspirates, and bronchoalveolar lavage (BAL) samples. In a more preferred embodiment, the sample is obtained from an individual suspected of COVID-19.

[0110] In some embodiments, the sample contains at least one pathogen. In some embodiments, the at least one pathogen is a fungus, a bacterium, a parasite, or a virus. In some embodiments, the at least one pathogen is a virus. In other embodiments, the virus is a respiratory virus. In some embodiments, the respiratory virus is from the Coronaviridae family. In more preferred embodiments, the virus is SARS-CoV-2. Other non-respiratory viruses, such as HIV, HCV, HBV, HAV, HEV, parvovirus, West Nile virus, Ustu virus, Chikungunya virus, Dengue virus, or Zika virus, among others, are also contemplated. In some embodiments, the at least one pathogen is a bacterium. In some embodiments, the at least one pathogen is a fungus. In some embodiments, the at least one pathogen is a parasite. Thus, other pathogens, such as Babesia or Plasmodium, are also contemplated within the scope of the present invention. EXAMPLES

[0111] It should be readily apparent to one of ordinary skill in the art that the examples disclosed herein below represent generalized examples only, and that other compositions and methods by which the invention can be reproduced are possible and encompassed by the present invention.

[0112] The following table summarizes exemplary compositions of the present invention used in the examples below.

[0113] [Table 1]

[0114] Samples analyzed in the embodiments of the present invention can be considered valid / invalid and / or reactive / non-reactive. A sample is considered valid if it is successfully pipetted by the Procleix Xpress system, introduced into the Procleix Panther system, where an RNA-based internal control (IC) is added, and analyzed using the Procleix SARS-CoV-2 assay or any other Procleix assay (Grifols Diagnostic Solutions, USA), thereby obtaining a positive result for the added IC. A valid sample can be either reactive (positive for SARS-CoV-2 or other pathogens) or non-reactive (negative for SARS-CoV-2 or other pathogens), depending on the presence or absence of the target nucleic acid of interest. Sensitivity, which is the proportion of true positives thus correctly identified (e.g., the percentage of infected patients correctly identified as having an infection), is also indicated when important.

[0115] Example 1 Comparison of compositions of the present invention for collection of swab samples One of the purposes of the compositions of the present invention is to be used for swab sample collection. Swab samples have a high viscosity that in some cases makes their processing by the test system more difficult than for other biological liquid samples such as serum or plasma. To investigate the suitability of the compositions of the present invention for collecting and further testing such samples, nasal swab samples were collected from healthy volunteers (tested negative for SARS-CoV-2) in compositions C1 and C3 (Table 1) and subsequently processed on the Panther system using the Procleix SARS-CoV-2 assay according to the manufacturer's instructions. Compositions C1 and C3 with different LLS concentrations (ranging from 3% to 10% (w / v)) were also tested.

[0116] Thus, the results shown in Table 2 demonstrate that compositions C1 and C3 are suitable for collection of swab samples, since all samples were found to be effective. In addition, the results also demonstrate that different concentrations of surfactants and other components of the compositions, particularly LiOH, do not affect the ability of the compositions to be used as collection media, since all compositions tested were found to be effective for sample collection and further analysis in the Panther.

[0117] [Table 2]

[0118] In further experiments, two novel nasal swab samples in 6% LLS C3 were spiked with a known amount of artificial virus (50 μL of AccuPlex SARS-CoV-2 (Seracare, ref 0505-0129) (61 copies / mL)) and run on the Panther system according to the Procleix SARS-CoV-2 assay instructions. Reactive results were obtained for both of them.

[0119] In conclusion, it has been demonstrated that the compositions of the present invention are suitable for collecting biological samples containing nucleic acids and for testing and detecting nucleic acids of interest by molecular diagnostic methods, even when the sample is a nasal sample.

[0120] Example 2 Comparison of VTM and the composition of the present invention with respect to sampling The objective of this study was to evaluate the sensitivity of collecting nasal samples using the compositions of the present invention and their stability over time. VTM was used for comparison since it is the standard medium for swab collection.

[0121] VTM was prepared according to the recommendations of the Centers for Disease Control and Prevention (SOP#: DSR-052-05). Briefly, 10 mL of inactivated fetal bovine serum (FBS) was added to a 500 mL bottle of sterile Hank's Balanced Salt Solution (HBSS), followed by 2 mL of gentamicin / amphotericin B mixture. This VTM was used for all experiments performed.

[0122] For the first comparative study, swabs from nasal samples were taken, spiked with a known amount of artificial virus, and then introduced into collection tubes containing either 3 mL of VTM or 3 mL of composition C2 (Table 1). In detail, swabs from nasal samples were taken from 17 healthy volunteers (tested negative for SARS-CoV-2) using flocked swabs. A known concentration of artificial virus (3 μL or 7.5 μL of AccuPlex SARS-CoV-2 (Seracare, ref 0505-0129) member 1 (100 copies / μL)) was spiked into each sample-containing swab, resulting in spiked swabs containing 0 copies / mL, 100 copies / mL, or 250 copies / mL. The spiked swabs were then immersed in the tube and rubbed against the tube wall for 15 seconds, after which the swabs were discarded. All samples were tested in triplicate within 2 hours of collection.

[0123] VTM collected samples were processed according to the Procleix SARS-CoV-2 assay instructions (GDSS-IFU-000047-EN v.4.0) which included vortexing the collection tube followed by transfer to a second tube containing extraction buffer and then loading the sample into the Panther system, while samples collected in composition C2 were loaded directly into the Panther system immediately after removing the cap (no swab inside).

[0124] Results for the negative control (0 copies / mL) and samples spiked with 100 and 250 copies / mL are shown in Table 3. Samples that tested positive for SARS-CoV-2 were considered reactive, and valid samples imply that the sample was suitable for processing with the Panther system.

[0125] [Table 3]

[0126] The limit of detection (LOD) using composition C2 for sample collection was comparable to the current standard VTM collection. In fact, the % reactivity in C2 was higher for a spike corresponding to 100 copies / mL, considering that the LOD is 80 copies / mL when spiked directly into the VTM. Since the LOD turned out to be comparable for the concentrations tested, a second experiment was performed using the same concentrations.

[0127] In a second experiment, to test the stability of the samples over time, swabs from nasal samples were taken into composition C2 and spiked with 750 and 300 copies of the artificial virus Accuplex SARS-CoV-2, resulting in 250 and 100 copies / mL, respectively. As a control, swabs from nasal samples were also taken into VTM and spiked with 300 copies (100 copies / mL).

[0128] In detail, nasal swabs from 37 healthy volunteers (tested negative for SARS-CoV-2) were collected using flocked swabs. From these, a set of 10 swab samples collected in VTM (experimental controls) were spiked with 300 copies to obtain 100 copies / mL. The other 27 swab samples were collected in composition C2, from which 10 swab samples were spiked with 300 copies to obtain 100 copies / mL, 10 swab samples were spiked with 750 copies to obtain 250 copies / mL, and 6 swab samples were spiked with 0 copies (negative controls). All samples were tested 2-3 hours after collection (t0) in duplicate and stored at RT for 48 hours (T48) without additional mixing to be retested once more. Samples were processed in Panther as previously described. The results are shown in Table 4.

[0129] [Table 4]

[0130] Comparative study results also confirmed comparable LODs for both collection methods, and comparable sensitivity to a previous study using Accuplex (80 copies / mL) added directly to the now standard VTM.

[0131] 48 hours after collection, all replicates were reactive. Therefore, considering that the comparison with the current method (VTM) produced similar percentages of reactivity and all results showed similar LODs, it can be concluded that the collection method does not significantly reduce the sensitivity of SARS-CoV-2 detection. However, the collection method using the composition of the present invention reduces the number of steps and improves sample traceability during sample preparation, since it is no longer necessary to transfer the sample to a second tube containing extraction buffer before loading the sample into the Panther system. Therefore, the composition of the present invention is suitable for both sample collection and storage of nucleic acids until molecular testing.

[0132] Further experiments were performed to evaluate the preservation of SARS-CoV-2 RNA at room temperature for longer periods of time. In particular, nasal swabs were collected from three healthy volunteers (tested negative for SARS-CoV-2) using flocked swabs. A known amount of artificial virus (100, 250 or (y)500 copies / ml of AccuPlex (Seracare)) was added to each sample-containing swab. The added swab was then immersed in a test tube containing 3 mL of composition C2 and rubbed against the test tube wall and bottom for 15 seconds, after which the swab was discarded. All samples were tested in triplicate immediately after collection. Samples were either loaded directly into the Panther system or stored at room temperature for 7 days.

[0133] The results show that all tested samples were reactive after 7 days of storage at room temperature, confirming that the compositions of the present invention preserve RNA even at room temperature for at least 7 days.

[0134] Example 3 Evaluation of viral inactivation by the compositions of the present invention The ability of composition C2 to inactivate SARS-CoV-2 was evaluated by a titration assay in Vero cells (Pasteur Institute. Simizu B. and Terasima T. 1988; Simizu B. et al., 1967).

[0135] The titration method is a quantitative assay in which virus titration is based on the detection of virus production in infected cells by observing a specific cytopathic effect. To evaluate the inactivation capacity of the compositions of the present invention, the compositions, particularly C2, were spiked with SARS-CoV-2 in a 1 / 10 ratio at room temperature and incubated for 2, 10 or 30 minutes (test samples T1, T2 and T3, respectively). As a positive control, the virus was spiked with stock medium (V1).

[0136] After incubation at room temperature, 0.5 mL of each sample in triplicate was diluted with dilution medium and ultracentrifuged. The pellet was resuspended in 25 mL titration medium and immediately titrated on Vero cells. Ultracentrifugation was shown to have no significant effect on the virus.

[0137] Test samples were then diluted with medium (8 replicates for each dilution) by serial 3-fold dilutions across a 96-well plate ("sample dilution plate"). Each well from the "sample dilution plate" was then inoculated into the corresponding well of a new plate ("sample titration plate"). A cell suspension was added to each well of the "sample titration plate" and the plate was then incubated at the appropriate temperature. After an incubation period allowing for virus replication and infection of neighboring cells, wells with foci were counted post-infection by observation under an inverted light microscope or a dye overlay (crystal violet) was added and the wells examined for cytopathic effects. Infected wells appear as clear areas while non-infected wells are stained (Figure 1).

[0138] 50% Tissue Culture Infectious Dose (TCID 50 The infectious titer [m(T)], expressed as 1000 μg / mL, was calculated using the Spearman-Karber formula. The viral load, defined by its mean value [m(L)] and its confidence interval, was calculated as the mean titer m(T) × Vt, where Vt is the total volume of the sample, which was 1 mL in all cases. The Log of the ratio of the viral load in the pre-treatment material (untreated) (Li) to the viral load in the post-treatment material (Lf) was calculated as 10 The viral reduction factor (R), defined as, [m(R)] = Log 10 (V1)-Log 10 (LS), where LS is the viral load of each sample T1, T2, or T3. The results are shown in Table 5.

[0139] [Table 5]

[0140] The resulting viral reduction coefficients (R) for composition C2 for all times tested in this study (2 min, 10 min and 30 min) were greater than 5.09. Thus, an additional 5 Log (99.999%) reduction titer of SARS-CoV-2 was demonstrated for the composition of the present invention.

[0141] Example 4 Inactivation of viral nucleic acids from viruses other than SARS-CoV-2 Clinical HIV-1, HIV-2, HCV, HBV and HEV plasma and seropositive samples (positive serological and / or NAT results) were obtained from the American Red Cross (Gaithersburg, MD), Japanese Red Cross (Tokyo, Japan), Boca Biolistics (Pompano Beach, FL), Medical Research Network (MRN) (New York, NY), SlieaGen (Austin, TX), Bioreclamation (Westbury, NY), Cerba (France), Discovery Live Sciences (Los Osos, CA) and Access Biologicals (San Diego, CA).

[0142] Plasma samples: 100 HIV-1, HCV, and HBV positive plasma samples, 100 HEV positive plasma samples, and 37 HIV-2 positive plasma samples were tested undiluted in single replicates using the Procleix UltrioPlex E assay (UPE). The same set of samples was tested in single replicates in pools of 16 and pools of 96. Pooled samples were made by mixing one portion of a positive sample with one portion from 15 (for the 16 pools) or 95 (for the 96 pools) different plasma normal donor samples.

[0143] Serum samples: 25 HIV-1, HIV-2, HCV, and HBV positive serum samples were tested undiluted in single replicates using the UPE assay. The same set of samples was tested in single replicates in pools of 16. Pooled samples were made by mixing one portion of a positive sample with one portion from 15 different serum normal donor samples.

[0144] The liquid samples were diluted in composition C1 in a ratio of 0.75:1 (C1:sample) in the first step of the assay.

[0145] [Table 6]

[0146] [Table 7]

[0147] The high sensitivity obtained (up to 1 copy of virus / mL) for serum (table 6) and plasma samples (table 7) demonstrated lysis of the samples, as lysis is necessary for detection of the virus, demonstrating that the compositions of the invention are suitable for inactivation of other viruses in liquid samples.

[0148] Example 5 Inactivation and preservation of SARS-CoV-2 RNA The objective of the first experiment was to evaluate the inactivation and preservation during storage of SARS-CoV-2 by the compositions of the invention. To this end, the following samples were prepared: - Contrived SeraCare positive samples were prepared by spiking SARS-CoV-2 positive material into CDC recommended viral transport fluid (VTM) at 123 copies / mL. The spiked concentration corresponded to approximately 3x the LOD for the SCV2 assay for the swab specimen type. - An artificial BEI SARS-associated coronavirus sample was prepared by spiking heat-inactivated positive material into CDC-recommended viral transport medium (VTM) at 63.87 c / mL.

[0149] The liquid samples were then mixed 1:1 with composition C1 and either i) stored at 30° C. and tested at 0 hours, 60 hours, 90 hours, 8 days and 17 days, or ii) stored at 4° C. and tested at 0 hours, 8 days and 17 days. The results for i) and ii) are shown in Tables 8 and 9, respectively.

[0150] [Table 8]

[0151] [Table 9]

[0152] Seracare-processed swab samples spiked with up to 123 copies / mL of SARS-CoV-2 positive material were 100% reactive at baseline and after 60 hours, 90 hours, 8 days, and 17 days of storage at 30° C. ± 3° C. Swab samples stored at 4° C. ± 3° C. were 100% reactive at baseline and after 8 and 17 days.

[0153] BEI heat inactivated swab samples spiked with up to 63.87 copies / mL of SARS-CoV-2 positive material were 100% reactive at baseline and after 60 hours, 90 hours, and 8 days of storage at 30°C ± 3°C. Swab samples stored at 4°C ± 3°C were 100% reactive at baseline and after 8 days.

[0154] The RNA added in these experiments was either artificial capsid (Seracare samples) or real capsid (BEI inactivated virus), but it was already demonstrated in Example 4 that the composition of the invention is able to lyse the samples and preserve the RNA even at 30°C.

[0155] In a second experiment, previous samples (SeraCare RNA (SC) or heat-inactivated virus (BEI) spiked at 3x LOD in VTM, followed by 1:1 C1 spike) were stored at either 30°C or 4°C for longer periods and subsequently retested at 3 and 6 months following the same Panther protocol. Results demonstrated that SeraCare RNA was stable at both 4°C and 30°C storage for at least 6 months, while BEI was stable at 4°C storage for at least 6 months. BEI at 30°C storage was stable for at least 3 months. Relevant results are shown in Figures 2-5.

[0156] In another experiment, the preservation of actual SARS-CoV-2 positive samples was also studied. For this, 35 nasopharyngeal samples taken in composition C2 from volunteers who tested positive for SARS-CoV-2 were retested after storage at temperatures between 2°C and 8°C for up to 18 days. All tested samples were reactive (positive) after testing in Panther, demonstrating that RNA is preserved even at refrigerated temperatures for up to 18 days.

[0157] Example 6 Long-term storage of pathogen RNA For this experiment, the following samples were prepared: - an in vitro synthetic transcript (IVT) for Babesia 18s ribosomal RNA at 500 c / mL, diluted in composition C1 to obtain 500, 100 or 30 copies / mL - Control: IVT against HIV-1 scrambled RNA sequence not found in any organism, diluted in composition C1

[0158] Babesia IVT and controls were stored at different temperatures and subsequently tested for the presence of target RNA using the Babesia Procleix assay and the Panther system (Grifols Diagnostic Solutions, USA).

[0159] In a first experiment, all samples were stored at 5° C.±3° C. instead of their intended storage temperatures of −15° C. to −35° C. Samples were subsequently tested at 0, 3, 6, 9, and 12 months. The results, shown in FIG. 6, demonstrate that the compositions of the present invention allow for the preservation of pathogen RNA for at least 9 months at 5° C.

[0160] In another experiment, samples were stored at their intended storage temperature of -20°C ± 5°C. Samples were subsequently tested at 0, 3, 6, 9, 12, 18, 24 and 26 months. At each time point, testing was performed on the day the kit was opened and 36 days after opening after being placed on the Panther system for 72 hours (OB stands for on-board). The results shown in Figure 7 demonstrate that the composition of the present invention allows for preservation of pathogen RNA for at least 26 months at -20°C, even with high content of RNA.

[0161] In conclusion, the composition of the invention is suitable for collection of samples such as biological, environmental or waste samples, in particular for collection of respiratory swab samples as demonstrated in Examples 1 and 2 or liquid samples as demonstrated in Example 4. The composition is also an effective collection medium to replace the current protocols using VTM, inactivating potential pathogens, making the sample safer for the person working with the sample (avoiding the use of safety cabinets for pathogens such as SARS-CoV-2), and reducing the steps for preparation of the sample for nucleic acid testing, since the same collection medium can lyse and inactivate pathogens present in the sample (as shown in Examples 3, 4 and 5), while successfully preserving the integrity of the nucleic acid until testing. In particular, the composition of the invention has been proven suitable for preserving samples containing nucleic acids during storage at temperatures ranging from 4°C to 30°C for a period of from a few hours up to 9 months (see Examples 5 and 6), and up to 26 months when stored at -20°C (see Example 6). Finally, the compositions of the present invention are less hazardous than other available inactivation media, which are often based on guanidine thiocyanate, and are compatible with test systems that use sodium hypochlorite in their cleaning process.

[0162] Example 7 Comparison of compositions of the present invention with Foam Ban for collecting swab samples Nasal swab samples were collected from healthy volunteers (tested negative for SARS-CoV-2) in compositions C1 and C3 (Table 1) containing different concentrations of LLS (ranging from 3% to 9% (w / v)) and Foam Ban at a concentration of 300 μL / L, and subsequently processed on the Panther system using the Procleix SARS-CoV-2 assay according to the manufacturer's instructions. Samples were collected using two different types of commercial swabs: Aptima® Multitest Swab Sampling Kit (Hologic, USA) and ViCUM® Floc (Deltalab, Spain).

[0163] All samples were effectively processed as shown in Table 10, demonstrating that compositions C1 and C3 containing Foam Ban are suitable for collecting swab samples. It was also demonstrated that the inclusion of different concentrations of surfactants and other components of the compositions, particularly LiOH, did not affect the ability of the compositions to be used as any collection medium, as all compositions tested were found to be effective in collecting samples and further analyzed in the Panther.

[0164] [Table 10]

[0165] In further experiments, nasal swab samples were collected from healthy volunteers (tested negative for SARS-CoV-2) in compositions C1 and C3 (Table 1) containing different concentrations of LLS (ranging from 3% to 10% (w / v)) and Foam Ban at a concentration of 300 μL / L. Samples were kept at 4 °C for 6 days and then processed on the Panther system using the Procleix SARS-CoV-2 assay according to the manufacturer's instructions. Samples were collected using two different types of commercial swabs: Aptima® Multitest Swab Sampling Kit (Hologic, USA) and ViCUM® Floc (Deltalab, Spain). As shown in Table 11, all samples were effectively processed, demonstrating that the composition of the present invention containing Foam Ban allows the collection and storage of samples at 4 °C for at least 6 days before processing. This case also demonstrates that different concentrations of surfactants and other components of the composition, particularly LiOH, do not affect the ability of the composition to be used as a collection medium.

[0166] [Table 11]

[0167] Example 8 Inactivation and Preservation of SARS-CoV-2 RNA Under Varying Storage Temperature Conditions Experiments were conducted to test the inactivation and preservation of SARS-CoV-2 RNA under alternating temperature storage conditions.

[0168] A clinical matrix was prepared by pooling surplus nasal samples collected from healthy volunteers (who tested negative for SARS-CoV-2). Each sample was collected by dipping a nasal swab into a tube containing 3 mL of composition C1, containing Foam Ban at a concentration of 300 μL / L, diluted to 50% (v / v) with 0.9% (w / v) sodium chloride, and rubbing the swab against the tube.

[0169] Twenty spiked samples of 3 mL at 2x LoD (low positive, 160 copies / mL), 10 spiked samples at 5x LoD (high positive, 400 copies / mL) and 10 negative samples to monitor false positives were prepared by spiking the virus using a recombinant virus containing SARS-CoV-2 RNA (AccuPlex SARS-CoV-2, LGC SeraCare) on a clinical matrix.

[0170] [Table 12]

[0171] Forty prepared test tubes containing 3 mL samples according to the panel in table 12 were cycled through temperature and time according to the following workflow: These samples were initially maintained at temperature condition A (table 13). At the end of the cycle, these samples were analyzed using the Procleix SARS-CoV-2 assay on the Panther system. The same samples (2.5 mL each) were then kept at temperature condition B (table 14). After the final cycle, samples were analyzed using the Procleix SARS-CoV-2 assay on a Panther system.

[0172] [Table 13]

[0173] [Table 14]

[0174] All experimental results were valid (Table 15), indicating that the composition of the present invention is capable of lysing samples and preserving RNA even under varying storage temperature conditions.

[0175] [Table 15]

[0176] Example 9 Detection of target nucleic acid from a sample Nasal samples from 42 healthy volunteers were collected using flocked swabs and by dipping each swab into a tube containing 3 mL of composition C1 with Foam Ban at a concentration of 300 μL / L, diluted to 50% (v / v) with 0.9% (w / v) sodium chloride. The swab was rubbed against the tube walls and bottom for 15 seconds, after which the swab was discarded, except for one tube in which the swab was not discarded.

[0177] All collected samples were stored for 24-72 hours and then tested to determine the presence of human inclusions and therefore verify the proper collection of the samples for further nucleic acid detection. Testing was performed by detecting the human RNase P gene in negative control samples and in collected clinical samples. Samples were first extracted using the QIAamp® MinElute® Virus Spin Kit according to the manufacturer's instructions, then evaluated using Nanodrop, and finally tested to detect human inclusions using the CDC 2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (IDT). This panel was designed for the specific detection of SARS-CoV-2 (two primer / probe sets), and the kit also contained an additional primer / probe set to detect the human RNase P gene, and assayed to detect human inclusions in the samples.

[0178] All samples tested positive for the human RNase P gene with Cts comparable to those obtained when analyzing fresh human samples of the same nature using other media, indicating that the compositions of the present invention are suitable for collecting and storing samples suspected of containing target nucleic acids for further nucleic acid testing, regardless of whether the target nucleic acid is pathogen-derived or specimen-derived target nucleic acid.

[0179] Example 10 Compositions of the Invention for Saliva Sample Collection and Preservation The purpose of this study was to evaluate the composition of the present invention as a medium for collection and storage of saliva samples.

[0180] For the first experiment, 2.5 mL saliva samples from healthy volunteers (tested negative for SARS-CoV-2) were collected and introduced into collection tubes containing 2.5 mL of composition C1 (Table 1) with Foam Ban at a concentration of 300 μL / L. The contents of each collection tube were divided into two equal aliquots of 2.5 mL. To one of the aliquots, a known amount of heat-inactivated virus (7.5 μL of SARS-associated coronavirus 2, isolate USA-WA1 / 2020; catalog number NR-52286, BEI Resources) was added to obtain a spiked sample at 30 copies / mL. The other aliquot was used as a negative control (0 copies / mL). Both aliquots were tested in quadruplicate within the next 2 hours after collection by directly loading each replicate onto the Panther system immediately after removing the cap. The results for the negative control (0 copies / mL) and spiked samples (30 copies / mL) are shown in Table 16. Samples that test positive for SARS-CoV-2 are considered reactive, and a valid sample implies that the sample was successfully processed by the Panther system.

[0181] [Table 16]

[0182] This experiment demonstrates that the compositions of the present invention are suitable for collecting saliva samples.

[0183] For the second experiment, 35 mL of saliva sample (pooled normal saliva from human donors, catalog number 991-05-P; Lee Biosolutions, Inc.) was spiked with a known amount of heat-inactivated virus (325 μL of SARS-associated coronavirus 2, isolate USA-WA1 / 2020; catalog number NR-52286, BEI Resources) to obtain a spiked sample at approximately 90 copies / mL. The spiked sample was then mixed with an equal amount of composition C1 (Table 1). Six aliquots of 1.5 mL each were stored at 30°C and tested for SARS-CoV-2 after 1, 2, 3, 5, 7, and 10 days of storage. Eight aliquots of 1.5 mL each were stored at 4°C and tested for SARS-CoV-2 after 1, 2, 3, 5, 7, 10, 13, and 15 days of storage. All aliquots were tested in quintuplicates by processing in the Panther system as previously described. One aliquot of 1.5 mL was tested on day 0 as a baseline. The results are shown in Table 17. Samples showing positive results for SARS-CoV-2 were considered reactive, and a valid sample implies that the sample was successfully processed by the Panther system.

[0184] [Table 17]

[0185] This experiment demonstrates that the compositions of the present invention preserve the RNA content of saliva samples for at least 10 days at 30°C and at least 15 days at 4°C.

Claims

1. i) an anionic surfactant in an amount of about 1% to 20% (w / v); ii) lithium hydroxide in an amount of about 1% to 5% (w / v); iii) a chelating agent selected from succinic acid, EGTA, EDTA, and combinations thereof, in an amount of about 0.5% to 5% (w / v); and iv) a first buffering agent, present in an amount sufficient to maintain a pH between 5 and 10; 1. An aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising a first component comprising: further comprising a diluent component selected from the group consisting of water, a second buffer, a transport fluid, sodium chloride (aq), and combinations thereof, preferably sodium chloride (aq); The aqueous composition, wherein the first component is diluted with a diluent component by up to about 50% (v / v), and further comprises an antifoaming agent, preferably in an amount of about 50 μL / L to 750 μL / L. The aqueous composition comprising:

2. i) an anionic surfactant in an amount of about 1% to 20% (w / v); ii) lithium hydroxide in an amount of about 1% to 5% (w / v); iii) a chelating agent selected from succinic acid, EGTA, EDTA, and combinations thereof, in an amount of about 0.5% to 5% (w / v); and iv) a first buffering agent, present in an amount sufficient to maintain a pH between 5 and 10; 1. An aqueous composition for storing a biological sample for subsequent nucleic acid testing comprising: An aqueous composition comprising an antifoam agent in an amount of about 50 μL / L to 750 μL / L.

3. 3. The aqueous composition of claim 1 or 2, wherein the antifoaming agent comprises a silicone polymer, a polysorbate, an organic polyether dispersion, or any combination thereof.

4. 4. The aqueous composition of any one of claims 1 to 3, wherein the anionic surfactant comprises a C5 to C20 alkyl sulfate anion, a C5 to C20 alkenyl sulfate anion, a C5 to C20 alkynyl sulfate anion, or any combination thereof, preferably the surfactant comprises a lauryl sulfate anion.

5. 5. The aqueous composition of claim 1, wherein the first buffer is selected from Tris, MES, Bis-Tris, HEPES, MOPS, citrate, sodium bicarbonate, sodium phosphate, and combinations thereof, preferably the first buffer comprises HEPES.

6. 6. The aqueous composition of claim 1, wherein the first buffering agent is present in an amount of about 5% to 30% (w / v).

7. 7. The aqueous composition of any one of claims 1 to 6, wherein the first buffering agent is present in an amount of about 10% to 25% (w / v), the anionic surfactant is present in an amount of about 2% to 15% (w / v), the chelating agent is present in an amount of about 1% to 4% (w / v), and the lithium hydroxide is present in an amount of about 1% to 4% (w / v).

8. 8. The aqueous composition of claim 1, further comprising a population of polynucleotides comprising RNA, DNA, or any combination thereof, preferably wherein the RNA comprises an in vitro synthetic transcription product.

9. 9. A method for obtaining a population of polynucleotides from a sample suspected of containing nucleic acids comprising the step of contacting the sample, preferably a biological or environmental sample, with an aqueous composition according to any one of claims 1 to 8 at a temperature in the range of 2°C to 40°C, preferably further comprising an amplification reaction for amplifying at least the target nucleic acid and detection of at least one target in the resulting amplification products.

10. A method for preserving the integrity of a population of polynucleotides in a sample comprising the step of contacting the sample, preferably a biological or environmental sample, with an aqueous composition according to any one of claims 1 to 8 at a temperature in the range of 4°C to 40°C, preferably (i) the sample after contact with the aqueous composition is stored at a temperature in the range of −25° C. to 40° C. for at least 1 hour up to 6 months, up to 9 months, up to 12 months, or up to 26 months; and / or (ii) the sample after contact with the aqueous composition is stored at −20° C., 4° C., RT or 30° C.; method.

11. 9. A method for inactivating a sample containing at least one pathogen, comprising contacting the sample, preferably a biological or environmental sample, with an aqueous composition according to any one of claims 1 to 8, wherein the aqueous composition represents at least 30% (v / v) of the resulting mixture, and preferably the sample is contacted with the aqueous composition at 4°C, RT or 30°C.

12. A method for obtaining a sample, comprising the step of contacting the sample, preferably a biological or environmental sample, with an aqueous composition according to any one of claims 1 to 8, wherein the aqueous composition represents at least 30% (v / v) of the resulting mixture, preferably (i) the sample is directly introduced into a collection device or collection vessel comprising an aqueous composition according to any one of claims 1 to 8; or (ii) A method in which the sample is collected using a swab and introducing it into a collection device or collection container comprising an aqueous composition according to any one of claims 1 to 8, preferably the swab being discarded after rubbing against the collection device or collection container.

13. A collection device or container comprising an aqueous composition according to any one of claims 1 to 8.

14. 9. Use of an aqueous composition according to any one of claims 1 to 8 for obtaining a sample, preferably a biological or environmental sample, suspected of containing a target nucleic acid.

15. The method according to any one of claims 9 to 12 or the use according to claim 14, wherein the sample comprises at least one pathogen, preferably the at least one pathogen is a fungus, a bacterium, a parasite or a virus, preferably if the pathogen is a virus it is SARS-CoV-2.

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