Systems and methods for nucleic acid storage

Abstract

The present disclosure relates to systems and methods for nucleic acid storage that can be used under ambient conditions economically. In one implementation, the system may include a cartridge. The cartridge may include a first surface and a second surface opposing the first surface. The cartridge may include a chamber disposed (i) parallel to the first surface and the second surface and (ii) between the first surface and the second surface. The chamber may be configured to receive a blotter substrate. The cartridge may also include at least one opening disposed in the first surface above and interfaces with the chamber. The opening may be configured to receive a permeable membrane.

Description

SYSTEMS AND METHODS FOR NUCLEIC ACID STORAGECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 573,890 filed April 3, 2024 and U.S. Provisional Application No. 63 / 515,172 filed July 24, 2023. The entirety of each of these applications is hereby incorporated by reference for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under EB027690 and All 51788 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Nucleic acids, such as Ribonucleic Acid (RNA), can be extracted from cells or viruses and used in various downstream molecular applications (e.g., transcriptome analysis using nextgeneration sequencing, northern analysis, array analysis, RT-PCR, etc.). One such use is molecular detection of viral pathogens, where, if present, RNA specific to the virus is amplified and detected. For many viruses, this is the most sensitive and specific diagnostic method. Accurate diagnosis is critical for clinical management and disease containment and relies on extracting high quality RNA from a sample.

[0004] Extracting RNA needs care because RNA is not stable at ambient temperature and is rapidly degraded if not protected from RNA degrading enzymes (RNases), which are ubiquitous. However, current protocols for RNA storage under ambient conditions can take several days to prepare and can be complicated, and thus are unusable in real-world settings and speak to the difficulty in accomplishing this task.SUMMARY

[0005] Thus, there is a need for systems and methods for nucleic acid storage that can be used under ambient conditions economically.

[0006] Systems and methods disclosed herein relate generally to hardware systems and reagents systems for nucleic acid extraction and / or storage that can be used under ambient conditions for downstream molecular applications. In some examples, this disclosure can relate to systems of nucleic acid extraction and / or storage.

[0007] In some examples, the system may include a cartridge. The cartridge may include a first surface, a second surface opposing the first surface; and a chamber. The chamber may be disposed (i) parallel to the first surface and the second surface and (ii) between the first surface and the second surface. The chamber may be configured to receive a blotter substrate. In some examples, at least one opening may be disposed in the first surface above and interfaced with the chamber. The chamber may be configured to receive a blotter substrate and the opening may be configured to receive a permeable membrane.

[0008] In some examples, this disclosure can relate to methods of nucleic acid extraction for storage and / or molecular applications, for examples, using reagents according to embodiments. In some examples, the methods may include a method that includes contacting a sample containing a nucleic acid with lysis solution providing a lysed solution (lysate). The nucleic acid may be microbial RNA or DNA, e.g., including viral, bacterial, and parasite. In some examples, the sample may be saliva, blood, nasal swab (e.g., nasopharyngeal, anterior nares, nasal mid-turbinate, etc.), oropharyngeal swab, tissue, wastewater, and / or stool samples.

[0009] In certain embodiments, the lysis solution may include sucrose, potassium chloride (KC1), proteinase K, and a nucleic acid carrier, such as a DNA carrier, RNA carrier, or poly A carrier RNA.

[0010] In certain embodiments, proteinase K may be conjugated to beads.

[0011] In some examples, RNA, DNA, or both, in the sample may non-specifically adhere to the nucleic acid carrier, e.g., RNA or DNA carrier. In certain embodiments, its contemplated that an RNA or DNA virus or other microbe may non-specifically adhere, absorb, or hybridize to the nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA providing a nucleic acid (RNA or DNA) bound nucleic acid carrier. In certain embodiments, a nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA and proteinase K in the lysis solution may be at a ratio by weight of 1 to 2. In certain embodiments, the duration of contacting may be for an incubation period providing an incubated sample.

[0012] In certain embodiments, the incubation period may be between about 10 to 60 minutes.

[0013] In certain embodiments, the method may include contacting the incubated sample with a pH buffered solution comprising arginine and ethanol. The method may also include providing a binding buffer solution with the RNA or DNA bound nucleic acid carrier.

[0014] In certain embodiments, the binding buffer solution may include arginine and magnesium (Mg) salt and potassium salt.

[0015] In certain embodiments, the method may include contacting the binding buffer solution with the RNA or DNA bound nucleic acid carrier with the permeable membrane providing an RNA or DNA bound membrane. The permeable membrane may be pre-washed or pre-treated.

[0016] Additional advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosure. The advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The disclosure can be better understood with the reference to the following drawings and description. The components in the figures are not necessarily to scale, the emphasis being placed upon illustrating the principles of the disclosure.

[0018] Figures 1 A-C show an example of a cartridge according to some embodiments. Figure 1A shows an orthogonal view of the cartridge; Figure IB shows a side view of the cartridge; and Figure 1C shows a top view of the cartridge.

[0019] Figures 2A-C show another example of a cartridge according to some embodiments. Figure 2A shows an orthogonal view of the cartridge; Figure 2B shows a side view of the cartridge; and Figure 2C shows a top view of the cartridge.

[0020] Figures 3A-I show another example of a cartridge according to some embodiments. Figure 3A shows a top orthogonal view of the cartridge; Figure 3B shows a bottom orthogonal view of the cartridge; Figure 3C shows a side view of the cartridge; Figure 3D shows a top view of a top member of the cartridge; Figure 3E shows a bottom view of the top member of the cartridge; Figure 3F shows a front and side views of the top member and a front view of a bottom member of the cartridge; Figure 3G shows a side view of the bottom member of the cartridge;Figure 3H shows a top view of the bottom member of the cartridge; and Figure 31 shows a bottom view of the bottom member of the cartridge.

[0021] Figure 4 shows another example of a cartridge according to embodiments.

[0022] Figures 5A-C show an example of a transfer tool according to some embodiments.Figure 5 A shows an orthogonal view of the transfer tool; Figure 5B shows a top view of the transfer tool; and Figure 5C shows a side view of the transfer tool.

[0023] Figures 6A-C show another example of a transfer tool according to some embodiments. Figure 6A shows an orthogonal view of the transfer tool; Figure 6B shows a top view of the transfer tool; and Figure 6C shows a side view of the transfer tool.

[0024] Figures 7A-D show an example of a basket according to some embodiments. Figure 7A shows an orthogonal view of the basket; Figure 7B shows a side view of the basket; Figure 7C shows a top view of the basket; and Figure 7D shows a bottom view of the basket.

[0025] Figures 8A-D show another example of a basket according to some embodiments. Figure 8A shows an orthogonal view of the basket; Figure 8B shows a side view of the basket; Figure 8C shows a top view of the basket; and Figure 8D shows a bottom view of the basket.

[0026] Figures 9A-D show an example of a basket according to some embodiments. Figure 9A shows an orthogonal view of the basket; Figure 9B shows a side view of the basket; Figure 9C shows a top view of the basket; and Figure 9D shows a bottom view of the basket.

[0027] Figure 10 shows an example of a method of nucleic acid extraction and / or storage method using the hardware and reagents according to embodiments.

[0028] Figures 11 A-D show examples of the results using an example of systems and methods according to embodiments. Figure 11A shows RNA stability in the lysate over 14 days; Figure 11B shows inactivation of chikungunya virus in cell culture after the sample is processed using the current protocol; Figure 11C shows a comparison of viral RNA (dengue virus) stability when stored in on membranes placed in drying tubes at 37C compared to control conditions; and Figure 1 ID shows dengue virus rRT-PCR Ct values following extraction with the protocol according to embodiments compared to a commercial extraction robot.DESCRIPTION OF THE EMBODIMENTS

[0029] In the following description, numerous specific details are set forth such as examples of specific components, devices, methods, etc., in order to provide a thorough understanding ofembodiments of the disclosure. It will be apparent, however, to one skilled in the art that these specific details need not be employed to practice embodiments of the disclosure. In other instances, well-known materials or methods have not been described in detail in order to avoid unnecessarily obscuring embodiments of the disclosure. While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that there is no intent to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure.

[0030] The disclosed embodiments relate to systems and methods for nucleic storage and / or extraction that can enable economical storage of nucleic acids at ambient temperatures. The systems and methods for nucleic acid extraction and / or storage may be used for stabilization of primary samples, nucleic acid extraction from a sample for downstream molecular applications (e.g., RT-PCR, sequencing, etc.), and / or ambient temperature storage of extracted nucleic acid. The sample may include but is not limited to a sample isolated from saliva, blood, nasal swab (e.g., nasopharyngeal, anterior nares, nasal mid-turbinate, etc ), oropharyngeal swab, tissue, wastewater, or stool samples. The nucleic acid may include but are not limited to RNA, DNA, or a combination thereof.

[0031] In some examples, the systems may include hardware and reagents, for example, as disclosed herein. The hardware may include any combination of components disclosed therein, as well as additional and / or alternative components. The reagents may include any combination of reagents disclosed therein, as well as additional and / or alternative reagents. It will also be understood that the hardware may be used with different and / or alternative reagents; and / or the reagents may be used with different and / or alternative hardware.

[0032] Hardware Examples

[0033] Figures 1-9B show examples of components of hardware systems according to embodiments. In some examples, a hardware system may include a cartridge, for example, as shown in Figures 1 A-4.

[0034] In some examples, the cartridge can allow the entirety of a buffer solution (e.g., buffer / EtOH mix) with the sample to be dispensed onto a permeable membrane all at once ascompared to conventional techniques that require one drop at a time to be dispensed from a pipette. Thus, simplifying the nucleic acid extraction process. Additionally, the cartridge may be reusable and can be configured to be decontaminated.

[0035] In some examples, the cartridge may be made of one or more materials. The one or more materials may include but is not limited to resin, polyamide, polyethylene, polycarbonate, polypropylene, polystyrene, thermoplastic elastomer, among others, or any combination thereof.

[0036] In some examples, the cartridge may include a chamber configured to receive a blotter substrate and a (first) opening on a top surface that interfaces with the chamber that is configured to receive a permeable membrane. In some examples, the permeable membrane may include but is not limited to a glass microfiber (GF / D) membrane. In some examples, the cartridge may include a (second) opening that interfaces with the chamber on a side surface.

[0037] In some examples, the chamber may be configured to allow the blotter substrate to slide in and out of the chamber. In use, the chamber can improve loading and reloading of the membrane. Also, the chamber can eliminate the need for centrifugation to move buffers across the membrane while also containing the used reagents. Thus, this can eliminate hazardous liquid waste thereby preventing contamination of the lab environment.

[0038] In some examples, the chamber may be sized to provide sufficient space for the blotter substrate while allowing air movement out to speed the buffer flow from the opening.

[0039] In some examples, the opening may be configured to allow for drop-in loading and pop-out unloading of permeable membrane. In use, this opening can be configured to guide the buffer through the membrane onto the blotter substrate. Thereby, user experience can be improved while increasing nucleic acid recovery. Although the openings are shown as circular in Figures 1 A-4, it would be understood that different shape openings may also be shown.

[0040] In some examples, the permeable membrane may be a single layer, matrix membrane. In some examples, the permeable membrane may be a glass microfiber membrane (also referred to as an “RNAES membrane”). In some examples, the permeable membrane may include but is not limited to a Whatman Fusion 5™ membrane, Whatman 3™ membrane, among others, or a combination thereof. The membrane may be pre-washed or pre-treated prior to use in the protocol. This can improve RNA and / or DNA binding. Hereinafter, these will be referred to as the “membrane” or “permeable membrane.”

[0041] In some examples, the cartridge may be unitary piece. Figures 1 A-2C show examples of cartridges that are a single, integrated component.

[0042] Figures 1A-C show an example of a cartridge 100. In this example, the cartridge 100 may be a unitary piece. In this example, the cartridge 100 may include a first surface 102 (also referred to as “top surface”) and a second surface 104 (also referred to as “bottom surface”) opposing the first surface 102. In some examples, the cartridge 100 may include a chamber 140 disposed (i) parallel to the first surface 102 and the second surface 104 and (ii) between the first surface 102 and the second surface 104. In some examples, the chamber 140 may be configured to receive a blotter substrate. In some examples, the cartridge 100 may include at least one (top) opening 130 disposed in the first surface 102 above and interfaces with the chamber 140. The opening 130 may be configured to receive a permeable membrane.

[0043] In some examples, the cartridge 100 may include a first side surface 112 that is disposed perpendicular to the first surface 102 and the second surface 104. In some examples, the cartridge 100 may include a second opening 142 disposed in the first side surface 112 and configured to interface with the chamber 140. The second opening 142 may be configured to receive the blotter substrate. In some examples, the second opening 142 may be used to load / unload the blotter substrate from the chamber 140.

[0044] In some examples, the first opening may have a tapered shape, for example, as shown in a cartridge 200 shown in Figures 2A-C. Like the cartridge 100, the cartridge 200 may be a unitary piece. In this example, like the cartridge 100, the cartridge 200 may include a first surface 202 (also referred to as “top surface”) and a second surface 204 (also referred to as “bottom surface”) opposing the first surface 202. In some examples, the cartridge 200 may include a chamber 240 disposed (i) parallel to the first surface 202 and the second surface 204 and (ii) between the first surface 202 and the second surface 204. In some examples, the chamber 240 may be configured to receive a blotter substrate. In some examples, the cartridge 200 may include at least one opening 230 disposed in the first surface 202 above and interfaces with the chamber 240. The opening 230 may be configured to receive a permeable membrane.

[0045] In this example, the opening 230 may have a tapered shape. For example, the opening 230 may have a first circumference 232 that is larger than the second circumference 234 so that its circumference tapers toward the chamber 240. In this example, the second circumference 234 maybe sized to correspond to a size of the permeable membrane. The second circumference 234 may be directly above the chamber 240.

[0046] In some examples, like the cartridge 100, the cartridge 200 may include a first side surface 212 that is disposed perpendicular to the first surface 202 and the second surface 204. In some examples, the cartridge 200 may include a second opening 242 disposed in the first side surface 212 and configured to interface with the chamber 240. The second opening 242 may be configured to receive the blotter substrate. In some examples, the second opening 242 may be used to load / unload the blotter substrate from the chamber 240.

[0047] In some examples, the cartridge may be two pieces that can be snapped together to form a single piece. Figures 3A-3I show an example of a cartridge 300 that includes a first (top) member 350 and a second (bottom member) 360.

[0048] Like the cartridges 100 and 200, the cartridge 300 may include a first surface 302 (also referred to as “top surface”) and a second surface 304 (also referred to as “bottom surface”) opposing the first surface 302. In some examples, the cartridge 300 may include a chamber 340 disposed (i) parallel to the first surface 302 and the second surface 304 and (ii) between the first surface 302 and the second surface 304. In some examples, the chamber 340 may be configured to receive a blotter substrate. In some examples, the cartridge 300 may include at least one opening 330 disposed in the first surface 302 above and interfaces with the chamber 340. The opening 330 may be configured to receive a permeable membrane.

[0049] In this example, the opening 330 may have a tapered shape like the cartridge 200. For example, the opening 330 may have a first circumference 332 that is larger than the second circumference 334 so that its circumference tapers toward the chamber 340. In this example, the second circumference 334 may be sized to correspond to a size of the permeable membrane. The second circumference 334 may be directly above the chamber 340.

[0050] In some examples, like the cartridges 100 and 200, the cartridge 300 may include a first side surface 312 that is disposed perpendicular to the first surface 202 and the second surface 304. In some examples, the cartridge 300 may include a second opening 342 disposed in the first side surface 312 and configured to interface with the chamber 340. The second opening 342 may be configured to receive the blotter substrate. In some examples, the second opening 342 may be used to load / unload the blotter substrate from the chamber 340 before the members 350 and 360 are snapped together.

[0051] In some examples, the first member 350 may include the opening 330 and the second member 360 may include the chamber 340 and the (second) opening 342. In some examples, each member may include one or more fastening members configured to allow for the members 350 and 360 to removably fit with each other. In this example, the fastening member(s) may be snap- fit member. For example, as shown in Figures 3E, the first member 350 may include fastening members 356 disposed on opposing side surfaces and the second member 360 may include fastening members 366 disposed on the opposing side surfaces and complimentary to the fastening members 356 to allow for the members 350 and 360 to snap-fit when pressed together.

[0052] In use, in this example, the blotter substrate may be loaded into the chamber 340 before snap-fitting the members 350 and 360. In some examples, the permeable membrane may be loaded into the opening 330 before the members 350 and 360 are snapped-fit.

[0053] In some examples, the cartridge may include more than one opening for a permeable membrane. By way of example, as shown in Figure 4, a cartridge 400 may include a plurality of openings 430 that interfaces with a blotter pad 470 disposed in the chamber 440 thereby enabling simultaneous extraction of a plurality of samples at a time. Like the cartridges 100 and 200, the blotter pad 470 may be loaded and unloaded using the opening 442. The cartridge 400 may include any number of openings 430 and is not limited to the number and / or configuration of channels and rows shown. For example, the number of openings 430, including the rows and columns, may be based on sample volume. By including a plurality of openings 430, unnecessary waste may be eliminated. The openings 430 may have a circular shape with a uniform and / or tapered circumference, for example, as shown in Figures 1-3. In other examples, the openings 430 may have a different shape.

[0054] In some examples, the system may include a transfer tool. In some examples, the transfer tool can be configured to allow for easy transfer of the membrane from the cartridge to a drying container (e.g., drying tube). This tool may be configured to be decontaminated and therefore reusable.

[0055] In some examples, the transfer tool may include a first section, a second section, and a third section. The second section may be disposed between the first section and the third section. In some examples, the first section may be a handle. In some examples, the third section may include one or more features configured to interface with the membrane and enable transferthereof. The third section may include a prong, flat surface, among others, or a combination thereof.

[0056] Figures 5A-6C show examples of transfer tools according to embodiments. Figures 5A-5C show an example of a transfer tool 500. In some examples, the transfer tool 500 may include a first section 510, a second section 520, and a third section 530. The second section 520 may be disposed between the first section 510 and the third section 530.

[0057] In some examples, the first section 510 may be a handle. In some examples, the third section 530 may be configured to interface with the membrane. In this example, the third section 530 may be a prong.

[0058] Figures 6A-6C show an example of a transfer tool 600. In some examples, the transfer tool 600 may include a first section 610, a second section 620, and a third section 630. The second section 620 may be disposed between the first section 610 and the third section 630.

[0059] In some examples, the first section 610 may be a handle. In some examples, the third section 630 may be configured to interface with the membrane. In this example, the third section 630 may be a flat surface.

[0060] In some examples, the system may also include a drying container. In some examples, the drying container may include a body having a chamber. In some examples, the chamber may be filled with a drying agent, such as silica beads. In some examples, the drying container may include a basket that is disposed onto the body above the chamber including the drying agent and configured to removably snap-close the chamber. In some examples, the basket may include a base surface configured to receive the permeable membrane and to separate the membrane from the drying agent. In some examples, the drying basket may be reusable so that the process can be more cost efficient.

[0061] In some examples, the basket may include one or more features configured to allow for air flow. In some examples, the one or more features may include spaced slots, one or more openings, among others, or a combination thereof.

[0062] In some examples, the basket may have a shape complimentary to the drying body. For example, if the body is a tube with a circular opening, the basket may also have a circular shape. Figures 7A-9D show examples of a basket according to embodiments.

[0063] Figures 7A-7D show an example of a basket 700 that includes slots 730. In some examples, the basket 700 may include a base surface 720 configured to receive the permeablemembrane and to separate the membrane from a drying agent. In some examples, the base surface 720 may be bounded by a wall 710. In this example, the basket 700 may have a circular shape.

[0064] In some examples the slots 730 may be disposed in the base surface 720 separated by openings 740 to allow for airflow.

[0065] Figures 8A-8D show an example of a basket 800 that includes an opening 830. In some examples, like the basket 700, the basket 800 may include a base surface 820 configured to receive the permeable membrane and to separate the membrane from a drying agent. In some examples, the base surface 820 may be bounded by a wall 810. In this example, the basket 800 may have a circular shape.

[0066] In some examples the basket 800 may include one or more openings 830 disposed in the base surface 820 to allow for airflow. In this example, the basket 800 includes one opening 830 disposed substantially in the center of the base surface 820. In other examples, the basket 800 may include more than one opening 830 disposed in a different position with respect to the base surface 820.

[0067] In some examples, the baskets may include one or more features configured to be used to remove the basket from the body of the drying container. In some examples, the baskets may include a pole and / or handle.

[0068] Figures 9A-9D show an example of a basket 900 that includes a pole 930. In some examples, the basket 900 may include a base surface 920 configured to receive the permeable membrane and to separate the membrane from a drying agent. In some examples, the base surface 920 may be bounded by a wall 910. In this example, the basket 900 may have a circular shape. In this example, the pole 930 may extend from the base surface 930 past the wall 910 so that it protrudes.

[0069] In some examples the basket 900 may include one or more features, such as openings and / or slots, like those shown and described in Figures 7A-8D, configured to allow for airflow between the permeable membrane and drying agent.

[0070] Reagents

[0071] In some examples, the reagents according to the disclosure can provide optimized buffers for stabilization of primary samples, nucleic acid extraction from a sample for downstreammolecular applications (e.g., RT-PCR, sequencing), and / or ambient temperature storage of extracted nucleic acid.

[0072] In some embodiments, the reagents may include a binding buffer solution, a lysis solution, an elution buffer, a wash buffer, among others, or any combination thereof. In some examples, the lysis solution may provide a lysed solution. In some examples, the lysis solution may include but is not limited to sucrose, potassium chloride (KC1), proteinase K, nucleic acid carrier, DNA carrier, RNA carrier, poly A carrier RNA, among others, or a combination thereof. In some examples, the nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA and proteinase K in the lysis solution may have a ratio by weight of 1 to 2. In certain embodiments, the wash buffer may contain glycerin hydrochloride at a pH below 5, 6 or 7. In certain embodiments, the pH buffer may be added after the sample is loaded onto the membrane.

[0073] In some examples, the binding buffer solution may be a pH-buffered solution. In some examples, the pH-buffered solution may include but is not limited to arginine, ethanol, among others, or a combination thereof. In some examples, the pH-buffered solution may provide a binding buffer solution. In some examples, the binding buffer solution may include but is not limited to arginine, magnesium (Mg) salt, potassium salt, among others, or a combination thereof.

[0074] In some examples, the elution buffer may include but is not limited to a TE buffer. In some examples, the TE buffer may have a pH of about 8.0. In some examples, the wash buffer may be a low pH buffer. In some examples, the wash buffer may include glycine hydrocholoride.

[0075] Methods For Nucleic Acid Storage And / or Extraction

[0076] In some examples, this disclosure relates to methods of nucleic acid extraction for storage and / or molecular applications. Figure 10 shows an example of a method 1000 for nucleic acid extraction and / or molecular applications using the reagents and hardware disclosed herein. It will be understood that the methods using the disclosed reagents is not limited to the hardware disclosed herein and may be used with different hardware.

[0077] In some embodiments, the method 1000 may include a step 1010 of contacting a sample containing a nucleic acid with a lysis solution providing a lysed solution, or a lysis sphere, for example, as shown in 1012. The nucleic acid may be viral, bacterial, or other microbial RNA or DNA. In some examples, the sample may be saliva, blood, nasal swab (e.g., nasopharyngeal, anterior nares, nasal mid-turbinate, etc.), oropharyngeal swab, tissue, wastewater, or stoolsamples. In certain embodiments, the lysis solution may include one or more lyophilized lysis reagents that, upon reconstitution with the sample, can form a lysate. In some examples, the lysis solution may include but is not limited to sucrose, KC1, proteinase K, a nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA, among others, or any combination thereof. In some examples, the proteinase K may be conjugated to beads.

[0078] For example, if the nucleic acid is viral, bacterial, or other microbial RNA, the viral bacterial or other microbial RNA in the sample may hybridize to the RNA carrier or poly A carrier RNA providing an RNA bound nucleic acid carrier or the RNA may non-specifically absorb or adhere to the RNA carrier, or poly A carrier RNA. In some examples, the RNA carrier or poly A carrier RNA and proteinase K in the lysis solution may be at a ratio by weight of 1 to 2.

[0079] For example, if the nucleic acid is viral, bacterial, or other microbial DNA, the DNA in the sample may hybridize to the DNA carrier providing a DNA bound nucleic acid carrier or the DNA may non-specifically absorb the DNA carrier. In some examples, the DNA carrier and proteinase K in the lysis solution may be at a ratio by weight of 1 to 2.

[0080] In some examples, the contacting may be for an incubation period providing an incubated sample. For example, the incubation period may be between about 10 to 60 minutes.

[0081] In some examples, the method 1000 may include a step 1020 of contacting the incubated sample with a pH buffered solution to provide a binding buffer solution, for example, with the RNA or DNA bound nucleic acid carrier. In some examples, the pH buffered solution may include arginine and ethanol. In some examples, the binding buffer solution may include arginine and Mg salt and potassium salt.

[0082] In some examples, the method 1000 may include a step 1030 of contacting the binding buffer solution with the RNA or DNA bound nucleic acid carrier with a permeable membrane (e g., glass fiber membrane) providing an RNA or DNA bound membrane. In some examples, the permeable membrane may be provided within a cartridge according to embodiments, for example, as shown in 1032. By way of example the cartridge may be the cartridge as disclosed and described with respect to Figures 1A-4.

[0083] In some examples, the method 1000 may include a step 1040 of washing the membrane with a washing solution.

[0084] Next, the method 1000 may include a step 1050 of removing the RNA or DNA bound membrane from the cartridge according to embodiments. In some examples, the method 1000 mayinclude a step 1060 of placing the membrane onto the base surface of the basket of the drying container filled with a drying agent according to embodiments, for example, as shown in 1062. For example, the basket may be the basket as disclosed and described with respect to Figures 7A- 9D.

[0085] In some examples, the transfer tool according to embodiments may be used to remove the membrane and transfer it to the basket. For example, the transfer tool may be the transfer tool as disclosed and described with respect to Figures 5A-6C. In other examples, other tools may be used. In some examples, before moving the RNA or DNA bound membrane to the cartridge, it may be washed with a wash buffer solution or ethanol.

[0086] In some examples, the RNA or DNA bound membrane may be stored in the drying container for a period of time under ambient conditions.

[0087] Next, when ready to perform molecular applications on the RNA or DNA bound membrane, the method 1000 may include a step 1070 of preparing the sample for one or more molecular applications. In some examples, the step 1070 may include transferring the RNA or DNA bound membrane from the drying container to a container with an elution reagent, for example, as shown in 1072.

[0088] Next, one or more molecular applications may be performed on the eluates in the container.

[0089] Figures 11 A-D shows A) RNA stability in the lysate over 14 days, B) inactivation of chikungunya virus in cell culture after the sample is processed using the current protocol, C) a comparison of viral RNA (dengue virus) stability when stored in on membranes placed in drying tubes at 37C compared to control conditions, and D) dengue virus rRT-PCR Ct values following extraction with the current protocol compared to a commercial extraction robot.

[0090] Nucleic Acid Extraction and / or Storage Kit

[0091] In some examples, the disclosure may relate to a kit that includes the one or more of the hardware components and / or reagents according to embodiments. In some examples, the kit may include a permeable membrane, the cartridge, blotter substrate, the drying container, and one or more reagents as disclosed herein. In some examples, the kit may further include the transfer tool as disclosed therein. In some examples, the kit may omit one or more of the disclosed hardware and / or reagents; and / or may include additional and / or alternative hardware and / or reagents and / orsolutions. For example, the kit may also include nuclease-free water, mylar bags, among others, or any combination thereof.

[0092] While the disclosure has been described in detail with reference to exemplary embodiments, those skilled in the art will appreciate that various modifications and substitutions may be made thereto without departing from the spirit and scope of the disclosure as set forth in the appended claims. For example, elements and / or features of different exemplary embodiments may be combined with each other and / or substituted for each other within the scope of this disclosure and appended claims.

Claims

CLAIMSWhat is claimed:

1. A nucleic acid storage system, comprising: a cartridge, the cartridge including: a first surface a second surface opposing the first surface; a chamber disposed (i) parallel to the first surface and the second surface and (ii) between the first surface and the second surface, the chamber being configured to receive a blotter substrate; and at least one opening disposed in the first surface above and interfaces with the chamber, the opening being configured to receive a permeable membrane.

2. The nucleic acid storage system according to claim 1, further comprising: the permeable membrane; wherein the permeable membrane is a glass microfiber membrane.

3. The nucleic acid storage system according to claims 1 or 2, wherein the cartridge further includes: a first side surface that is disposed (i) perpendicular to the first surface and the second surface and (ii) between the first surface and the second surface; and a second opening disposed in the first side surface and configured to interface with the chamber; the second opening being configured to receive the blotter substrate.

4. The nucleic acid storage system according to any of claims 1-4, wherein the at least opening is circular.

5. The nucleic acid storage system according to any of claims 1-5, wherein the at least opening has tapered diameter.

6. The nucleic acid storage system according to any of claims 1-5, wherein the cartridge includes a plurality of second openings.

7. The nucleic acid storage system according to any of claims 1-6, further comprising: a drying container, the drying container including: a body including a chamber, the chamber filled with a drying agent; and a basket disposed within the chamber above the drying agent and configured to removably snap-close the chamber.

8. The nucleic acid storage system according to claim 7, wherein the basket includes a base surface configured to receive the permeable membrane and to separate the permeable membrane from the drying agent.

9. The nucleic acid storage system according to claims 7 or 8, wherein the base surface is slotted.

10. The nucleic acid storage system according to any of claims 7-9, wherein the basket includes one or more openings within the base surface.

11. The nucleic acid storage system according to any of claims 7-10, wherein: the body is a tube and the basket is circular; and the drying agent is silica beads.

12. The nucleic acid storage system according to any of claims 1-11, further comprising: a transfer tool, the transfer tool including a first section, a second section, and a third section, the second section being disposed between the first section and the third section; wherein the transfer tool is configured to transfer the permeable membrane between and from the cartridge and / or the drying container.

13. The nucleic acid storage system according to claim 12, wherein the first section is a handle.

14. The nucleic acid storage system according to claims 12 or 13, wherein the third section is a prong or a flat surface.

15. The nucleic acid storage system according to any of claims 12-14, wherein the third section is configured to remove the permeable membrane from the blotter substrate when disposed in the cartridge.

16. The nucleic acid storage system according to any of claims 1-15, further comprising: one or more reagents, the one or more reagents including a lysis solution, among others, or a combination thereof.

17. A nucleic acid extraction method for storage and / or molecular applications, comprising: contacting a sample containing RNA or DNA with a buffer reagent, to provide a binding buffer solution with an RNA or DNA bound nucleic acid carrier; contacting the binding buffer solution with the RNA or DNA bound nucleic acid carrier with the permeable membrane disposed within the cartridge according to any of claims 1-6; removing the permeable membrane from the cartridge using the transfer tool according to any of claims 12-15; and using the transfer tool according to any of claims 12-15, placing the permeable membrane onto the base surface of the basket of the drying container according to any of claims 7-11 .

18. The nucleic acid extraction method according to claim 17, wherein the sample is isolated from saliva, blood, nasal swab, oropharyngeal swab, tissue, wastewater, or stool.

19. The nucleic acid extraction method according to claims 17 or 18, further comprising: contacting the sample containing a nucleic acid with a lysis solution providing a lysed solution for an incubation period; wherein the incubated sample is contacted with a pH buffered solution comprising arginine and ethanol.

20. The nucleic acid extraction method according to claim 19, wherein lysis solution comprises sucrose, KC1, proteinase K, and nucleic acid carrier, DNA carrier, RNA carrier, or poly A carrier RNA.

21. The nucleic acid extraction method according to claim 20, wherein: the sample contains RNA or DNA virus, bacteria, or other microbe; and when the sample is contacted with a lysis solution, the RNA or DNA non-specifically absorbs or hybridizes to the nucleic acid carrier, DNA carrier, RNA carrier or poly A carrier RNA providing an RNA or DNA bound nucleic acid carrier.

22. The nucleic acid extraction method according to claim 21, wherein the nucleic acid carrier, DNA carrier, RNA carrier or poly A carrier RNA and proteinase K in the lysis solution are at a ratio by weight of 1 to 2.

23. The nucleic acid extraction method according to any of claims 17-22, further comprising: storing the permeable membrane in the drying container for a period of time under ambient conditions.

24. The nucleic acid extraction method according to any of claims 17-23, further comprising: removing the permeable membrane from the drying container; placing the permeable membrane into a container with an elution reagent; removing the permeable membrane from the container; and performing a molecular application on the eluates in the container.

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