Nucleic acid extraction and separation with thermally labile silanes and chemically modified solid supports

Abstract

Compositions and methods for isolating and detecting nucleic acids in a biological sample are provided. Compositions and methods utilize a modified solid support comprising amine or amide groups.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 337,014, filed on April 29, 2022, which is incorporated herein by reference for all purposes. Technical Field

[0003] The present invention relates generally to the field of molecular biology. In certain embodiments, the present invention provides devices, kits and methods related to the separation and detection of nucleic acids. Background Art

[0004] Isolation of nucleic acids is typically the first step in most molecular biology studies, including polymerase chain reaction (PCR), DNA hybridization, restriction enzyme digestion, DNA sequencing, and array-based experiments. Therefore, there is a need for simple and reliable methods for isolating nucleic acids, particularly for isolating high-quality nucleic acids. Various techniques for isolating nucleic acids from samples have been described, the most common of which involves cleaving a nucleic acid source in a chaotropic substance (e.g., guanidine salts, urea, and sodium iodide) in the presence of a DNA-binding solid phase (e.g., glass beads or fibers). The released nucleic acids are bound to the solid phase in a one-step reaction, in which the solid phase is washed to remove any remaining contaminants. As an example, a glass fiber disk with a 30 mm diameter and a 0.7 μm pore size has been shown to capture 150 μg of plasmid DNA (binding capacity approximately 30 μg / cm) with a 2M guanidine hydrochloride (GuHCl) lysate. 2 ). Kim, YC and Morrison, SLP LoS ONE (2009) 4, 11, e7750. Although these methods have been shown to be rapid, they result in moderate DNA shearing and a degree of contamination. Residual GuHCl or GuSCN can poison downstream PCR reactions and must be removed by extensive washing. A rapid, economical, and high-yield method for isolating nucleic acids from a sample is needed.

[0005] In order to improve the sensitivity of nucleic acid detection, large sample volumes can be prepared. However, the preparation of large volumes is in conflict with the fluid system for automatic lysis, processing and / or analysis of biological samples. In particular, the automated market products currently used to process samples (e.g., whole blood for detecting pathogenic targets) are limited to only tolerate hundreds of microliters of blood per test. The prior art that can process multi-milliliter blood samples for pathogen detection involves a laborious process with multiple manual steps, which must be correctly followed by the user, including centrifugation, decantation, vortexing and DNA precipitation and purification based on glass columns. After using the existing sample processing package products, the user is still responsible for preparing a PCR device that can accurately analyze the sample produced by the sample package products. There is a demand for automatic sample processing of multi-milliliter blood in a single device on the market. If the method minimizes the required operation of the sample and can be performed using a single device, the method will be useful. Certain embodiments of the present invention described herein provide such methods. Other embodiments of the present invention described herein provide devices and package products that can be used to separate nucleic acids from samples. Other embodiments of the present invention also provide detection of nucleic acids in samples. Summary of the invention

[0006] Described herein are compositions, systems and methods for isolating and purifying nucleic acids from biological samples. The compositions, systems and methods utilize modified solid supports comprising DNA binding ligands as separation materials, thereby reducing and / or eliminating the amount of lysis reagents and nucleic acid binding agents conventionally used in PCR processes.

[0007] In some aspects, compositions and systems for isolating nucleic acids from a biological sample comprise: a compound bonded to a solid support, the compound being derived from a structure represented by the formula:

[0008] Y-(L) y -SiX3

[0009] wherein Y is a DNA binding ligand selected from an alkylamine, a cycloalkylamine, an alkoxyamine, a polyamine moiety, an intercalator, a minor groove binder (e.g., a bisbenzimide minor groove binder), a peptide, an amino acid, an arylamine, or a combination thereof; L is a linker selected from an alkyl group, a heteroalkyl group, an olefin group, a heteroolefin group, a polyacrylic acid, a Diels-Alder adduct, or a combination thereof; each X is independently selected from a hydrolyzable group, an alkyl group, a heteroalkyl group, an alkenyl group, or two or three Xs are combined to form one or more cyclic groups, or one X is combined with Y to form a cyclic azasilane; and y is 0 or 1.

[0010] The DNA binding ligand or substituent Y may comprise a plurality of amine groups; a plurality of amide groups; or a combination thereof. For example, the DNA binding ligand or Y may comprise at least two, at least three, at least four, at least five, at least six amine or amide groups or a combination thereof. In some embodiments, the DNA binding ligand or Y comprises an alkylamine group, an imidazole group or a combination thereof. Representative examples of amine groups include spermine, methylamine, ethylamine, propylamine, ethylenediamine, diethylenetriamine, 1,3-dimethyldipropyldiamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium hydrochloride, tris(2-aminoethyl)amine or a combination thereof.

[0011] In some aspects of the compounds disclosed herein, a linker L is present (or y is 1). The linker may be selected from an alkylene oxide group, an alkylene group, or a Diels-Alder adduct.

[0012] The group X facilitates the attachment of the compounds disclosed herein to a solid support. For example, Figure 4 Shown how to be incorporated into the solid support material by bonding with the solid support material and / or with adjacent silane molecules. In some embodiments, each X can be independently selected from the part that promotes hydrogen bonding, electrostatic attraction, covalent bonding, horizontal and / or vertical polymerization at each occurrence, wherein the functional group is present at the interface or solid support material and / or exists with adjacent silanes. In some aspects of the composition and system, at least two X can be independently selected from halogen, alkoxy, dialkylamino, triflate, or combined with the Si atom to which they are connected to form oligosilane or polysilane, silazotricycle, cyclic siloxane, polysilsesquioxane or silazane. For example, at least two X can be independently selected from alkoxy groups (e.g., ethoxy or methoxy).

[0013] In other aspects, compositions and systems for separating nucleic acids from biological samples include Diels-Alder adducts, which include DNA binding ligands, wherein the Diels-Alder adducts are optionally bonded to a solid support. As defined herein, the DNA binding ligand can include an amine group, an intercalator, a minor groove binder, a peptide, an amino acid, or a combination thereof. In some examples, the DNA binding ligand is selected from an alkylamine, a cycloalkylamine, an alkoxyamine, an arylamine, a polyamine moiety, or a combination thereof. The Diels-Alder adducts described herein can be derived from unsaturated cyclic imide groups.

[0014] In some embodiments, the compound or Diels-Alder adduct bound to the solid support can be derived from a structure represented by the following general formula, its isomers, salts, tautomers, or combinations thereof:

[0015]

[0016] , wherein Y' is a DNA binding ligand, and L, X and y are as defined herein. For example, Y' may include an alkylamine group, an imidazole group, or a combination thereof. In some examples, Y' may further include more than one DNA binding ligand, for example, Figure 8 In, it is shown that the alkylamine group is further connected to the minor groove DNA binding ligand. L optionally exists and can be selected from alkylene oxide group, alkylene group, cyanuric chloride, alkylamine or its combination. At least two X can be independently selected from halogen, alkoxy, dialkylamino, triflate, or form silazotricycle, cyclic siloxane, polysilsesquioxane or silazane together with the Si atom to which they are connected.

[0017] In some examples, the compound or Diels-Alder adduct can be derived from one of the following structures:

[0018]

[0019] 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, aminoalkylsilazotricyclo, 3-(2-aminoethyl)aminopropyltriethoxysilane, 3-(2-aminoethyl)aminopropyltrimethoxysilane, or a combination thereof, and wherein n is an integer from 0 to 10, from 1 to 10, or from 1 to 5.

[0020] The solid support described herein can include a material selected from silica, glass, ethylene skeleton polymers, mica, polycarbonate, zeolite, titanium dioxide, magnetic beads, glass beads, cellulose filters, polycarbonate filters, polytetrafluoroethylene filters, polyvinyl pyrrolidone filters, polyethersulfone filters, glass fiber filters, or combinations thereof. In some examples, the solid support is a glass fiber filter. The glass fiber filter can have a selected pore size to accommodate beads of corresponding sizes to promote mechanical lysis. For example, the glass fiber filter can have a pore size of 0.2 μm to 3 μm, 0.2 μm to 2 μm, 0.5 μm to 1.0 μm, or 0.6 μm to 0.8 μm. In addition, the glass fiber filter can have a pore size of 35 g / m 2 Up to 100g / m 2 Basis weight, preferably 50 g / m 2 Up to 85g / m 2 , or 70g / m 2 Up to 80g / m 2The glass fiber filter may have a fiber diameter of 1 μm to 100 μm, preferably 1 μm to 50 μm, or 1 μm to 25 μm. The glass fiber filter may have a thickness of 250 μm to 2,000 μm, 300 μm to 1,500 μm, 300 μm to 1,000 μm, 300 μm to 750 μm, or 350 μm to 500 μm. As described herein, the glass fiber filter may accommodate beads to facilitate mechanical lysis. The beads may include glass beads, silica beads, or a combination thereof. The compound or Diels-Alder adduct may be bonded to the solid support via a siloxane bridge, a carboxylate bridge, an ester bridge, an ether bridge, or a combination thereof.

[0021] Also disclosed herein are separation materials for nucleic acid separation and purification. The separation material may include a compound or composition disclosed herein, including a DNA binding ligand. For example, the separation material may include a glass fiber solid support and a compound bonded to the glass fiber solid support. As described herein, the compound may be derived from a structure represented by the following formula: Y-(L) y -SiX3, wherein Y, L, X and y are as defined herein. In other examples, the separation material may include a glass fiber solid support, the glass fiber solid support comprising a Diels-Alder adduct with a DNA binding ligand, wherein the adduct is chemically bonded to the glass fiber solid support. The glass fiber solid support may further comprise a polymer binder. In some examples, the glass fiber solid support may be in the form of a glass fiber disk of 500 microns to 2000 microns thick, the glass fiber disk having an effective pore size of 0.5 microns to 1 micron.

[0022] Also provided is a system including compounds, compositions and separation materials disclosed herein. The system can be a sample box, preferably an automated sample box. Therefore, disclosed herein is a system including a sample box for separating and detecting nucleic acids from biological samples. The sample box can include: a) a box body having a plurality of chambers defined therein, wherein the plurality of chambers are fluidly connected by a fluid path of the box, and wherein at least one chamber is configured to receive a biological sample, b) a reaction vessel configured to amplify nucleic acids by thermal cycling, and c) a filter disposed in a fluid path between the plurality of chambers and the reaction vessel, wherein the filter includes a compound, separation material or composition as disclosed herein, wherein the plurality of chambers and the reaction vessel independently include reagents for releasing nucleic acids from the biological sample and primers and probes for detecting nucleic acids. In other aspects, the sample box may include a) a box body having a plurality of chambers therein, wherein the plurality of chambers include: a sample chamber having at least a fluid outlet fluidically connected to another chamber in the plurality of chambers; and a lysis chamber fluidically connected to the sample chamber, the lysis chamber including a reagent for releasing nucleic acid, optionally wherein the sample chamber and the lysis chamber are the same; b) a reaction vessel fluidically connected to the plurality of chambers of the box body and configured for amplifying nucleic acid, and ii) detecting a plurality of amplification products; c) a filter disposed in a fluid path between the lysis chamber and the reaction vessel, wherein the filter includes a solid support modified with a DNA binding ligand selected from alkylamines, cycloalkylamines, alkoxyamines, polyamine moieties, intercalators, minor groove binders (e.g., bisbenzimide minor groove binders), peptides, amino acids, proteins, arylamines, or combinations thereof, and d) a plurality of primers and / or probes disposed in one or more chambers in the plurality of chambers or the reaction vessel for detecting nucleic acid. The sample box is configured to perform non-isothermal amplification, as well as isothermal amplification, by, for example, thermal cycling, gradient (temperature difference) or temperature oscillation.

[0023] The sample cartridge may further include a removable syringe to facilitate the flow of fluid into and out of the lysis chamber via fluctuations in pressure.

[0024] In some aspects of the sample box, the lysis chamber can further include a valve body and a valve cap, wherein the valve body and the valve cap are connected to define a lysis chamber therebetween, and wherein the filter is retained within the lysis chamber fixed between the valve body and the valve cap. The lysis chamber can have a fluid flow path between an inlet in the cap and an outlet in the valve body, the fluid flow path being fluidly connected to a fluid displacement region of the valve body, wherein the fluid displacement region can be depressurized by movement of the syringe to introduce fluid into the fluid displacement region, and can be pressurized by movement of the syringe to expel fluid from the fluid displacement region.

[0025] The lysis chamber optionally includes a lysis reagent selected from a chaotropic agent, a chelating agent, a buffer, a detergent or a combination thereof to promote chemical lysis. The box body may further include an ultrasonic, piezoelectric, magnetostrictive or electrostatic transducer, for example in the lysis chamber to promote mechanical lysis. The box body in one or more of the multiple chambers may further include a binding agent.

[0026] The disclosed compositions and systems provide improved performance of the sample boxes disclosed herein by helping to reduce and / or eliminate the amount of lysing agents and binding agents conventionally used in box designs. In particular, the sample boxes disclosed herein reduce the amount of chaotropic agents and / or PEG contained in lysing buffer, washing buffer, elution buffer, and binding agents. In addition, the volumes of lysing buffer, washing buffer, elution buffer, and binding agents stored in the box can be reduced.

[0027] In some previous experiments, the maximum fluid volume that can be processed with a conventional box is 300 μL, at which pressure suspension occurs about 50% of the time (at 100 psi or higher), so the volume in the conventional box is reduced and limited to 125 μL to avoid pressure suspension. The current box allows higher sample volumes (e.g., 300 μL, 700 μL, 1,000 μL, up to 5,000 μL) without reaching or exceeding the maximum allowable pressure (100 psi). The properties of the box that allows the processing of higher volumes are related to the reduction of chaotropic agents and binding agents and the filter material. For conventional boxes, the flow rate of these experiments is 10 μL / second. The flow rate is limited by the pressure in the sample box, but the compounds and compositions in the sample box of the present disclosure allow flow rates up to at least 100 μL / second. Therefore, compared with conventional boxes, more total samples can be processed in a shorter time using the box of the present disclosure while keeping a feasible pressure below 100 psi. In summary, compared to conventional boxes, the sample box of the present disclosure can allow a higher flow rate of up to about 100 μL / sec, such as about 10 μL / sec to about 100 μL / sec, together with reagents. The sample box of the present disclosure can allow pressures below 100 psi, below 80 psi, or below 60 psi together with reagents. The sample box of the present disclosure can allow sample volumes up to 5000 μL, such as 300 μL to 5,000 μL, 300 μL to 3,000 μL, 300 μL to 2,000 μL, or 300 μL to 1,000 μL.

[0028] The box can be a single-use box. In some embodiments, the box is an automated box.

[0029] Also provided is a method for separating nucleic acids from a biological sample. The method may include (a) contacting the nucleic acid with a compound bonded to a solid support provided in the compositions and systems disclosed herein, and (b) eluting the nucleic acid from the solid support. In other aspects, the method for separating nucleic acids from a biological sample includes (a) contacting the nucleic acid with a composition comprising a Diels-Alder adduct, the Diels-Alder adduct including a DNA binding ligand disclosed herein, and (b) concentrating the nucleic acid onto a solid support. In some aspects, the method for detecting nucleic acids in a biological sample obtained from an object may include a) contacting nucleic acids from a biological sample with a primer set and an optional probe in a sample box as described herein; b) subjecting nucleic acids, primer pairs, and optional probes to amplification conditions; c) detecting the presence of an amplification product, optionally by real-time PCR, melting curve analysis, or a combination thereof, and d) detecting the presence of nucleic acids in a biological sample based on the detection of an amplification product. Exposure of nucleic acid from the sample to the primer set and optional probe in the sample box may include placing the biological sample in a box, the box including a box body having a plurality of chambers in fluid communication, a reaction vessel having one or more reaction chambers and configured for nucleic acid amplification, a fluid path between the plurality of chambers and the reaction vessel, and a filter in the fluid path, and if the biological sample contains cells, lysing the cells in the biological sample with one or more lysis reagents present in at least one of the plurality of chambers. Subjecting the nucleic acid, primer pair, and optional probe to amplification conditions may include amplifying the nucleic acid with primers and probes in a solution present in at least one of the plurality of chambers. Subjecting the nucleic acid, primer pair, and optional probe to amplification conditions may include amplifying the nucleic acid with primers and probes in a solution present in at least one of the plurality of chambers.

[0030] The method is used to separate and purify nucleic acids from biological samples. The biological sample can be selected from blood, plasma, serum, semen, spinal fluid, tissue, tears, urine, feces, saliva, smear specimens, respiratory samples, nasopharyngeal samples, vaginal swabs, vaginal mucus samples, vaginal tissue samples, vaginal cell samples, bacterial cultures, mammalian cell cultures, viral cultures, human cells, bacteria, extracellular fluid, pancreatic juice, cell lysates, PCR reaction mixtures or in vitro nucleic acid modification reaction mixtures. In some embodiments, the biological sample is blood, plasma, respiratory samples or vaginal swabs. In some examples, the biological sample comprises a nucleic acid selected from genomic DNA, total RNA, short DNA, small DNA, tumor-derived nucleic acid, methylated DNA, microbial nucleic acid, bacterial nucleic acid, viral nucleic acid, cell-free nucleic acid or a combination thereof. In some embodiments, the biological sample comprises cell-free nucleic acid.

[0031] In the methods disclosed herein, before or simultaneously with step a) contacting the nucleic acid with the composition or the compound bound to the solid support, the biological sample may be contacted with a buffer. The buffer may be a lysis buffer and may comprise one or more of a chaotropic agent, a salt, a buffer, a surfactant, a defoaming agent, a binding agent, a precipitant, or a combination thereof. The chaotropic agent may be selected from guanidine thiocyanate, guanidine hydrochloride, an alkali metal perchlorate, an alkali metal iodide, urea, formamide, or a combination thereof. Compared with conventional lysis assays, a chaotropic agent may be used at a lower concentration. For example, a chaotropic agent may be used at a concentration of less than 4.5 M, less than 2 M, or less than 1 M in a lysis buffer. In some embodiments, the methods disclosed herein do not use a chaotropic agent or a lysis buffer.

[0032] When a chaotropic agent or lysis buffer is not used in the methods disclosed herein, the biological sample can be contacted with a buffer comprising saline (inorganic salts, such as CaCl2, MgSO4, KCl, NaHCO3, NaCl, etc.), phosphate buffer, Tris buffer, 2-amino-2-hydroxymethyl-1,3-propanediol, HEPES, PBS, citrate buffer, TES, MOPS, PIPES, cacodylate, SSC, MES, sugar or disaccharide or a combination thereof. For example, the buffer can be a commercially available buffer, such as Hanks balanced salt solution available from Sigma Aldrich or TE buffer available from Fisher BioReagents.

[0033] In some embodiments, the method further includes contacting the nucleic acid with a binding agent, a filtering agent, a washing agent, or a combination thereof while concentrating or before eluting the nucleic acid. A binding agent (such as PEG or a salt) can promote the binding of the nucleic acid to the filter while removing non-target materials. The filter and / or the washing agent can contain a binding agent. Compared with conventional cleavage assays, the binding agent can be used at a lower concentration. For example, the binding agent can be used at a concentration of less than 40% v / v, less than 30% v / v, less than 20% v / v, or less than 10% v / v of the filter and / or the washing agent. In some embodiments, the compositions, systems, and methods disclosed herein do not use a binding agent. In some cases, neither a lysis buffer (or a chaotropic agent) nor a binding agent is used in the methods disclosed herein. In such a case, proteinase K or mechanical treatment (such as sonication) can be used to release nucleic acids from the sample, which are then combined with a modified filter.

[0034] Elution can include, in the presence of an eluent, heating the nucleic acid on the filter to 100°C or less, 95°C or less, 85°C or less, 75°C or less, 65°C or less, 55°C or less; sonicating the nucleic acid; photochemically cleaving the compound / composition; or a combination thereof. In some embodiments, the method for separating and purifying nucleic acids can include eluting the nucleic acid with an eluent. The eluent can have a pH greater than about 9, greater than about 10, greater than about 11, or greater than about 12. The eluent can have a pH greater than about 10. The eluent can have a pH of about 10 to about 13. In some examples provided herein, 50mM KOH (pH 12.7) is used to elute the nucleic acid. The use of a high pH to elute nucleic acids (e.g., DNA) is particularly unique to the cartridges herein and provides improved speed and performance of the methods disclosed herein. Speed ​​is provided by rapid neutralization of acidic ammonium ions by high concentrations of hydroxide ions. Alkylamines have a pKa of about 10 to 11 and are immediately deprotonated at pH 12.7 to form a neutral free base on the solid surface and release cationic DNA. A further advantage of high pH is the denaturation of KOH to captured DNA or RNA. The acidic functional groups in the heterocyclic bases of DNA or RNA are immediately deprotonated and cannot form Watson-Crick bonds. Double-stranded structures and other secondary structures are destroyed, but can be renatured when, for example, neutralized with Tris HCl. This chemical denaturation of captured genomic DNA is advantageous for isothermal determinations of conventional thermal denaturation without PCR. The box provided herein allows the eluted DNA or RNA to be rapidly neutralized in KOH, and then reacts with Tris to produce a final pH of about 8.5 for downstream PCR or other nucleic acid determinations. In some embodiments, the eluent may have a pH of less than about 9, less than about 8.5, or less than about 8. This lower pH elution in conjunction with DNA or RNA can be an advantage, particularly for devices that do not contribute to rapid neutralization of KOH solutions. RNA is known to be hydrolyzed at high pH, ​​so a short exposure to KOH is important for high quality RNA. In some examples, the eluent comprises a polyanion, a polycation, ammonia, or an alkali metal hydroxide. For example, the eluent may comprise a polyanion, such as carrageenan, a carrier nucleic acid, or a combination thereof.

[0035] Also disclosed is a method for detecting nucleic acids in a biological sample. The method may comprise (a) isolating nucleic acids from the biological sample using a method as defined herein; (b) eluting the nucleic acids from the solid support with an eluent; and (c) detecting the nucleic acids. Detecting the nucleic acids may comprise amplifying the nucleic acids by polymerase chain reaction. The polymerase chain reaction may be selected from nested PCR, isothermal PCR, qPCR or RT-PCR.

[0036] In other embodiments, the method for detecting nucleic acids in a biological sample may include placing the biological sample in a cartridge as disclosed herein, optionally lysing the cells with one or more lysis reagents present in at least one of the plurality of chambers, and capturing nucleic acids released therefrom; and amplifying the nucleic acids using primers and probes for detecting the presence of nucleic acids. Nucleic acids in biological samples may be detected within 75 minutes or within 60 minutes of collecting the sample from the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0038] Figure 1A The attachment of a DNA binder to glass is shown. The image on the left shows the use of silane reagents to attach reactive groups such as amines, carboxylic acids, and epoxides to the glass surface. Aliphatic amines can directly bind to DNA. The silane density, thickness, crosslinking, reactive groups, surface charge, or surface density can be varied to facilitate faster extraction, exclude / reduce the amount of binding agents (e.g., PEG), or chaotropes (e.g., guanidine thiocyanate). The type of rea can also be varied. The image on the right shows other DNA binding ligands conjugated to glass via linkers. The type of DNA binder, linker type, loading density, surface charge, or surface density can be varied to facilitate faster extraction, exclude / reduce the amount of binding agents (e.g., PEG), or chaotropes (e.g., guanidine thiocyanate).

[0039] Figure 1B Scanning electron micrographs (SEM) of glass fiber filters (GFF) are shown. GFFs of specified pore size (0.7 μm) and thickness (0.43 mm) are standard for leaching assays in compliance with the EPA. The filter material was supplied as discs of various diameters and was used as received from the manufacturer for the silanization method described herein.

[0040] Figure 2 Shown is a scheme for the synthesis of PFP ester reagents and the DMT assay for measuring surface alkylamine groups. Accessible surface amino groups on the glass surface react with PFP esters containing DMT (dimethoxytrityl). Treatment with acid releases the DMT cation and the absorbance is measured.

[0041] Figure 3 Measurements of surface amine density in aminopropyl (AP) coated glass fiber filters (AP-GFF) are shown. AP-GFF was prepared using Method B as detailed in the Examples. For kinetic experiments, 0.33 mm thick, 1 μm pore size AP-GFF filters were treated with 0.1 M PFP ester.

[0042] Figure 4 APTES-derivatized solid support layer is shown: individual silane molecules can be integrated into the layer through (a) hydrogen bonding, (b) electrostatic attraction, (c) covalent bonding to the substrate, (d) horizontal and (e) vertical polymerization with neighboring silanes, and (f) oligo / polymeric silanes can also react / interact with functionalities present at the interface. The figure shows multilayer coating, cross-linking, and non-covalent bonding occurring at the surface.

[0043] Figure 5 It is shown that the rigid linker in the CL silane increases (e.g., doubles) the amine density relative to the aliphatic amine. The structure of the flexible aliphatic DETA and the rigid CL54 linker structure are also shown in Figure 5 Both linkers can form three ionizable alkylammonium groups to ionically bind polyanionic DNA. The structure of the ionically "trapped" DNA dinucleotide illustrates the relative size of the silanized glass surface.

[0044] Figure 6 A comparison of EDA and APTES coated GFFs (Method A) is shown. These data show the efficiency of DNA extraction using the two amine coating types. 2% and 4% APTES showed slightly lower capture efficiency. In this example, EDA retained DNA better at all concentrations. Amine loading values ​​(nmol / cm 2 ) are as follows: APTES: 2% at 32.5, 4% at 33.3, 6% at 31, 8% at 35. EDA: 0.5% at 48, 1% at 50, 2% at 45, and 4% at 47.

[0045] Figure 7 EDC coupling of BisTris to succ-AP-GFF is shown. Aminopropyl GFF was first succinylated to provide a carboxylic acid coated surface. EDC activation and coupling to Bis-Tris produced an ester coating. At pH 6, the unprotonated amines of Bis-Tris can also react.

[0046] Figure 8 AP-GFF coated with a minor groove binding DNA ligand is shown. Bisbenzimide with attached hexylamine linker (BB-NH2) was prepared and conjugated to AP-GFF using a novel cyanuric chloride (CC) activated surface (CC-GFF). BB-CC-AP-GFF binds DNA efficiently but does not elute at high pH.

[0047] Fig. 9Thermal release (top) and alkaline release (bottom) of DNA are shown. A glass fiber filter is first silanized with a cleavable linker silane (CL53) to produce CL53-GFF (top). The amine coating is released from the GFF by heating at 95°C for a few minutes. GL53-GFF is further coated with a bisbenzimide ligand. DNA capture / thermal release is demonstrated. Alternatively, GFF is first silanized with APTES (bottom). The coating binds DNA at pH <10 and releases DNA at pH>12.

[0048] FIG. 10A to FIG. 10C An overview of a sample cartridge having a valve assembly according to some embodiments of the present invention is shown, which is configured to perform various sample processing including chemical lysis of a target configured for integrated nucleic acid analysis of an immuno-PCR and an optional target assay panel. Fig. 10A A sample cartridge with reaction vessels is shown, Fig. 10B An exploded view of the sample box is shown, and Fig. 10C Components of a valve assembly according to some embodiments are shown.

[0049] Fig.11A Various valve assemblies A, B, C and universal boxes are shown, each of which is suitable for one or more types of target lysis, any of which can be used in the corresponding sample box. According to the embodiment, each box includes a filter material having a surface modified with a DNA binding ligand. Box A only performs mechanical lysis on harder targets and includes a filter membrane modified with a DNA binding ligand. Boxes B and C only perform chemical lysis on viruses, free NA or more fragile targets and include filter columns modified with DNA binding ligands. The universal box includes a modified filter (40), preferably a modified glass fiber filter, and its size is suitable for being fixed between the valve cap and the valve body.

[0050] Fig. 11B A universal valve assembly according to some embodiments is shown, which uses glass beads on a modified glass fiber filter suitable for mechanical lysis of certain types of targets compared to conventional assemblies in conventional sample boxes. In this embodiment, the modified filter 40 is formed of glass fiber and has a pore size of 0.7 μm. In contrast, box A uses a filter formed as a disc of a polymer film (i.e., PCTE), which is suitable for mechanical lysis but not for chemical lysis. By using a filter with a pore size of 0.7 μm, the filter is suitable for receiving glass beads of appropriate size for mechanical lysis. The use of glass fiber to form a filter promotes affinity bonding of free nucleic acids released by chemical lysis. Therefore, the filter is suitable for both mechanical and chemical lysis.

[0051] Fig.12Shown is a comparison of glass fiber filters modified with 2% APTES by chemical vapor deposition (CVD1-4) versus unmodified glass fiber filters (85A and RCC). DETAILED DESCRIPTION

[0052] definition

[0053] To facilitate understanding of the present disclosure, a number of terms and phrases are defined below:

[0054] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes mixtures of compounds, reference to "an amine group" includes mixtures of two or more such amine groups, and the like.

[0055] Herein, ranges may be expressed as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from a particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each range are meaningful with respect to and independently of the other endpoint.

[0056] Throughout this specification and the appended claims, reference will be made to a number of terms which are defined to have the following meanings:

[0057] The term "alkyl" includes "unsubstituted alkyl" and "substituted alkyl", the latter refers to an alkyl group having one or more substituents replacing hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxy, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphoric acid, phosphonic acid, phosphinic acid, amino, amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclic, aralkyl or aromatic or heteroaromatic moieties.

[0058] Unless the number of carbons is otherwise specified, "alkyl" as used herein means an alkyl group as defined above, but having 1 to 20 carbons in its backbone structure, more preferably 1 to 10 carbons. Likewise, "alkenyl" and "alkynyl" have similar chain lengths.

[0059] The alkyl group may also contain one or more heteroatoms within the carbon backbone. Examples include oxygen atoms, nitrogen atoms, sulfur atoms, and combinations thereof. In certain embodiments, the alkyl group contains 1 to 4 heteroatoms.

[0060] As used herein, the term "heteroalkyl" refers to a straight or branched chain, or a cyclic carbon-containing free radical or a combination thereof, comprising at least one heteroatom. Suitable heteroatoms include, but are not limited to O, N, Si, P, Se, B and S, wherein phosphorus atoms and sulfur atoms are optionally oxidized, and nitrogen heteroatoms are optionally quaternized. Heteroalkyl can be substituted as defined above for alkyl groups.

[0061] As used herein, "alkenyl" and "alkynyl" refer to groups containing one or more double or triple bonds of similar length (e.g., C2 to C 30 ) and may substitute the unsaturated aliphatic groups mentioned above for the alkyl groups.

[0062] As used herein, "aryl" refers to 5-, 6- and 7-membered aromatic rings. The ring can be a carbocyclic ring, a heterocyclic ring, a fused carbocyclic ring, a fused heterocyclic ring, a dicarbocyclic ring or a diheterocyclic ring system, optionally substituted as described above for alkyl. Broadly defined, "Ar" as used herein includes 5-, 6- and 7-membered monocyclic aromatic groups, which can include 0 to 4 heteroatoms. Examples include, but are not limited to benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. Those aryl groups having heteroatoms in the ring structure can also be referred to as "heteroaryl", "aryl heterocycle" or "heteroaromatic". The aromatic ring can be substituted at one or more ring positions by substituents as described above, for example, by halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, sulfhydryl, imino, amide, phosphonic acid, phosphinic acid, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, -CF3 and -CN. The term "Ar" also includes polycyclic ring systems having two or more cyclic rings, wherein two or more carbons are common to two adjacent rings (the rings are "fused rings"), wherein at least one of the rings is an aromatic ring, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocycle, or both rings are aromatic.

[0063] As used herein, "alkylaryl" or "aryl-alkyl" refers to an alkyl group substituted with an aryl group (eg, aromatic or heteroaromatic).

[0064] As used herein, "heterocycle" or "heterocyclic" refers to a cyclic radical attached through a ring carbon or ring nitrogen of a monocyclic or bicyclic ring, wherein the monocyclic and bicyclic rings contain 3 to 10 ring atoms, preferably 5 to 6 ring atoms, carbon and 1 to 4 radicals each selected from non-peroxide oxygen, sulfur and N(Y) (wherein Y is absent or Y is H, O, (C 1-4)alkyl, phenyl or benzyl) heteroatoms, and optionally contain one or more double bonds or triple bonds, and are optionally substituted by one or more substituents. The term "heterocycle" also includes substituted and unsubstituted heteroaryl rings. Examples of heterocyclic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, benzopyranyl, cinnolinyl, decahydroquinolinyl, 2H, 6H-1,5,2-dithiazinyl, dihydrofuran [2,3-b] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolyl (indolenyl), dihydroindolinyl, indolizinyl, indolyl, 3H-indolyl, isatinyl, isobenzofuranyl, isobenzodihydrofuranyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methoxydioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidine 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thienyl, and xanthenyl.

[0065] As used herein, "heteroaryl" refers to a monocyclic aromatic ring containing 5 or 6 ring atoms containing carbon and 1, 2, 3 or 4 heteroatoms each selected from non-peroxide oxygen, sulfur and N(Y), wherein Y is absent or is H, O, (C1-C8)alkyl, phenyl or benzyl. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, triazolyl, triazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridinyl (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide), and the like. The term "heteroaryl" may include radicals of orthogonally fused bicyclic heterocycles of about 8 to 10 ring atoms derived from heteroaryl, particularly benzo derivatives or radicals of bicyclic heterocycles derived by fusing a propylene, trimethylene or tetramethylene diradical to a heteroaryl. Examples of heteroaryl include, but are not limited to, furanyl, imidazolyl, triazolyl, triazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridinyl (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide), and the like.

[0066] As used herein, the term "alkoxy" or "alkoxy" refers to an alkyl group as defined above with an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, etc. "Ether" is two hydrocarbons covalently linked by an oxygen atom. Therefore, the substituent of the alkyl that makes the alkyl ether is an alkoxy or similar to an alkoxy, such as can be represented by-O-alkyl,-O-alkenyl and-O-alkynyl. Aryloxy can be represented by-O-aryl or O-heteroaryl, wherein aryl and heteroaryl are defined as follows. Alkoxy and aryloxy can be substituted as described above about alkyl.

[0067] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines, such as moieties that can be represented by the general formula: -NR9R 10 or NR9R 10 R' 10 , where R9, R 10 and R' 10 Each independently represents hydrogen, alkyl, alkenyl, -(CH2) m -R'8, or R9 and R 10 Together with the N atom to which they are attached, they form a heterocyclic ring having 4 to 8 atoms in the ring structure; R'8 represents aryl, cycloalkyl, cycloalkenyl, heterocyclic or polycyclic; and m is 0 or an integer in the range of 1 to 8. In some embodiments, R9 or R 10 Only one of them can be a carbonyl group, such as R9, R10 and nitrogen do not form an imide. In some embodiments, the term "amine" does not include amides, for example, where R9 and R 10 In some embodiments, R9 and R 10 (and optionally R' 10 ) each independently represents hydrogen, alkyl or cycloalkyl, alkenyl or cycloalkenyl or alkynyl. Thus, as used herein, the term "alkylamine" means an amine group as defined above having a substituted (as described above for alkyl) or unsubstituted alkyl group attached thereto, i.e., R9 and R 10 At least one of the groups is an alkyl group.

[0068] The term "amido" or "amide" is art-recognized as an amino-substituted carbonyl group and includes amino groups of the general formula -CONR9R 10 Indicates the part where R9 and R 10 As defined above.

[0069] As used herein, "halogen" refers to fluorine, chlorine, bromine or iodine.

[0070] As used herein, "hydroxy" refers to -OH.

[0071] The term "carbonyl" is art-recognized and includes a group that can be represented by the general formula -CO-XR 11 or -X-CO-R' 11 represents a moiety wherein X is a bond or represents oxygen or sulfur, and R 11 represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl or alkynyl, R' 11 represents hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl or alkynyl. When X is oxygen and R 11 or R' 11 When X is oxygen and R 11 As defined above, this moiety is referred to herein as a carboxyl group, and in particular when R 11 When X is oxygen and R' 11 When X is sulfur and R is hydrogen, the formula represents a "formate". Generally, when the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. 11 or R' 11 When X is sulfur and R 11 When X is sulfur and R' 11 When X is a bond and R is hydrogen, the formula represents a "thioformate". On the other hand, when X is a bond and R is 11 When X is a bond and R 11When is hydrogen, the above formula represents an "aldehyde" group.

[0072] As used herein, the term "substituted" refers to all permissible substituents of the compounds described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Exemplary substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic group containing any number of carbon atoms (preferably, 1 to 14 carbon atoms), and optionally include one or more heteroatoms, such as oxygen, sulfur or nitrogen groups in the form of linear, branched or cyclic structures. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxy, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amide, substituted amide, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C 20 Ring, substituted C3-C 20 Cycles, heterocycles, substituted heterocycles, amino acids, peptides and polypeptide groups.

[0073] It should be understood that "substitution" or "substituted" includes the implicit proviso that such substitution is consistent with the allowed valencies of the substituted atom and the substituent and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation (e.g., by rearrangement, cyclization, elimination, etc.).

[0074] As used herein, the term "hydrolyzable" refers to a group or part that can be hydrolyzed or solvolyzed. For example, a hydrolyzable group can be hydrolyzed (that is, converted into a hydrogen group) by being exposed to water or a protic solvent at ambient temperature or an elevated temperature, or close to ambient temperature or an elevated temperature, and at atmospheric pressure or an elevated pressure or close to atmospheric pressure or an elevated pressure. In some cases, a hydrolyzable group can be hydrolyzed by being exposed to acidic or alkaline water or an acidic or alkaline protic solvent. Typical hydrolyzable groups include but are not limited to alkoxy, aryloxy, aralkoxy, acyloxy or halogen. As used herein, the term is often used to refer to one or more groups bonded to the silicon atom in a silyl group.

[0075] As used herein, the terms "detect," "detecting," or "detection" can describe the general act of finding or distinguishing or the specific observation of a detectably labeled composition.

[0076] The term "nucleic acid" refers to a nucleotide polymer and, unless otherwise limited, includes analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides (e.g., hybridization). The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or viral RNA or by amplification; DNA molecules synthesized or produced by amplification; mRNA; and non-coding RNA. The term nucleic acid includes double-stranded or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In a double-stranded or triple-stranded nucleic acid complex, the nucleic acid chains need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both chains).

[0077] The term nucleic acid also includes any modification thereof, for example by methylation and / or by capping. Nucleic acid modifications can include the addition of chemical groups that bind additional charge, polarity, hydrogen bonding, electrostatic interactions, and functionality to individual nucleic acid bases or to the nucleic acid as a whole. Such modifications can include base modifications, such as 2'-sugar modifications, 5-pyrimidine modifications, 8-purine modifications, modifications at the amine outside the cytosine ring, 5-bromouracil substitutions, sugar-phosphate backbone modifications, abnormal base pairing combinations, such as the isobases isocytidine and isoguanidine, etc. More specifically, in some embodiments, nucleic acids can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids (PNAs)) and polymorpholino polymers (see, e.g., Summerton and Weller (1997) "Morpholino Antisense Oligomers: Design, Preparation, and Properties," Antisense & Nucleic Acid Drug Dev. 7: 1817-195; Okamoto et al. (20020) "Development of electrochemically gene-analyzing method using DNA-modified electrodes," Nucleic Acids Res. Supplement No. 2: 171-172), and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, such as those found in DNA and RNA. The term nucleic acid also includes locked nucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, the disclosures of which are incorporated herein by reference in their entirety. Nucleic acids can be derived from a completely chemical synthesis process (e.g., solid phase-mediated chemical synthesis), from a biological source (e.g., by isolation from any species that produces nucleic acids), or from a process involving the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of these processes.

[0078] As used herein, the terms "oligonucleotide", "polynucleotide" and the like refer to molecules containing nucleic acids, including but not limited to DNA or RNA. The term "oligonucleotide" is used to refer to relatively short nucleic acids, usually shorter than 500 nucleotides, particularly shorter than 200 nucleotides, more particularly shorter than 100 nucleotides, and most particularly shorter than 50 nucleotides. Typically, an oligonucleotide is a single-stranded DNA molecule. The term includes sequences containing any known base analogs of DNA and RNA, including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5 -methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine and 2,6-diaminopurine.

[0079] In some embodiments, the oligonucleotide length is 8 to 200 nucleotides, 8 to 100 nucleotides, 12 to 200 nucleotides, 12 to 100 nucleotides, 12 to 75 nucleotides, or 12 to 50 nucleotides. Oligonucleotides can be represented by their length, for example, an oligonucleotide of 24 residues can be referred to as a "24-mer".

[0080] The term "nucleic acid amplification" includes any means of replicating at least a portion of at least one target nucleic acid, generally in a template-dependent manner, including, but not limited to, a wide range of techniques for linearly or exponentially amplifying nucleic acid sequences. Exemplary means for performing the amplification step include polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiple ligation probe amplification (MLPA), Q-replicase amplification after ligation, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-dependent amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), recombinase polymerase amplification, etc., including multiple formats and combinations thereof, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction-CCR), digital amplification, etc. Descriptions of such techniques can be found in, among other sources, Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, and U.S. Pat. No. 5,686,243.

[0081] As used herein, "sample" or "biological sample" includes samples of tissues, cells or liquids separated from a subject containing various nucleic acids (e.g., DNA and / or RNA), including but not limited to, for example, whole blood, buffy coat, plasma, serum, immune cells (e.g., monocytes or macrophages) and saliva. In some embodiments, the sample comprises a buffer, such as an anticoagulant and / or a preservative. In some embodiments, whole blood is mixed with heparin in a lithium heparin blood collection tube. The sample can be from any body fluid, tissue or cell containing expressed biomarkers. Biological samples can be obtained from a subject by conventional techniques. For example, according to methods well known in the art, blood can be obtained by venipuncture or fingertip capillary blood sampling, and solid tissue samples can be obtained by surgical techniques. In some aspects, the blood sample is placed in a tube designed specifically for the assay.

[0082] "Reagents" broadly refer to any reagent used in a reaction other than an analyte (e.g., a nucleic acid being analyzed). Illustrative reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example, substrates, coenzyme factors, buffers, metal ions, inhibitors, and activators.

[0083] As used herein, the term "detection" refers to "determining the presence of" an item, such as a nucleic acid sequence, such as the sequence indicating the presence of a coronavirus. Detection can include determining the presence of a coronavirus without determining the identity of the coronavirus; determining the presence of one or more coronaviruses belonging to a class of coronaviruses; determining the presence of a specific, known coronavirus strain; or determining the presence of a new (previously undescribed) coronavirus strain.

[0084] As used herein, "Clinical Laboratory Improvement Amendments (CLIA)" refers to the Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations that were effective on the original filing date of this application. CLIA regulations include federal standards for all U.S. facilities or sites that test human samples for health assessment or diagnosis, prevention or treatment of disease. A "CLIA-compliant" test is a test that complies with these rules. A "CLIA-exempt" test includes a test that does not comply with all of these regulations. For example, CLIA-exempt tests include test systems approved by the U.S. Food and Drug Administration for home use and those tests approved for exemption under CLIA standards.

[0085] (a) Solid support

[0086] Provided herein is a solid support for separating and purifying nucleic acids from samples containing nucleic acids, the solid support comprising a DNA binding ligand. In some aspects, the DNA binding ligand is chemically bonded to the surface of the solid support. For example, the DNA binding ligand can be covalently bonded to the solid support (e.g., by a siloxane bridge). In other examples, the DNA binding ligand is chemically bonded to the solid support by a connexon (e.g., by a low polyethylene connexon or a PEG oligomer). In other aspects of the compositions and methods disclosed herein, the DNA binding ligand is not chemically bonded to the solid support.

[0087] As used herein, the term "solid support" refers to any substrate selected from paramagnetic particles, gels, fibers, controlled pore glass, magnetic beads, microspheres, nanospheres, capillaries, filter membranes, columns, cloths, wipes, paper, planar supports, porous plates, porous membranes, porous monoliths, wafers, combs, or any combination thereof. The solid support may include any suitable material, including but not limited to glass, silica, titanium oxide, aluminum oxide, iron oxide, ethylene backbone polymers, polypropylene, polyethylene, polystyrene, ceramics, cellulose, nitrocellulose, magnetic silica particles (e.g., MagneSil available from Promega Corporation) or any combination thereof. TMIn some embodiments, the solid support includes a material selected from polystyrene, glass, ceramic, polypropylene, polyethylene, silicon dioxide, mica, titanium dioxide, polycarbonate, latex, PMMA, zeolite, polyethersulfone, carboxymethyl cellulose, cellulose and a combination thereof. Examples of solid supports include magnetic beads, glass beads, polystyrene beads, cellulose filters, polystyrene filters, polycarbonate filters, polyethersulfone filters, polytetrafluoroethylene filters, polyvinyl pyrrolidone filters or glass fiber filters. The solid support may further include a polymer binder for binding particles or fibers to the solid support. Exemplary polymer binders include acrylate polymers. In the disclosure provided herein, the solid support typically includes surface functional groups that can interact and / or react with DNA binding ligands (e.g., compounds with silane groups).

[0088] In some embodiments, the solid support is a fiber material, preferably a glass fiber filter (GFF). Fiber filters (such as glass fiber filters) provide several advantages that are superior to other porous supports (such as glass beads). Porous glass fiber filters have a surface area that is much larger than a flat glass surface, but smaller than porous glass beads. Different from glass beads, the thin, paper-like sheet of glass fiber filters is easy to process in aqueous or organic solvents. The mechanical stability of glass fiber filters allows ribbons and sheets to be used for high-throughput manufacturing. Different from beads, glass fiber filters simplify the fluid flow during filtering, and do not need to include sintered bodies. This feature of GFF allows the construction of multilayer devices, in which several modified glass fiber filters can be stacked on each other in the inside of a cylindrical flow-through housing. If more DNA binding capacity is needed, the effective thickness of the filter can be adjusted by stacking multiple discs.

[0089] The fibrous material can be characterized by fiber diameter, pore size, basis weight, thickness, and / or specific surface area. The fibers in the fibrous material can have an average diameter of 1 micron or more, 1.5 microns or more, 2 microns or more, 2.5 microns or more, 3 microns or more, 3.5 microns or more, 4 microns or more, 4.5 microns or more, 5 microns or more, 5.5 microns or more, 6 microns or more, 6.5 microns or more, 7 microns or more, 7.5 microns or more, 8 microns or more, 9 microns or more, 10 microns or more, 12 microns or more, 15 microns or more, 16 microns or more, 18 microns or more, 19 microns or more, or 20 microns or more. In certain embodiments, the fibers in the fibrous material may have an average diameter of 25 microns or less, 24 microns or less, 22 microns or less, 20 microns or less, 19 microns or less, 18 microns or less, 16 microns or less, 15 microns or less, 14 microns or less, 13 microns or less, 12 microns or less, 10 microns or less, 9 microns or less, 8 microns or less, 7.5 microns or less, 7 microns or less, 6 microns or less, 5.5 microns or less, or 5 microns or less. In certain embodiments, the fibers in the fibrous material may have an average diameter of 1 micron to 20 microns, 2 microns to 20 microns, 2 microns to 18 microns, 2.5 microns to 15 microns, 2.5 microns to 12 microns, 2.5 microns to 10 microns, 3 microns to 20 microns, 3 microns to 18 microns, 3 microns to 15 microns, 3 microns to 12 microns, 4 microns to 20 microns, 4 microns to 15 microns, or 5 microns to 20 microns.

[0090] The fiber material can have an effective pore size (or average pore size) of 0.20 micrometers or greater, 0.30 micrometers or greater, 0.40 micrometers or greater, 0.50 micrometers or greater, 0.55 micrometers or greater, 0.60 micrometers or greater, 0.65 micrometers or greater, 0.70 micrometers or greater, 0.75 micrometers or greater, 0.80 micrometers or greater, 0.85 micrometers or greater, 0.90 micrometers or greater, 0.95 micrometers or greater, 1.0 micrometers or greater, or 1.1 micrometers or greater. In certain embodiments, the fiber material can have an average pore size of 3.0 microns or less, 2.0 microns or less, 1.9 microns or less, 1.8 microns or less, 1.7 microns or less, 1.6 microns or less, 1.5 microns or less, 1.4 microns or less, 1.3 microns or less, 1.2 microns or less, 1.1 microns or less, 1.05 microns or less, 1.0 microns or less, 0.95 microns or less, 0.90 microns or less, 0.85 microns or less, 0.80 microns or less, 0.75 microns or less, or 0.70 microns or less. In certain embodiments, the fiber material may have an average pore size of 0.2 μm to 3 μm, 0.20 micron to 2.0 micron, 0.20 micron to 1.5 micron, 0.40 micron to 1.5 micron, 0.40 micron to 1.3 micron, 0.40 micron to 1.2 micron, 0.50 micron to 1.5 micron, 0.50 micron to 1.3 micron, 0.50 micron to 1.2 micron, 0.60 micron to 1.5 micron, 0.60 micron to 1.3 micron, 0.60 micron to 1.2 micron, 0.70 micron to 1.5 micron, 0.70 micron to 1.3 micron, 0.70 micron to 1.2 micron, or 0.70 micron to 1.0 micron.

[0091] The fiber material, such as a glass fiber filter, can have a size selected to accommodate beads of corresponding size to facilitate mechanical lysis. The beads can include glass beads, silica beads, or a combination thereof.

[0092] The thickness of the fiber material can be 100 microns or more, 150 microns or more, 200 microns or more, 250 microns or more, 300 microns or more, 350 microns or more, 400 microns or more, 450 microns or more, 500 microns or more, 550 microns or more, 600 microns or more, 650 microns or more, 700 microns or more, 750 microns or more, 800 microns or more, 900 microns or more, 1,000 microns or more, 1,200 microns or more, 1,500 microns or more, 1,600 microns or more, 1,800 microns or more, 1,900 microns or more, or 2,000 microns or more. In certain embodiments, the fiber material can have a thickness of 2,500 microns or less, 2,400 microns or less, 2,200 microns or less, 2,000 microns or less, 1,900 microns or less, 1,800 microns or less, 1,600 microns or less, 1,500 microns or less, 1,400 microns or less, 1,300 microns or less, 1,200 microns or less, 1,000 microns or less, 900 microns or less, 800 microns or less, 750 microns or less, 700 microns or less, 600 microns or less, 550 microns or less, or 500 microns or less. In certain embodiments, the fiber material can have a thickness of 100 to 2,000 microns, 200 to 1,500 microns, 200 to 1,200 microns, 250 to 1,200 microns, 250 to 1,000 microns, 250 to 800 microns, 300 to 2,000 microns, 300 to 1,800 microns, 300 to 1,500 microns, 300 to 1,200 microns, 400 to 2,000 microns, 400 to 1,500 microns, or 500 to 2,000 microns.

[0093] The basis weight of the fiber material can be 10 g / m 2 or larger, 15g / m 2 or larger, 20g / m 2 or larger, 25g / m 2 or larger, 30g / m 2 or larger, 35g / m 2 or larger, 40g / m 2 or larger, 45g / m 2 or larger, 50g / m 2 or larger, 55g / m 2 or larger, 60g / m 2 or larger, 65g / m 2 or larger, 70g / m 2 or larger, 75g / m 2or larger, 80g / m 2 or larger, 90g / m 2 or larger, 100g / m 2 or larger, 120g / m 2 or larger, 150g / m 2 or larger, 160g / m 2 or larger, 180g / m 2 or larger, 190g / m 2 or larger, or 200g / m 2 In certain embodiments, the fiber material may have a basis weight of: 250 g / m 2 or smaller, 240g / m 2 or smaller, 220g / m 2 or smaller, 200g / m 2 or smaller, 190g / m 2 or smaller, 180g / m 2 or smaller, 160g / m 2 or smaller, 150g / m 2 or smaller, 140g / m 2 or smaller, 130g / m 2 or smaller, 120g / m 2 or less, 100g / m 2 or smaller, 90g / m 2 or smaller, 80g / m 2 or smaller, 75g / m 2 or smaller, 70g / m 2 or smaller, 60g / m 2 or smaller, 55g / m 2 or less, or 50g / m 2 In certain embodiments, the fiber material may have a basis weight of: 10 g / m 2 Up to 200g / m 2 , 20g / m 2 Up to 150g / m 2 , 20g / m 2 Up to 120g / m 2 , 25g / m 2 Up to 120g / m 2 , 25g / m 2 Up to 100g / m 2 , 25g / m 2 Up to 90g / m 2 , 30g / m 2 Up to 200g / m 2 , 30g / m 2 Up to 180g / m 2, 30g / m 2 Up to 150g / m 2 , 30g / m 2 Up to 100g / m 2 , 40g / m 2 Up to 150g / m 2 , 40g / m 2 Up to 100g / m 2 , or 50g / m 2 Up to 90g / m 2 .

[0094] In some examples, unmodified solid supports (which may be modified to contain DNA binding ligands) are available from Pall Corporation with varying pore sizes and thicknesses: for example, Type A / E, Type A / B, and Type A / C glass fiber filters are available from Pall Corporation with thicknesses of 0.33 mm, 0.66 mm, and 0.25 mm, respectively, and all with a nominal pore size of 1 μm. Other filter types are described below. Pall TCLP (Toxicity Characteristic Leaching Program) products have similar pore sizes to Figure 3 The Whatman (Cytiva) filters were of the same dimensions as described in (0.7 μm pore size, 0.4 mm thickness).

[0095] Table A. Description of Exemplary Glass Fiber Filters Available from Pall Corporation

[0096]

[0097] In a specified embodiment, the solid support is a glass fiber filter having a thickness of 400 to 2000 microns and a pore size of 0.5 to 1 micron.

[0098] In other embodiments, the solid support is a particulate material, such as silica gel or silicon dioxide, having a pore size of about 30 angstroms to about 1000 angstroms, a particle size of about 2 microns to about 300 microns, and a particle size of about 35 microns. 2 / g to about 1000m 2 / g specific surface area. In some embodiments, the particulate material can have a pore size of about 40 angstroms to about 500 angstroms, about 60 angstroms to about 500 angstroms, about 100 angstroms to about 300 angstroms, and about 150 angstroms to about 500 angstroms. In some embodiments, the particulate material can have a particle size of about 2 microns to about 25 microns, about 5 microns to about 25 microns, about 15 microns, about 63 microns to about 200 microns, and about 75 microns to about 200 microns; and about 100 microns. 2 / g to about 350m 2 / g, about 100m 2 / g to about 500m 2 / g, about 65m 2 / g to about 550m 2 / g, about 100m 2 / g to about 675m 2 / g, and about 35 to about 750m 2 / g specific surface area.

[0099] As described herein, solid supports include DNA binding ligands, such as amino compounds, and can be used as separation materials for nucleic acid separation. In particular, the DNA binding ligands on the solid support surface provide high nucleic acid binding capacity for separating nucleic acids from samples. Therefore, separation materials for nucleic acid separation are disclosed herein, including solid supports (e.g., glass fiber solid supports) containing DNA binding ligands. In some embodiments, the separation materials for nucleic acid separation are selected from columns, capillaries or boxes comprising modified solid supports disclosed herein. In some embodiments, separation materials can be used for, for example, separation, separation and purification of nucleic acids from biological samples or chemical reaction mixtures.

[0100] The separation material including the solid support may have a concentration of 10 nmol compound / cm 2 or greater (e.g., 15 nmol / cm 2 or greater, 20nmol / cm 2 or greater, 25nmol / cm 2 or greater, 35nmol / cm 2 or greater, 40nmol / cm 2 or greater, 45nmol / cm 2 or greater, 50nmol / cm 2 or greater, 55nmol / cm 2 or greater, 60nmol / cm 2 or greater, 65nmol / cm 2 or greater, 70nmol / cm 2 or greater, 75nmol / cm 2 or greater, 80nmol / cm 2 or greater, 85nmol / cm 2 or greater, 90nmol / cm 2 or greater, 95nmol / cm 2 or greater, 100nmol / cm 2 or greater, 10nmol / cm 2 Up to 100 nmol / cm 2 , 15nmol / cm 2Up to 80 nmol / cm 2 、30nmol / cm 2 Up to 100nmol / cm 2 or 30 nmol / cm 2 Up to 90 nmol / cm 2 ). The high density of DNA binding ligands on the surface of the solid support provides a high nucleic acid binding capacity for isolating nucleic acids from a sample. Also disclosed herein is an assay for measuring the density of surface amine or amide groups on a solid support (eg, GFF).

[0101] The separation material including the solid support may have a pH of at least 10 μg / cm 2 DNA binding capacity (e.g., 15 μg / cm 2 or greater, 20 μg / cm 2 or greater, 25 μg / cm 2 or greater, 35 μg / cm 2 or greater, 40 μg / cm 2 or greater, 45 μg / cm 2 or greater, 50 μg / cm 2 or greater, 55 μg / cm 2 or greater, 60 μg / cm 2 or greater, 65 μg / cm 2 or greater, 70 μg / cm 2 or greater, 75 μg / cm 2 or greater, 80 μg / cm 2 or greater, 85 μg / cm 2 or greater, 90 μg / cm 2 or greater, 95 μg / cm 2 or greater, 100 μg / cm 2 or greater, 30 μg / cm 2 Up to 100 μg / cm 2 or 30 μg / cm 2 Up to 90 μg / cm 2 Of course, separation materials comprising solid supports with higher DNA binding capacity are preferred, such materials having a DNA binding capacity of at least 30 μg / cm 2 DNA binding capacity.

[0102] Compound

[0103] As described herein, solid supports include DNA binding partners. The term "DNA binding partners" is widely used in this article. However, other types of nucleic acids except DNA are also relevant. Therefore, usually the above terms can be replaced by the terms "nucleic acid binding partners" or "nucleic acid binding molecules". Nucleic acids are typically RNA or DNA, double-stranded or single-stranded, or have secondary (single-stranded DNA or RNA can form internal double-stranded regions, i.e., secondary structures) or tertiary structures.

[0104] DNA binding ligands include any molecule capable of binding or associating with DNA. Such binding or association can be via covalent bonding, via ionic bonding, via hydrogen bonding, via van der Waals bonding, or via any other type of reversible or irreversible association. As used herein, the term "ligand" refers to any atom, ion, molecule, macromolecule (e.g., polypeptide) or combination of such entities. The term "ligand" is used interchangeably with the term "molecule".

[0105] The DNA binding ligand can be selected from amine-containing compounds, such as alkylamines, cycloalkylamines, alkoxyamines, polyamines or arylamines), intercalators (e.g., furanocoumarins, coumarins, anthracyclines, phenanthridines, psoralen derivatives, acridines, ellipticines, actinomycins, anthraquinones and Tris compounds), groove binders (e.g., pyrrolo(1,4)benzodiazepines (PBDs), cytomegalovirus, chrysomycin, fusobacterium, distomycin, calicheamicin, CC-1065 and Hoechst 33258), polypeptides, amino acids (histidine), proteins (e.g., zinc finger proteins, homeodomains, leucine zipper proteins, helix-loop-helix proteins or β-sheet motifs) or combinations thereof.

[0106] The solid supports described herein may comprise a compound derived from a structure represented by the following formula:

[0107] Y-(L) y -SiX3

[0108] in,

[0109] Y is a DNA binding ligand selected from an alkylamine, a cycloalkylamine, an alkoxyamine, a polyamine moiety, an intercalator, a minor groove binder, a peptide, an amino acid, an arylamine, or a combination thereof,

[0110] L is a linker selected from an alkyl group, a heteroalkyl group, an olefin group, a heteroolefin group, a polyacrylic acid, a Diels-Alder adduct, or a combination thereof,

[0111] Each X is independently selected at each occurrence from a hydrolyzable group, an alkyl group, a heteroalkyl group, an alkenyl group, or two or three Xs are combined to form one or more cyclic groups, or one X is combined with Y to form a cyclic azasilane, and

[0112] y is either 0 or 1.

[0113] In some aspects of the compounds disclosed herein, the DNA binding ligand or Y may comprise a plurality of amine groups (or polyamines), a plurality of amide groups (or polyamides), or a combination thereof (polyamine-amides). For example, the DNA binding ligand or Y may comprise at least two, at least three, at least four, at least five, at least six amine or amide groups, or a combination thereof. In other examples, the DNA binding ligand or Y comprises a single amine or amide group. The amine or amine group may be a primary amine, a secondary amine, or a tertiary amine. In some embodiments, the DNA binding ligand or Y comprises a quaternary ammonium group.

[0114] In some aspects of the compounds, the DNA binding ligand or Y comprises C1-C 16 Alkylamines (e.g., C1-C 12 Alkylamine, C1-C 10 Alkylamine, C1-C8 alkylamine, C2-C8 alkylamine or C2-C6 alkylamine), C3-C 12 Cycloalkylamines (e.g., C3-C 10 Cycloalkylamine, C3-C8 cycloalkylamine, C3-C6 cycloalkylamine, C4-C8 cycloalkylamine or C4-C6 cycloalkylamine), C1-C 16 Alkoxyamines (e.g., C1-C 12 Alkoxyamine, C1-C 10 Alkoxyamine, C1-C8 alkoxyamine, C2-C8 alkoxyamine or C2-C6 alkoxyamine), C6-C 12 Arylamines (e.g., C6-C 10 Arylamine, C6-C8 arylamine), C6-C 12 Imidazole group, C3-C 14 Heterocycloalkylamines (e.g., C3-C 10 heterocycloalkylamine, C3-C8 heterocycloalkylamine, C3-C6 heterocycloalkylamine, C4-C8 heterocycloalkylamine or C4-C6 heterocycloalkylamine), and C2-C 20 Heteroalkylamines (e.g., C1-C 12 Heteroalkylamine, C1-C 10 In some embodiments, the DNA binding ligand or Y comprises an alkylamine group, an imidazole group or a combination thereof.

[0115] In some examples of compounds, the DNA binding ligand or Y is selected from spermidine, spermine, methylamine, ethylamine, propylamine, cadaverine, putrescine, ethylenediamine, diethylenetriamine, 1,3-dimethyldipropylenediamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium hydrochloride, tris(2-aminoethyl)amine, or a combination thereof. The DNA binding ligand or Y is optionally substituted with one or more groups, such as C1-C6 alkyl, heteroalkyl or amino groups.

[0116] In some aspects of the compounds of the present disclosure, there is a linker L, i.e., y is 1. The linker can be selected from an alkyleneoxy (e.g., C2-C4 alkyleneoxy) group, an alkylene (e.g., C2-C4 alkylene or C2-C3 alkylene) group, or a heteroalkylene (e.g., C4-C6 heteroalkylene). In some examples, L is a bond. In other examples, L can be derived from a Diels-Alder adduct. The Diels-Alder adduct can be derived from an unsaturated cycloimino group.

[0117] In some aspects of the compounds disclosed herein, each X can be independently selected from halogen (e.g., Cl, Br, I), C1-C6 alkoxy, dialkylamino, triflate or C1-C6 straight chain, branched or cycloalkyl. Preferably, at least two X include hydrolyzable groups independently selected from halogen, alkoxy, dialkylamino, triflate, or they are combined with the Si atom to which they are connected to form a tricyclic azosilane, cyclic siloxane, polysilsesquioxane or silazane. In some examples, two X can be independently selected from halogen (e.g., Cl, Br, I), C1-C6 alkoxy (e.g., ethoxy, methoxy, acetoxy), dialkylamino or triflate, and one X is selected from C1-C6 straight chain, branched or cycloalkyl.

[0118] Also disclosed herein are compositions comprising Diels-Alder adducts, wherein the Diels-Alder adducts include a DNA binding ligand. As defined herein, the DNA binding ligand may comprise an amine group, an intercalator, a minor groove binder, a peptide, an amino acid, a protein, or a combination thereof. In some examples, the DNA binding ligand is selected from an alkylamine, a cycloalkylamine, an alkoxyamine, an arylamine, a polyamine moiety, or a combination thereof. The Diels-Alder adduct may be represented by the following general formula, its isomers, salts, tautomers, or combinations thereof:

[0119]

[0120] and

[0121] wherein Y' is a DNA binding ligand as defined herein, and L, X and y are also as defined herein. The Diels-Alder adduct may be covalently or non-covalently bound to the solid support.

[0122] Specific examples of compounds and compositions comprising DNA binding ligands described herein may include aminosilylated compounds selected from the group consisting of:

[0123]

[0124]

[0125] 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, aminoalkylsilazotricyclo, 3-(2-aminoethyl)aminopropyltriethoxysilane, 3-(2-aminoethyl)aminopropyltrimethoxysilane, or a combination thereof, and wherein n is an integer from 0 to 10, from 1 to 10, or from 1 to 5.

[0126] In some cases, the compound may comprise a functional group, such as a silanized group or a moiety other than a silanized group, which may facilitate binding to a solid support. For example, the compound can include an ether, a silyl ether, a siloxane, a carboxylate, a sulfonate, an aminosulfate, a sulfate, a phosphonate, a phosphinate, a phosphate, a carboxylic acid silyl ester, a silyl sulfonate, a silyl sulfinate, a silyl sulfate, a silyl phosphonate, a silyl phosphinate, a silyl phosphate, an oxide, a sulfide, a carbocycle, a heterocycle having at least one oxygen atom, a heterocycle having at least one nitrogen atom, a heterocycle having at least one sulfur atom, a heterocycle having at least one silicon atom, a carbodiimide (e.g., DCC and EDCI), a phosphine or an imine (e.g., BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH), a heterocycle derived from a "click" reaction, a carbocycle derived from a Diels-Alder reaction, a heterocycle derived from a Diels-Alder reaction, an amide, an imide, a sulfide, a thiolate, a metal thiolate, a urethane, an oxime, a hydrazide, a hydrazone, a physically or chemically adsorbed or otherwise non-covalently attached moiety, or a combination thereof. In certain embodiments, compound comprises the following functional group selected from: maleimide, acrylate, acrylamide, epoxide, aziridine, thioethane, aldehyde, ketone, azide, alkyne, disulfide, anhydride, carboxylic acid phosphate, phosphonate, sulfate, sulfonate, nitrate, amidine, silane, siloxane, cyanate, acetylene, cyanide, halogen, acetal, ketal, amino, carbonyl, carboxyl, biotin, cyclodextrin, adamantane or the vinyl group that can be connected to solid support.In some embodiments, solid support is the glass comprising silanol and siloxane groups.Such silanol groups on glass surface can react with the compound comprising silane and siloxane to provide the surface with the compound chemically bonded via siloxane bridge.The method that the compound comprising amino group is covalently connected to functionalized surface and solid surface is known in the art.

[0127] box

[0128] In some embodiments, the solid support is incorporated into an automated cassette, e.g. In one aspect, the invention relates to sample cartridges utilizing a valve body platform that allows for detection of both enveloped and free nucleic acid targets. In some embodiments, the valve body includes a sample processing region or lysis chamber that provides mechanical and / or chemical lysis. This allows a single cartridge to provide lysis for many different types of targets and can therefore be considered an "assay panel cartridge."

[0129] The sample box can be any device configured to perform one or more processing steps related to the preparation and / or analysis of a biological fluid sample according to any method described herein. In some embodiments, the sample box is configured to perform at least sample preparation. The sample box is further configured to perform additional processes by using a reaction vessel connected to the box, such as detecting target nucleic acids in a nucleic acid amplification test (NAAT), such as a polymerase chain reaction (PCR) assay. In some embodiments, the reaction vessel extends from the body of the sample box. The preparation of fluid samples generally involves a series of processing steps, which may include chemical, electrical, mechanical, thermal, optical or acoustic processing steps according to a specified protocol. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, the combination of analytes and the combination of unwanted materials.

[0130] Cartridges suitable for use with the present invention include one or more transfer ports through which a prepared fluid sample can be transported to a connected reaction vessel for analysis. Fig. 10AAn exemplary assay panel box 100 suitable for sample preparation and analytical testing by PCR when received in an instrument module according to some embodiments is shown. The sample box is connected to a reaction vessel 116 (also referred to as a "reaction tube" or "PCR tube"), which is suitable for analyzing a fluid sample processed in the sample box 100. In some embodiments, the reaction vessel extends from the box body. Such a sample box 100 includes various components, including a main housing 102 with one or more chambers 108 for processing fluid samples, and the processing generally includes sample preparation before analysis. The instrument module facilitates the processing steps required for sample preparation, and the prepared sample is transported to the fluid conduit of the reaction vessel 116 of the housing connected to the sample box 100 by one of a pair of transfer ports. The prepared biological fluid sample is then transported to the reaction chamber of the reaction vessel, in which the biological fluid sample performs nucleic acid amplification. In some embodiments, amplification is polymerase chain reaction. In some embodiments, simultaneously with the amplification of the biofluid sample, the excitation tool and optical detection tool of the module are used to detect optical emissions indicating the presence or absence of a target nucleic acid analyte of interest (e.g., bacteria, viruses, pathogens, toxins, tumors, or other target analytes). It should be understood that such a reaction vessel may include various chambers, conduits, or microwell arrays for detecting target analytes. The box is provided with tools for preparing biofluid samples before being transported to the reaction vessel. Any chemical reagents required for cell lysis, or tools for binding or detecting analytes of interest (e.g., reagent beads) may be contained in one or more chambers of the box and may therefore be used for sample preparation. U.S. Provisional Application No. 63 / 217,672, filed on July 1, 2021, further describes the assay box of valve assembly D in detail and is incorporated herein by reference in its entirety.

[0131] Exemplary uses of reaction vessels for analyzing biological fluid samples are described in commonly assigned U.S. Patent No. 6,818,185, filed May 30, 2000, entitled "Cartridge for Conducting a Chemical Reaction," which is incorporated herein by reference in its entirety for all purposes. Examples of sample cartridges and associated modules are shown and described in: U.S. Patent No. 6,374,684, entitled “Fluid Control and Processing System,” filed on August 25, 2000, U.S. Patent No. 8,048,386, entitled “Fluid Processing and Control,” filed on February 25, 2002, U.S. Patent Application No. 63 / 218,672, entitled “Universal Assay Cartridge and Methods of Use,” filed on July 1, 2021; U.S. Provisional Application No. 63 / 319,993, entitled “Unitary Cartridge Body and Associated Components and Methods of Manufacture,” filed on March 15, 2022; and U.S. Patent No. 10,562,030, entitled “Molecular Diagnostic Assay System,” filed on July 22, 2016; the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0132] like Fig. 10A As shown, the assay panel 100 includes a body 102 containing a plurality of chambers 108 for reagents or buffers and sample handling. The chambers are arranged around a central syringe barrel 106, which is in fluid communication with a valve body 110 (see Figure 1B and Fig. 10C) and sealed with gasket 104. Valve body 110 can include cap 112, and the whole box body can be supported on box base 101. Valve body generally includes one or more channels or chambers (chamber 114), which can include filters or separation materials that can play the role of binding and eluting nucleic acids as described herein. In some embodiments, the box further includes one or more temperature-controlled channels or can be used as a chamber of a thermal cycle chamber in certain embodiments. Unshown "piston" can be operated to suck fluid into syringe barrel 106, and the rotation of valve body 110 provides various reagent chambers 108 and channels, reaction chambers, mixing chambers and any optional temperature control area. Therefore, each reagent chamber 108, reaction chamber, matrix material and temperature-controlled chamber or channel are selectively fluidly connected by the rotation of the piston, and the "injection" action of the piston in the valve assembly operates the reagent movement (for example, chamber loading or unloading). Fig.11A and Fig. 11B Shows the available Fig. 10A Different valve assemblies in sample boxes of the present invention. Valve assembly A performs only mechanical lysis and is suitable for lysis of hard targets (e.g., some bacteria, spores). Valve assemblies B and C perform only chemical lysis and are suitable for lysis of less hard targets (e.g., viruses, free NA, some spores, some bacteria and yeast). Universal valve assemblies can perform mechanical and chemical lysis on all types of targets. In all such boxes, the valve assembly includes a syringe tube, a valve body (VB) and a valve cap. The ability of the valve assembly of the sample box depends on the filter or separation material described herein, and the specific workflow sequence performed by the instrument interface of the module. For example, valve assembly A has a valve body shaped as a circular cavity to support a filter disc to filter a sample, and the cap has a sonication dome feature, which interfaces with the sonication angle of the box receiving module to sonicate the target. In contrast, valve assemblies B and C have a valve body having an elliptical filter recess in which a filter (e.g., a glass fiber filter column) is received, and the filter is configured to optionally bind to the nucleic acid released from the target by chemical lysis. The universal valve assembly has a design more similar to valve assembly A, with a cap with a sonication dome, and a valve body with a circular cavity for supporting a disc filter, however this design uses a glass fiber filter as described herein. Forming the filter with a DNA binding ligand and a filter material (e.g., modified glass fiber) facilitates affinity binding to free nucleic acids released from a biological sample, optionally by chemical cleavage.

[0133] Although the methods described herein are primarily based on Cepheid Inc. cassette (Sunnyvale, Calif.) is described (see, e.g., Fig. 10A), it will be appreciated that, in view of the teachings provided herein, the methods may be implemented on other cartridges / microfluidic systems, including alternative cartridge designs having a valve assembly involving multiple interface components, and a cartridge body defined by multiple interface components to form multiple chambers of the cartridge, such as those described in Korean Application No. 102293717B1, cartridges for lysing cells in a biological sample using ultrasound, such as those described in International Application No. WO2021 / 245390A1, cartridges and systems for droplet operations using electrowetting grids, and electrical sensor arrays configured to detect analytes of interest, such as those described in International Application No. WO2016 / 077341A2 Those described in the International Application No. WO2012 / 145730A2, a cartridge that facilitates the movement of nucleic acids from one chamber to the next by opening a vent bag, for example, a multiplex assay system comprising multiple thermal cycling units so that a single chamber can be heated, cooled and / or compressed to mix the fluid within the chamber or to push the fluid within the chamber into another chamber, for example, those described in the International Application No. WO2015 / 138343A1, and a system for rapid amplification of nucleic acids facilitated by alignment of flexible portions of a sample cartridge to complete temperature cycling for nucleic acid amplification, for example, those described in the International Application No. WO2017 / 147085A1. Such cartridge / microfluidic systems may include, for example, microfluidic systems implemented using soft lithography, micro / nano-fabricated microfluidic systems implemented using hard lithography, and the like. In addition, it should be understood that the assay panel methods described herein (i.e., identifying multiple conditions based on comparative levels of multiple targets obtained from a single sample) can be further implemented in completely different systems, including: isothermal nucleic acid amplification systems, digital RT-PCR, electrochemical PCR, lateral flow test kits, electrochemical sensors, nucleic acid sequencing, CRISPR / Cas-based technologies, chemiluminescence, and nanoparticle-based colorimetric detection.

[0134] In some embodiments, the sample box may include: a) a box body having a plurality of chambers defined therein, wherein the plurality of chambers are fluidically connected via a fluid path of the box, and wherein at least one chamber is configured to receive a biological sample, b) a reaction vessel configured for amplifying nucleic acids by thermal cycling, and c) a filter disposed in the fluid path between the plurality of chambers and the reaction vessel, wherein the filter includes a separation material as disclosed herein, wherein the plurality of chambers and the reaction vessel independently include reagents for releasing nucleic acids from the biological sample, and primers and probes for detecting nucleic acids.

[0135] In some embodiments, the sample box may include a) a box body having a plurality of chambers therein, wherein the plurality of chambers include: a sample chamber having at least a fluid outlet in fluid communication with another of the plurality of chambers; and a lysis chamber in fluid communication with the same sample chamber, the lysis chamber including a reagent for releasing nucleic acid, optionally wherein the sample chamber and the lysis chamber are the same; b) a reaction vessel, which is fluidically connected to the plurality of chambers of the box body and is configured for amplifying nucleic acid; c) a filter disposed in a fluid path between the lysis chamber and the reaction vessel, wherein the filter includes a solid support modified with a DNA binding ligand, the DNA binding ligand being selected from alkylamines, cycloalkylamines, alkoxyamines, polyamine moieties, intercalators (e.g., tris compounds), minor groove binders, peptides, amino acids (histidine), arylamines, or combinations thereof, and d) a plurality of primers and / or probes disposed in one or more of the plurality of chambers or reaction vessels for detecting nucleic acid. The compound used to modify the filter may be as described herein.

[0136] The lysis chamber optionally includes a lysis reagent selected from a chaotropic agent, a chelating agent, a buffer and a detergent. The lysis chamber may further include a valve body and a valve cap, wherein the valve body is connected to the valve cap to define the lysis chamber therebetween, and wherein the filter is retained in the lysis chamber fixed between the valve body and the valve cap.

[0137] The lysis chamber has a fluid flow path between an inlet in the cap and an outlet in the valve body, the fluid flow path being fluidly connected to a fluid displacement region of the valve body, wherein the fluid displacement region can be depressurized by movement of a syringe to introduce fluid into the fluid displacement region, and can be pressurized by movement of a syringe to expel fluid from the fluid displacement region. The sample box together with the reagents can allow a flow rate of up to about 100 μL / sec, such as about 10 μL / sec to about 100 μL / sec. The sample box together with the reagents can allow a pressure of less than 100 psi, less than 80 psi, or less than 60 psi. The sample box can allow a sample volume of up to 1000 μL, such as 300 μL to 1000 μL.

[0138] The cartridge may further include ultrasonic, piezoelectric, magnetostrictive or electrostatic sensors, for example in the lysis chamber to facilitate mechanical lysis. The sample cartridge may further include a removable syringe to facilitate fluid flow into and out of the lysis chamber by fluctuations in pressure.

[0139] The box can be a single-use box. In some embodiments, the box is an automated box.

[0140] In order to improve the sensitivity of nucleic acid detection, large volume samples can be prepared. However, as described herein, the preparation of large volumes is in conflict with the microfluidic system for automatic cracking, processing and / or analysis of biological samples. Therefore, it is necessary to allow the preparation of large volume samples by, for example, filtering, and to provide the separated nucleic acid to the microfluidic system with a small volume through the microfluidic interface. The sample box including a modified filter (preferably a modified glass fiber filter) and the method described herein are used to realize this need. The box and method allow detection of target nucleic acids (such as DNA) from various sample types (including whole blood, plasma, serum, semen, spinal fluid, tissue, tears, urine, feces, saliva, respiratory samples, nasopharyngeal samples, vaginal swabs, vaginal mucus samples, vaginal tissue samples, vaginal cell samples, bacterial cultures, mammalian cell cultures, viral cultures, human cells, bacteria, extracellular fluid, pancreatic juice, cell lysates, PCR reaction mixtures or in vitro nucleic acid modification reaction mixtures), without requiring the user to perform too many sample processing steps.

[0141] Methods for processing large volume samples include introducing the biological sample into a sample box. In some cases, the sample can be mixed with a reagent before it is introduced into the box to destroy particles present in the sample. However, the sample introduced into the sample box can also be destroyed in the sample box only when processed.

[0142] In order to be able to process large volumes, the volume of the sample chamber and / or lysis chamber within the sample box can in particular be at least 300 μL, at least 500 μL, at least 1,000 μL, at least 1,500 μL, at least 2,000 μL, at least 2,500 μL, at least 3,000 μL, at least 3,500 μL, at least 4,000 μL, at least 4,500 μL, at least 5,000 μL, at least 5,500 μL, at least 6,000 μL, at least 6,500 μL, at least 7,000 μL, at least 7,500 μL, at least 8,000 μL, at least 8,500 μL, at least 9,000 μL, at least 9,500 μL or at least 10,000 μL. Preferably, the sample cartridge can purify and process nucleic acids from a liquid sample having a volume of up to 10,000 μL, such as 300 μL to 5,000 μL, 300 μL to 3,000 μL, 300 μL to 2,000 μL, or 300 μL to 1,000 μL.

[0143] In some cases, it is possible to destroy the biological sample in the sample box with a lysis buffer, i.e., a solution. In other cases, the biological sample is destroyed before the sample box is introduced. Generally, it is desirable to treat the sample with an enzyme such as lysozyme, proteinase K, and / or a leukocyte reduction agent before mixing the sample with a chemical lysis buffer. In addition, it may be desirable to have more than one lysis buffer and more than one washing buffer. Similarly, aspects of the instrument that can be used to improve the extraction and purification efficiency of large-volume samples are not shown. For example: 1) a primary filter for capturing particulate matter in the sample before or after lysis, but before the lysate is sent to a modified nucleic acid binding filter; 2) an element that involves heating the sample during / before lysis, 3) an element that involves sonicating or shearing the sample during lysis, 4) an element that involves sending heated or dehumidified air on a nucleic acid binding matrix to improve drying, and similar features are not shown, but it can be inferred that these elements are included to improve the overall performance of the instrument. In some embodiments, multiple rounds of the following operations can be completed: extracting the sample, and then directing the "filtered" sample to waste until all samples or sample containers remain empty.

[0144] method

[0145] Preparation of solid support

[0146] The preparation of the solid supports or separation materials described herein can be achieved in any suitable manner. Typically, the solid support comprises reactive groups, such as silanol, epoxide, aldehyde, ketone, or activated ester groups, so that the DNA binding ligands disclosed herein can be attached to the solid support by derivatization reactions, non-covalent coating, or a combination thereof. In some embodiments, the compound comprising the DNA binding ligand can be covalently attached to the solid support by cycloaddition, nucleophilic or electrophilic substitution, or any other mechanism known in the art. For example, Figure 8 Alkylamine-coated solid supports reacted with cyanuric chloride are shown, and in turn, the stable, electrophilic surface is used to couple amine-modified DNA binding ligands. Cyanuric chloride, aminopropyl-modified glass fiber filters (CC-AP-GFF) can be used for other applications to immobilize various ligands, such as amine-modified oligonucleotides for the preparation of DNA microarrays.

[0147] As described herein, the solid support can include glass fibers comprising silanol groups. Such glass fiber solid supports can be reacted with silanized groups to obtain separation materials disclosed herein. Thus, the silanol groups of the glass fibers can react with the silanol groups of the formula Y-(L) y-SiX3, wherein each X is independently selected from halogen, alkoxy, dialkylamino, triflate, or linear, branched or cycloalkyl; L is an optional linker, such as an alkylene, heteroalkylene linker, cyanuric chloride, alkylamine or a combination thereof, and the linker can be optionally substituted; and Y is a DNA binding ligand, as described herein. The reaction of the glass fiber with the compound described herein provides a DNA binding group on the surface of the glass fiber.

[0148] The density of surface DNA binding ligands can be determined using any suitable method, such as the DMT assay provided herein. In the DMT assay, amino or amido groups exposed on the surface of a solid support (e.g., GFF) are reacted with a pentafluorophenyl (PFP) ester containing a dimethoxytrityl (DMT) reporter group, such as Figure 2 As shown. In short, the method can include treating a modified solid support with a PFP ester containing DMT. After washing away the excess PFP ester, the spatially accessible amino or amido groups on the surface of the solid support are covered with DMT groups. Treatment of the solid support with an acid can release the orange trityl cation. The absorbance at 497 nm can be measured with a spectrophotometer. The known DMT extinction coefficient (ε 497 =76,000M -1 cm -1 ) and Beer's law can be used to calculate the DMT concentration. Amine density is expressed in nmol / cm 2 Report.

[0149] Isolation of nucleic acids

[0150] Also provided herein is a method for separating and purifying nucleic acids from samples containing nucleic acids using solid supports disclosed herein. Samples containing nucleic acids can be selected from blood, plasma, serum, semen, vaginal swabs, vaginal mucus samples, vaginal tissue samples, vaginal cell samples, spinal fluid, tissue, tears, urine, feces, saliva, coating specimens, bacterial cultures, mammalian cell cultures, viral cultures, human cells, bacteria, extracellular fluid, PCR reaction mixtures, paraffin-embedded tissue samples, cell lysates, or in vitro nucleic acid modification reaction mixtures. In a specific embodiment, the sample (biological sample) containing nucleic acids can be a fixed paraffin-embedded sample (e.g., from FFPET samples), which can be used to identify the presence and / or expression level of a gene, and / or the mutation state of a gene. In some embodiments, the sample containing nucleic acids can be a liquid biopsy sample for detecting cancer, such as prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, esophageal cancer, ovarian cancer, bile duct cancer, gastric cancer, and liver cancer. In some embodiments, the sample containing nucleic acids can be a respiratory sample for detecting infectious diseases. The sample comprising nucleic acids may include human, bacterial, fungal, animal or plant material. In other embodiments, the sample comprising nucleic acids may be obtained from a nucleic acid modification reaction or a nucleic acid synthesis reaction.

[0151] Nucleic acid includes any synthetic or naturally occurring nucleic acid, such as DNA or RNA, in any possible configuration (i.e., double-stranded nucleic acid, single-stranded nucleic acid, aptamer, or any combination thereof). Nucleic acid can be DNA, including dsDNA, ssDNA, and their hybrids. Nucleic acid can also be RNA, such as mRNA, non-coding RNA, total RNA, etc. Nucleic acid can be a synthetic nucleic acid. In some embodiments, nucleic acid separation using the methods described herein is very suitable for diagnostic methods, prognostic methods, monitoring treatment (e.g., cancer treatment) methods, etc. Therefore, the target nucleic acid can include genomic DNA, total RNA, short DNA, small DNA, tumor-derived nucleic acid (including circulating tumor DNA), methylated DNA, microbial nucleic acid, bacterial nucleic acid, viral nucleic acid, cell-free nucleic acid, or a combination thereof. In some embodiments, nucleic acid separated using the methods described herein is used to detect the presence, and / or copy number, and / or expression level, and / or mutation status of one or more cancer markers.

[0152] A method for separating nucleic acids from a sample containing nucleic acids may include (a) contacting the nucleic acids with a solid support containing a compound having a DNA binding ligand as disclosed herein, and (b) eluting the nucleic acids from the modified solid support. As described herein, the nucleic acids may exist as part of a biological sample. In some embodiments, the biological sample is contacted with a lysis solution before contacting the solid support, thereby lysing the cells contained in the biological sample and releasing the nucleic acids into the solution. The lysis solution may include a chaotropic agent, such as guanidine thiocyanate, guanidine hydrochloride, perchloric acid base, iodine base, urea, formamide, and combinations thereof. In some embodiments, the lysis solution may include a salt, such as sodium chloride or calcium chloride salt. In some examples, the lysis buffer includes one or more of a chaotropic agent, a salt, a buffer, a surfactant, a defoaming agent, or a combination thereof. Before contacting the sample with the solid support, the sample may be lysed by contacting the sample with a lysis buffer, and then precipitating the nucleic acids. In some cases, the lysis solution includes one or more proteases. Suitable proteases include, but are not limited to, serine proteases, threonine proteases, cysteine ​​proteases, aspartic proteases, metalloproteases, glutamic acid proteases, metalloproteases, and combinations thereof. Exemplary suitable proteases include, but are not limited to, proteinase K (a broad-spectrum serine protease), subtilisin, chymotrypsin, pepsin, papain, etc. Other proteases can be obtained by one skilled in the art using the teachings and examples provided herein.

[0153] In some embodiments, the methods disclosed herein do not require the use of chaotropic agents or high salt concentrations for lysing samples containing nucleic acids. In some embodiments, the methods disclosed herein may require lower concentrations of chaotropic agents or salts for lysing samples containing nucleic acids compared to conventional lysis assays. For example, chaotropic agents can be used at a concentration of less than 4.5M, less than 2M, or less than 1M. In some embodiments, the methods disclosed herein do not require the use of lysis buffers.

[0154] The method for separating nucleic acid can further include filtering, centrifuging, precipitating and / or washing nucleic acid to concentrate nucleic acid before elution. Usually, after nucleic acid cleavage, lysate is filtered on solid support in the presence of binding agent (e.g., PEG) to bind nucleic acid to solid support. Binding agent can include one or more of the following: alcohol (e.g., methanol, ethanol, propanol, isopropanol), alkanediol or alkanetriol with 2 to 6 carbon atoms, monocarboxylic acid ester or dicarboxylic acid diester with 2 to 6 carbon atoms in acidic component and 1 to 4 carbon atoms in alcohol component; (poly) ethylene glycol and ether derivatives and ester derivatives thereof, and poly (4-styrene sulfonic acid-to-maleic acid). For example, the binding agent may include one or more of the following: 1,2-butanediol, 1,2-propylene glycol, 1,3-butanediol, 1-methoxy-2-propanol acetate, 3-methyl-1,3,5-pentanetriol, DBE-2, DBE-3, DBE-4, DBE-5, DBE-6, diethylene glycol monoethyl ether (DGME), triethylene glycol monoethyl ether (TGME), diethylene glycol monoethyl ether acetate (DGMEA), ethyl lactate, ethylene glycol, poly (2-ethyl-2-oxazoline), poly (4-styrenesulfonic acid-co-maleic acid) sodium salt solution, tetraethylene glycol (TEG), tetraethylene glycol, tetrahydrofurfuryl polyethylene glycol 200, tri (ethylene glycol) divinyl ether, anhydrous triethylene glycol and triethylene glycol monoethyl ether. In some embodiments of the methods disclosed herein, no binding agent is required, or a lower concentration of binding agent can be used compared to conventional assays. For example, binding agents, such as PEG, can be used at a concentration of less than 40% v / v, less than 30% v / v, less than 20% v / v, or less than 10% v / v in a filter and / or detergent. The solid supports described herein are coated with DNA affinity ligands (i.e., DNA binding ligands). Therefore, the solid supports disclosed herein allow selective capture of nucleic acids (RNA and DNA) from biological matrices. In fact, modified solid supports (e.g., modified glass fibers) can capture free circulating nucleic acids and nucleic acids from cells without using lysis buffers, salts, or binding agents, or using very low concentrations of lysis buffers, salts, or binding agents. It is important to point out that the present invention described herein includes capturing complex genomic DNA or RNA from various organisms in biological samples. For example, modified glass microscope slides are typically used to fix DNA or RNA for microarray imaging. Typically, these flat, modified surfaces have very low surface areas and are not suitable for separating DNA or RNA from a large number of complex samples.

[0155] The bound nucleic acids may optionally be washed on the solid support, for example, to remove components of the lysis buffer or unwanted components from the biological sample.The concentrated (bound) nucleic acids may be washed in a buffer compatible with the PCR reaction.

[0156] The nucleic acid is subsequently eluted from the solid support with elution buffer. The elution of nucleic acid from the solid support can be achieved by increasing the pH of the elution mobile phase or eluent stepwise or in a gradient manner. In some embodiments, the bonded nucleic acid can be eluted from the solid support by contacting with an alkaline solution. The alkaline solution can include ammonia or an alkali metal hydroxide, ammonium hydroxide, NaOH or KOH, and its concentration is enough to destroy the combination of the nucleic acid and the compound on the solid support. In some embodiments, the eluent has an alkaline pH. In some embodiments, the eluent has a pH greater than about 9, greater than about 10, greater than about 11, about 9 to about 12, about 9.5 to about 12, about 10 to about 12, or about 9 to about 11. Preferably, the pH of the eluent is higher than 10. Exemplary eluents include 1% or more ammonia, 15mM or more KOH (e.g., 25mM KOH, 35mM KOH, 40mM KOH, or 50mM KOH), or 15mM or more NaOH (e.g., 25mM NaOH, 35mM NaOH, 40mM NaOH, or 50mM NaOH). As described herein, the use of high pH to elute nucleic acids, such as DNA, is unique, particularly for the boxes described herein, and provides improved speed and performance of the disclosed methods. Speed ​​is provided by rapidly neutralizing acidic ammonium ions with high concentrations of hydroxide ions. A further advantage of high pH is the denaturing effect of KOH on captured DNA or RNA. Double-stranded structures and other secondary structures are destroyed, but can be renatured when, for example, neutralized with Tris HCl. The boxes described herein allow for rapid neutralization of eluted DNA or RNA in KOH / NaOH. A separate Tris reagent (e.g., in the form of beads) can be provided for immediate reaction with a KOH / NaOH eluent to produce a final pH of about 8.5 for downstream PCR or other nucleic acid assays. In some embodiments, the eluent has a pH of less than about 9, less than about 8.5, or less than about 8.

[0157] In some embodiments, the eluent comprises a polyanion. The polyanion is typically a polymer comprising a plurality of anionic groups. In some embodiments, the anionic group is a phosphate, phosphonate, sulfate or sulfonate group or a combination thereof. In some embodiments, the polyanion is a polymer with a negative charge at pH higher than about 7. Both synthetic polyanions and naturally occurring polyanions can be used in the methods disclosed herein. In some embodiments, the polyanion is carrageenan. In other embodiments, the polyanion is a carrier nucleic acid. As used herein, the carrier nucleic acid is a nucleic acid that does not interfere with subsequent concentrated nucleic acid detection (e.g., by PCR). Exemplary carrier nucleic acids include poly-rA, poly-dA, herring sperm DNA, salmon sperm DNA, and other nucleic acids well known to those skilled in the art. In some embodiments, the eluent comprises carrageenan and an alkali metal hydroxide, such as NaOH or KOH.

[0158] In general, nucleic acid chains including DNA and RNA are easily captured on the solid support surface and washed free of impurities at pH 5 or higher. For alkylamine-modified glass fiber filters, for example, nucleic acids can be efficiently eluted with high pH buffers (8.5 to 12.5) or with at least 50 mM KOH, as demonstrated by the PCR assay described herein.

[0159] For solid supports modified with Diels-Alder adducts, the nucleic acids can be optionally released by photochemical or thermal cleavage of the adducts. In some cases, the method of isolating nucleic acids includes eluting the nucleic acids from the solid support in the presence of an eluent, including heating the concentrated (bound) nucleic acids to a temperature of 100° C. or less, 95° C. or less, 85° C. or less, 75° C. or less, 65° C. or less, 55° C. or less; sonicating the nucleic acids; photochemically cleaving the compounds; or a combination thereof.

[0160] The nucleic acids separated using the methods and solid supports described herein have suitable amplification quality to detect and / or quantify one or more target nucleic acid sequences in a sample. In fact, the nucleic acid separation methods and solid supports described herein can be applied to basic research aimed at discovering gene expression profiles related to the diagnosis and prognosis of a disease. The methods are also applicable to the diagnosis and / or prognosis of a disease, the determination of a specific treatment regimen, and / or the monitoring of the effectiveness of treatment.

[0161] Nucleic acid detection

[0162] The methods described herein simplify the separation of nucleic acids from biological samples and effectively produce nucleic acids that are very suitable for separation in RT-PCR systems. In some embodiments, nucleic acids separated from samples containing nucleic acids using the methods described herein can be detected by any suitable known nucleic acid detection method. Therefore, a method for detecting nucleic acids in biological samples is disclosed. The detection method may include nucleic acid amplification. In some embodiments, after eluting nucleic acids from a solid support with an eluent, the method for detecting nucleic acids may include combining the eluate with a PCR reagent that can be present in a box as freeze-dried particles. In some embodiments, PCR uses a Taq polymerase with a hot start function, such as AptaTaq (Roche, Switzerland). The polymerase chain reaction can be nested PCR, isothermal PCR, gradient PCR, qPCR, reverse transcriptase PCR, real-time PCR, multiplex PCR, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), rolling circle amplification (RCA) or strand displacement amplification (SDA).

[0163] In certain embodiments, a method for detecting nucleic acids in a biological sample obtained from a subject may include placing the biological sample in a cartridge as described herein, wherein the cartridge includes a plurality of fluidically connected chambers, reaction tubes configured for amplifying nucleic acids by thermal cycling, and filters in the fluid paths between the plurality of chambers and the reaction tubes; lysing cells with a lysis reagent present in at least one of the plurality of chambers and capturing DNA released therefrom; and amplifying the DNA with primers and probes for detecting the presence of nucleic acids.

[0164] In some embodiments, a target nucleic acid, e.g., a coronavirus such as an alphacoronavirus, a betacoronavirus, or SARS-CoV-2, an adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella spp., parainfluenza virus, and respiratory syncytial virus (RSV), hantavirus, cytomegalovirus, coxsackievirus, herpes simplex virus, echovirus, influenza virus C, Streptococcus pneumoniae, Chlamydia pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus parainfluenzae, Group A Streptococcus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas spp., Neisseria spp., Pneumocystis jiroveci, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Candida spp., Aspergillus spp., Mucor spp., Cryptococcus neoformans, or Pneumocystis carinii biomarkers and / or optional controls can be detected. The target nucleic acid can be detected by the following methods: (a) contacting nucleic acid from the sample with a primer set and optionally a probe for detecting the presence of the desired target nucleic acid, (b) subjecting the nucleic acid, primers, and optional probe to amplification conditions; (c) detecting the presence of any amplification product, optionally by real-time PCR, melting curve analysis, or a combination thereof, and (d) optionally identifying the presence of the target nucleic acid in the sample based on the detection of the amplification product or its absence.

[0165] In some embodiments, the amplification method includes an initial denaturation at about 90°C to about 100°C for about 1 to about 10 minutes, followed by a cycle comprising denaturation at about 90°C to about 100°C for about 1 to about 30 seconds, annealing at about 55°C to about 75°C for about 1 to about 30 seconds, and extension at about 55°C to about 75°C for about 5 to about 60 seconds. In some embodiments, the cyclic denaturation step is omitted for the first cycle after the initial denaturation. The specific time and temperature will depend on the specific nucleic acid sequence being amplified and can be easily determined by a person of ordinary skill in the art.

[0166] In some embodiments, separation and detection of nucleic acid are carried out in an automatic sample handling and / or analysis platform. In some embodiments, commercially available automatic analysis platforms are utilized. For example, in some embodiments, the GeneXpert system (Cepheid, Sunnyvale, Calif.) is used. However, the present invention is not limited to specific detection methods or analysis platforms. Those skilled in the art recognize that any number of platforms and methods can be utilized. The GeneXpert system utilizes a self-contained disposable box. Sample extraction, amplification, and detection of nucleic acid can all be carried out in this independent "laboratory in a box".

[0167] Examples of other methods that can be used in the methods described herein include bead-based flow cytometry. See Lu J. et al. (2005) Nature 435:834-838, which is incorporated herein by reference for this specification. An example of a bead-based flow cytometric assay is Luminex, Inc.'s Technology. See www.luminexcorp.com / technology / index.html. Another method uses microfluidic devices and single molecule detection. See U.S. Patent Nos. 7,402,422 and 7,351,538 to Fuchs et al, US Genomics, Inc., each of which is incorporated herein by reference in its entirety. Yet another method is simple gel electrophoresis and detection with labeled probes (e.g., probes labeled with radioactive or chemiluminescent tags), such as by Northern blotting.

[0168] Although in some embodiments the extracted nucleic acids are used for amplification reactions, other uses are also contemplated. Thus, for example, the isolated nucleic acids or their amplification products can be used in various sequencing or hybridization schemes, including but not limited to nucleic acid-based microarrays and next generation sequencing.

[0169] Methods that are easily automated are of great interest. Commercially available nucleic acid amplification systems can be used to perform the methods described herein in a substantially automated manner. Exemplary non-limiting nucleic acid amplification systems that can be used to perform the methods of the invention include system, Infinity System and Xpress system (Cepheid, Sunnyvale, Calif.). In some embodiments, the amplification system can be used at the same location as the individual to be tested, such as a health care provider's office, clinic, or community hospital, so that processing is not delayed by transporting the sample to another facility. Determinations according to the methods described herein can be performed in less than 3 hours, in some embodiments less than 2 hours, in some embodiments less than 1 hour, in some embodiments less than 45 minutes, in some embodiments less than 35 minutes, and in some embodiments less than 30 minutes using an automated system (e.g., system) completed. Utilize independent, disposable box.As described herein, sample extraction, amplification and detection can all be carried out in this independent sample box.

[0170] Before the sample is subjected to the amplification reaction, one or more sample preparation operations are performed on the sample. Typically, these sample preparation operations will include such operations as extracting intracellular materials such as nucleic acids from whole cell samples, viruses, etc. to form a crude extract, additional processing to prepare the sample for subsequent operations, such as denaturation, purification, filtration, desalting, etc. of contaminating (e.g., DNA binding) proteins. The release of nucleic acids from sample cells or viruses and the denaturation of DNA binding proteins can usually be performed by chemical, physical, or electrolytic lysis methods. For example, chemical methods typically use lysis agents to destroy cells and extract nucleic acids from cells, and then treat the extract with chaotropic salts (e.g., guanidine isothiocyanate or urea) to denature any contaminating and potentially interfering proteins. Typically, in the case of using chemical extraction and / or denaturation methods, appropriate reagents can be incorporated into the sample preparation chamber, a separate accessible chamber, or can be introduced from the outside. Preferably, sample preparation is performed in only one step or no more than two steps. As described herein, the method simplifies the separation of nucleic acids from biological samples and effectively produces nucleic acids that are very suitable for separation in RT-PCR systems.

[0171] The method for detecting nucleic acid described herein can be implemented without transporting the sample from the location where the sample is collected. For example, the method can be performed at a POC diagnostic location. The location for POC diagnosis includes a patient care environment, preferably a hospital, an emergency care center, an emergency room, a physician's office, a health clinic, or a home. In some cases, the presence or absence of nucleic acid in a biological sample can be detected within 75 minutes or within 60 minutes of collecting the sample from the object.

[0172] Implementation

[0173] In one embodiment, a method for isolating nucleic acid from a biological sample, the method comprising: (a) contacting the nucleic acid with a compound bonded to a glass fiber filter, the compound being derived from a structure represented by the following formula:

[0174] Y-(L) y -SiX3

[0175] wherein Y is a DNA binding ligand selected from an alkylamine, a cycloalkylamine, an alkoxyamine, a polyamine moiety, an arylamine, an intercalator, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof,

[0176] L is a linker selected from an alkyl group, a heteroalkyl group, an olefin group, a heteroolefin group, a polyacrylic acid, a Diels-Alder adduct, or a combination thereof,

[0177] Each X is independently selected at each occurrence from a hydrolyzable group, an alkyl group, a heteroalkyl group, an alkenyl group, or two or three Xs are combined to form one or more cyclic groups, or one X is combined with Y to form a cyclic azasilane, and

[0178] y is 0 or 1; and

[0179] (b) Elution of nucleic acids from glass fiber filters.

[0180] In one embodiment, a method for isolating nucleic acids from a biological sample comprises: (a) contacting the nucleic acids with a composition comprising a Diels-Alder adduct, wherein the Diels-Alder adduct comprises a DNA binding ligand, and (b) concentrating the nucleic acids onto a solid support, wherein the Diels-Alder adduct is optionally bonded to the solid support.

[0181] In one embodiment, a method for detecting nucleic acids in a biological sample comprises: (a) isolating nucleic acids from the biological sample using a method as defined in any one of the above embodiments; (b) eluting the nucleic acids from the solid support with an eluent; and (c) detecting the nucleic acids.

[0182] In one embodiment, the separation material for nucleic acid separation comprises: a glass fiber solid support and a compound bonded to the glass fiber solid support, wherein the compound is derived from a structure represented by the following formula:

[0183] Y-(L) y -SiX3

[0184] wherein Y is a DNA binding ligand selected from an alkylamine, a cycloalkylamine, an alkoxyamine, a polyamine moiety, an arylamine, an intercalator, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof,

[0185] L is a linker selected from an alkyl group, a heteroalkyl group, an olefin group, a heteroolefin group, a polyacrylic acid, a Diels-Alder adduct, or a combination thereof,

[0186] Each X at each occurrence is independently selected from a hydrolyzable group, an alkyl group, a heteroalkyl group, an alkenyl group, or two or three Xs are combined to form one or more cyclic groups, and

[0187] y is either 0 or 1.

[0188] In one embodiment, the separation material for nucleic acid isolation comprises: a glass fiber solid support comprising a Diels-Alder adduct with a DNA binding ligand, cyanuric chloride, or a combination thereof, wherein the adduct or cyanuric chloride is chemically bonded to the glass fiber solid support, optionally via a linker.

[0189] In one embodiment, a sample box for separating and detecting nucleic acids from a biological sample comprises: a box body having a plurality of chambers defined therein, wherein the plurality of chambers are fluidically connected via a fluid path of the box, and wherein at least one chamber is configured to receive the biological sample, a reaction vessel, and a filter, wherein the reaction vessel is configured to amplify nucleic acids by thermal cycling, wherein the filter is disposed in the fluid path between the plurality of chambers and the reaction vessel, wherein the filter comprises a separation material according to any one of the embodiments herein, wherein the plurality of chambers and the reaction vessel independently comprise reagents for releasing nucleic acids from the biological sample, and primers and probes for detecting nucleic acids.

[0190] In one embodiment, a sample box for separating and detecting nucleic acids from a biological sample comprises: a box body having a plurality of chambers therein, wherein the plurality of chambers comprises: a sample chamber having at least a fluid outlet fluidically connected to another chamber of the plurality of chambers; and a lysis chamber fluidically connected to the sample chamber, the lysis chamber comprising a reagent for releasing nucleic acids, optionally wherein the sample chamber and the lysis chamber are the same; a reaction vessel fluidically connected to the plurality of chambers of the box body and configured for ii) amplifying nucleic acids, and detecting a plurality of amplification products; a filter disposed in a fluid path between the lysis chamber and the reaction vessel, wherein the filter comprises a solid support modified with a DNA binding ligand selected from alkylamines, cycloalkylamines, alkoxyamines, polyamine moieties, aromatic amines, intercalators, DNA groove binders, peptides, amino acids, proteins or combinations thereof; and a plurality of primers and / or probes disposed in one or more of the plurality of chambers or the reaction vessel for detecting nucleic acids.

[0191] In one embodiment, a method for detecting nucleic acids in a biological sample obtained from a subject, the method comprising: placing the biological sample in a sample box according to any one of the embodiments herein; optionally lysing cells with one or more lysis reagents present in at least one of a plurality of chambers and capturing nucleic acids released therefrom; and amplifying the nucleic acids with primers and probes for detecting the presence of the nucleic acids.

[0192] In one embodiment, a method for detecting nucleic acids in a biological sample obtained from a subject, the method comprising: a) contacting nucleic acids from the biological sample with a primer set and optional probes in a sample kit according to any one of the embodiments herein; b) subjecting the nucleic acids, primer pairs, and optional probes to amplification conditions; c) detecting the presence of amplification products, optionally by real-time PCR, melting curve analysis, or a combination thereof, and d) detecting the presence of nucleic acids in the biological sample based on the detection of the amplification products.

[0193] In any of the above embodiments, wherein the DNA binding ligand comprises an amine group, an intercalator, a minor groove binder, a peptide, an amino acid, a protein, or a combination thereof.

[0194] In any of the above embodiments, wherein the DNA binding ligand is selected from an alkylamine, a cycloalkylamine, an alkoxyamine, an arylamine, a polyamine moiety, or a combination thereof.

[0195] In any of the above embodiments, wherein the DNA binding ligand comprises a plurality of amine groups.

[0196] In any of the above embodiments, wherein the DNA binding ligand comprises at least two, at least three, at least four, at least five, at least six amine groups, or a combination thereof.

[0197] In any of the above embodiments, wherein the DNA binding ligand comprises an alkylamine group, an imidazole group, a bisbenzimide minor groove binder, or a combination thereof.

[0198] In any of the above embodiments, the DNA binding ligand is selected from spermine, methylamine, ethylamine, propylamine, ethylenediamine, diethylenetriamine, 1,3-dimethyldipropylenediamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium chloride, tris(2-aminoethyl)amine, or a combination thereof.

[0199] In any of the above embodiments, wherein the Diels-Alder adduct is derived from an unsaturated cyclic imino group.

[0200] In any of the above embodiments, wherein the compound or Diels-Alder adduct is derived from a structure represented by the following general formula, its isomers, salts, tautomers, or combinations thereof:

[0201]

[0202] wherein Y' is a DNA binding ligand, and L, X and y are as defined in any of the embodiments herein.

[0203] In any of the above embodiments, wherein linker L is present (or y is 1).

[0204] In any of the above embodiments, wherein the linker is selected from an alkylene oxide group, an alkylene group, cyanuric chloride, an alkylamine, or a combination thereof.

[0205] In any of the above embodiments, at least two X are independently selected from halogen, alkoxy, dialkylamino, triflate, or together with the Si atom to which they are attached, form a silatricycle, cyclic siloxane, polysilsesquioxane or silazane.

[0206] In any of the above embodiments, wherein at least two X are independently selected from alkoxy groups (eg, ethoxy or methoxy).

[0207] In any of the above embodiments, wherein the compound or Diels-Alder adduct is derived from one of the following structures:

[0208]

[0209] 3-aminopropyltrimethoxysilane, aminoalkylsilazotricyclo, 3-(2-aminoethyl)aminopropyltriethoxysilane, 3-(2-aminoethyl)aminopropyltrimethoxysilane, or a combination thereof, and wherein n is an integer from 0 to 10, from 1 to 10, or from 1 to 5.

[0210] In any of the above embodiments, wherein at least two X are independently selected from alkoxy groups (eg, ethoxy or methoxy).

[0211] In any of the above embodiments, wherein the compound or Diels-Alder adduct is derived from 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, or a combination thereof.

[0212] In any of the above embodiments, the solid support comprises a material selected from silica, glass, vinyl backbone polymers, mica, polycarbonate, zeolite, titanium dioxide, magnetic beads, glass beads, cellulose filters, polycarbonate filters, polytetrafluoroethylene filters, polyvinyl pyrrolidone filters, polyether sulfone filters, glass fiber filters or combinations thereof.

[0213] In any of the above embodiments, wherein the solid support is a glass fiber filter.

[0214] In any of the above embodiments, wherein the compound or Diels-Alder adduct is bonded to the solid support via a siloxane bridge, a carboxylate bridge, an ester bridge, an ether bridge, or a combination thereof.

[0215] In any of the above embodiments, wherein the glass fiber filter has a pore size selected to accommodate beads of a corresponding size to facilitate mechanical lysis.

[0216] In any of the above embodiments, wherein the glass fiber filter has an effective pore size of 0.2 μm to 3 μm, 0.2 μm to 2 μm, preferably 0.5 μm to 1.0 μm or 0.6 μm to 0.8 μm.

[0217] In any of the above embodiments, wherein the glass fiber filter has a thickness of 35 g / m 2 Up to 100g / m 2 , preferably 50g / m 2 Up to 85g / m 2 , or 70g / m 2 Up to 80g / m 2 The basis weight.

[0218] In any of the above embodiments, wherein the glass fiber filter has a fiber diameter of 1 μm to 100 μm, preferably 1 μm to 50 μm, or 1 μm to 25 μm.

[0219] In any of the above embodiments, wherein the glass fiber filter has a thickness of 250 μm to 2,000 μm, 300 μm to 1,500 μm, 300 μm to 1,000 μm, 300 μm to 750 μm, or 350 μm to 500 μm.

[0220] In any of the above embodiments, wherein the beads are selected from glass beads, silica beads, or a combination thereof.

[0221] In any of the above embodiments, the biological sample is blood, plasma, serum, semen, cerebrospinal fluid, tissue, tears, urine, feces, saliva, a smear specimen, a respiratory sample, a nasopharyngeal sample, a vaginal swab, a vaginal mucus sample, a vaginal tissue sample, a vaginal cell sample, a bacterial culture, a mammalian cell culture, a viral culture, a human cell, bacteria, extracellular fluid, pancreatic juice, a cell lysate, a PCR reaction mixture, or an in vitro nucleic acid modification reaction mixture.

[0222] In any of the above embodiments, wherein the biological sample is blood, plasma, a breath sample, or a vaginal swab.

[0223] In any of the above embodiments, the biological sample comprises a nucleic acid selected from genomic DNA, total RNA, short DNA, small DNA, tumor-derived nucleic acid, methylated DNA, microbial nucleic acid, bacterial nucleic acid, viral nucleic acid, cell-free nucleic acid, or a combination thereof.

[0224] In any of the above embodiments, wherein the biological sample comprises cell-free nucleic acid.

[0225] In any of the above embodiments, the biological sample is contacted with a buffer prior to or simultaneously with step a) contacting the nucleic acid with the composition or compound bound to a solid support.

[0226] In any of the above embodiments, the buffer is a lysis buffer comprising one or more of a chaotropic agent, a salt, a buffering agent, a surfactant, a defoaming agent, a binding agent, or a combination thereof.

[0227] In any of the above embodiments, wherein the lysis buffer comprises a chaotropic agent selected from guanidine thiocyanate, guanidine hydrochloride, base perchlorate, base iodide, urea, formamide, or a combination thereof.

[0228] In any of the above embodiments, wherein the chaotropic agent is used at a concentration of less than 4.5 M, less than 2 M, or less than 1 M in the lysis buffer.

[0229] In any of the above embodiments, wherein the method does not use a chaotropic agent or a lysis buffer.

[0230] In any of the above embodiments, the buffer comprises saline (inorganic salts, such as CaCl2, MgSO4, KCl, NaHCO3, NaCl, etc.), phosphate buffer, Tris buffer, 2-amino-2-hydroxymethyl-1,3-propanediol, HEPES, PBS, citrate buffer, TES, MOPS, PIPES, cacodylate, SSC, MES, a sugar or a disaccharide, or a combination thereof.

[0231] In any of the above embodiments, wherein the nucleic acid is contacted with a binding agent, a filtering agent, a washing agent, or a combination thereof simultaneously with concentration or prior to elution of the nucleic acid.

[0232] In any of the above embodiments, wherein the filtration reagent and / or the wash reagent comprises a binding agent.

[0233] In any of the above embodiments, the binding agent comprises a polyalkylene oxide (eg, PEG200) or a salt.

[0234] In any of the above embodiments, the binding agent is used in a concentration of less than 40% v / v, less than 30% v / v, less than 20% v / v or less than 10% v / v in the filter and / or detergent.

[0235] In any of the above embodiments, wherein the method does not use a binding agent, or the filtration reagent and / or the washing agent does not contain a binding agent.

[0236] In any of the above embodiments, wherein the method comprises eluting the nucleic acid with an eluting agent.

[0237] In any of the above embodiments, elution comprises heating the nucleic acid to a temperature of 100°C or less, 95°C or less, 85°C or less, 75°C or less, 65°C or less, 55°C or less in the presence of an eluent; sonicating the nucleic acid; photochemically cleaving the compound / composition; or a combination thereof.

[0238] In any of the above embodiments, wherein the eluent has a pH greater than about 9, greater than about 10, or greater than about 11.

[0239] In any of the above embodiments, wherein the eluent has a pH greater than about 10.

[0240] In any of the above embodiments, wherein the eluent has a pH of about 10 to about 13.

[0241] In any of the above embodiments, wherein the eluent is neutralized with a buffer.

[0242] In any of the above embodiments, wherein the eluent is neutralized with an acidic buffer.

[0243] In any of the above embodiments, wherein the eluent is neutralized with Tris HCl.

[0244] In any of the above embodiments, wherein the eluent has a pH of less than about 9, less than about 8.5, or less than about 8.

[0245] In any of the above embodiments, wherein the eluent comprises a polyanion, a polycation, ammonia, or an alkali metal hydroxide (eg, NaOH or KOH).

[0246] In any of the above embodiments, wherein the eluent comprises a polyanion.

[0247] In any of the above embodiments, wherein the polyanion is carrageenan, a carrier nucleic acid, or a combination thereof.

[0248] In any of the above embodiments, wherein the method is performed in a cassette, preferably in an automated cassette.

[0249] In any of the above embodiments, wherein detecting the nucleic acid comprises amplifying the nucleic acid by polymerase chain reaction.

[0250] In any of the above embodiments, wherein the polymerase chain reaction is nested PCR, isothermal PCR, qPCR or RT-PCR.

[0251] In any of the above embodiments, wherein the glass fiber solid support further comprises a polymer binder.

[0252] In any of the above embodiments, wherein the sample chamber and the lysis chamber are the same.

[0253] In any of the above embodiments, the reaction vessel comprises one or more reaction chambers for detecting multiple amplification products.

[0254] In any of the above embodiments, wherein each reaction chamber is configured to detect a single amplification product.

[0255] In any of the above embodiments, each reaction chamber is configured to detect multiple amplification products.

[0256] In any of the above embodiments, wherein the cartridge is configured to simultaneously detect multiple amplification products present in solution in a single reaction chamber.

[0257] In any of the above embodiments, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA) compliant cartridge.

[0258] In any of the above embodiments, wherein the box is configured to perform isothermal amplification.

[0259] In any of the above embodiments, wherein the cartridge is configured to achieve non-isothermal, optionally by thermal cycling, gradient (temperature differential), or temperature oscillation.

[0260] In any of the above embodiments, wherein the sample cartridge further comprises: a syringe movable to facilitate fluid flow into and out of the lysis chamber by fluctuations in pressure.

[0261] In any of the above embodiments, wherein the dissolution chamber comprises: a valve body and a valve bonnet, wherein the valve body contacts the valve bonnet to define a lysis chamber therebetween, and wherein the filter is held within the lysis chamber fixed between the valve body and the valve bonnet.

[0262] In any of the above embodiments, the lysis chamber has a fluid flow path between an inlet in the cap and an outlet in the valve body, and the fluid flow path is fluidly coupled to a fluid displacement region of the valve body, wherein the fluid displacement region can be depressurized by movement of the syringe to introduce fluid into the fluid displacement region, and can be pressurized by movement of the syringe to expel fluid from the fluid displacement region.

[0263] In any of the above embodiments, wherein the sample cartridge together with the reagents allows a flow rate of up to about 100 μL / sec, such as about 10 μL / sec to about 100 μL / sec.

[0264] In any of the above embodiments, wherein the sample cartridge together with the reagents allows pressures below 100 psi, below 80 psi, or below 60 psi.

[0265] In any of the above embodiments, wherein the sample cartridge allows for sample volumes of up to 1000 μL, such as 300 μL to 1,000 μL.

[0266] In any of the above embodiments, the lysis chamber further comprises an ultrasonic, piezoelectric, magnetostrictive or electrostatic transducer to facilitate mechanical lysis.

[0267] In any of the above embodiments, wherein the lysis chamber comprises a lysis reagent selected from the group consisting of a chaotrope, a chelator, a buffer, and a detergent to facilitate chemical lysis.

[0268] In any of the above embodiments, the chaotropic agent is selected from guanidine thiocyanate, guanidine hydrochloride, base perchlorate, base iodide, urea, formamide, or a combination thereof.

[0269] In any of the above embodiments, wherein the lysis reagent comprises a guanidinium compound, sodium hydroxide, EDTA, a buffer, and a detergent.

[0270] In any of the above embodiments, wherein the box does not contain a chaotropic agent.

[0271] In any of the above embodiments, wherein the filter is configured to bind the nucleic acid to be analyzed.

[0272] In any of the above embodiments, wherein the box further comprises a binding reagent, a washing reagent, an elution reagent, or a combination thereof.

[0273] In any of the above embodiments, wherein the binding agent comprises a polyalkylene oxide polymer (eg, PEG 200) or a salt.

[0274] In any of the above embodiments, the binding agent is used in a concentration of less than 40% v / v, less than 30% v / v, less than 20% v / v or less than 10% v / v in the filter and / or detergent.

[0275] In any of the above embodiments, wherein the kit does not comprise a binding agent or PEG.

[0276] In any of the above embodiments, wherein the box is an automated box.

[0277] In any of the above embodiments, the cartridge is a single-use cartridge.

[0278] In any of the above embodiments, wherein the amplification is performed by a real-time PCR multiplex assay.

[0279] In any of the above embodiments, wherein: a) contacting the nucleic acid from the sample with the primer set and optional probe in the sample box includes: placing the biological sample in a box, the box including a box body having multiple chambers connected by fluid, a reaction container having one or more reaction chambers and configured for amplifying nucleic acid, a fluid path between the multiple chambers and the reaction container, and a filter in the fluid path; and if the biological sample contains cells, the cells in the biological sample are lysed with one or more lysis reagents present in at least one of the multiple chambers; b) subjecting the nucleic acid, primer pair and optional probe to amplification conditions includes amplifying the nucleic acid with primers and probes in a solution present in at least one of the multiple chambers; and c) subjecting the nucleic acid, primer pair and optional probe to amplification conditions includes amplifying the nucleic acid with primers and probes in a solution present in at least one of the multiple chambers.

[0280] In any of the above embodiments, the biological sample is blood, plasma, serum, semen, cerebrospinal fluid, tissue, tears, urine, feces, saliva, a smear specimen, a respiratory sample, a nasopharyngeal sample, a vaginal swab, a vaginal mucus sample, a vaginal tissue sample, a vaginal cell sample, a bacterial culture, a mammalian cell culture, a viral culture, a human cell, bacteria, extracellular fluid, pancreatic juice, a cell lysate, a PCR reaction mixture, or an in vitro nucleic acid modification reaction mixture.

[0281] In any of the above embodiments, wherein the biological sample is blood, plasma, a breath sample, or a vaginal swab.

[0282] In any of the above embodiments, the method for detecting nucleic acids is used to determine the presence or absence of one or more target polynucleotides in a biological sample.

[0283] In any of the above embodiments, the one or more target polynucleotides are selected from genomic DNA, total RNA, short DNA, small DNA, tumor-derived nucleic acid, methylated DNA, microbial nucleic acid, bacterial nucleic acid, viral nucleic acid, cell-free nucleic acid, or a combination thereof.

[0284] In any of the above embodiments, wherein the one or more target polynucleotides comprise cell-free nucleic acid.

[0285] In any of the above embodiments, wherein the one or more target polynucleotides are infectious pathogen nucleic acids, preferably selected from respiratory pathogens or selected from coronaviruses, such as alpha-coronavirus, beta-coronavirus or SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A, influenza B, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella spp., parainfluenza virus and respiratory syncytial virus (RSV), hantavirus, cytomegalovirus, coxsackievirus, herpes simplex virus, echovirus, influenza virus C, Streptococcus pneumoniae, Chlamydia pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus parainfluenzae, Group A Streptococcus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas spp., Neisseria spp., Pneumocystis jiroveci, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Candida spp., Aspergillus spp., Mucor spp., Cryptococcus neoformans or Pneumocystis carinii.

[0286] In any of the above embodiments, wherein the biological sample is contacted with a lysate reagent, the lysate reagent comprises one or more of a chaotropic agent, a salt, a buffer, a surfactant, a defoaming agent, a binder, a precipitant, or a combination thereof.

[0287] In any of the above embodiments, the chaotropic agent is selected from guanidine thiocyanate, guanidine hydrochloride, base perchlorate, base iodide, urea, formamide, or a combination thereof.

[0288] In any of the above embodiments, wherein the chaotropic agent is used at a concentration of less than 2.0 mol / mL in the lysate.

[0289] In any of the above embodiments, wherein the method does not comprise the use of a chaotropic agent or a lysis buffer.

[0290] In any of the above embodiments, wherein the method further comprises contacting the nucleic acid with a binding agent, a filtering agent, and / or washing to promote binding of the nucleic acid to the filter while removing non-target material.

[0291] In any of the above embodiments, wherein the filter and / or wash agent comprises a binding agent (eg, PEG or a salt).

[0292] In any of the above embodiments, wherein the binding agent is used at a concentration of less than 30% v / v in the filter and / or detergent.

[0293] In any of the above embodiments, wherein the filter and / or the wash agent does not comprise a binding agent.

[0294] In any of the above embodiments, wherein the method comprises eluting the nucleic acid with an eluting agent.

[0295] In any of the above embodiments, wherein the nucleic acid in the biological sample is detected within 75 minutes or within 60 minutes of collecting the sample from the subject.

[0296] In any of the above embodiments, wherein the amplification is performed by a real-time PCR multiplex assay.

[0297] The following examples are for illustrative purposes only and are not intended to be limiting in any way.

[0298] Examples (a) Example 1: Coated glass fiber filters for DNA purification

[0299] Methods for coating glass fiber filters with DNA affinity ligands are illustrated. Porous glass fiber filters (GFF) are easily manufactured with various DNA affinity coatings to allow selective capture of nucleic acids (RNA and DNA) from biological matrices. Silanization and surface modification methods are illustrated. Strands of DNA and RNA are easily captured on affinity-modified glass surfaces and washed away impurities at pH 5. For alkylamine-modified GFF, NAs are effectively eluted with high pH buffers (8.5 to 12.5). For other affinity capture surfaces (or lower pH elutions), cleavable linkers (e.g., esters), photolyzable or thermally cleavable linkers are used. A reliable assay for measuring the density of surface alkylamine groups on GFF has been developed. A surface density of 30 nmol alkylamine / cm 2 Up to 90 nmol alkylamine / cm 2 The coated GFFs had good DNA binding ability and effectively released DNA with 50 mM KOH, as demonstrated by PCR assays.

[0300] The method for chemically conjugating GFF to DNA-binding ligands is shown in Figure 1. A reliable assay for measuring the density of surface alkylamine groups on GFF was developed as Figure 2 As shown. As described herein, PFP ester reagent is a novel compound prepared from succinic acid. PCR assays were developed to measure DNA extraction performance and are described herein. Briefly, a 1 / 2 inch diameter coated GFF was assembled into a syringe holder and moistened with 0.5 mL volume of dsDNA or RNA solutions of various concentrations at low pH. The amount of DNA passed was measured using GeneXpert, and the passed solution was subsequently purified in a separate box. The GFF was then eluted with 1 mL of high pH buffer and the DNA content was measured. The DNA extraction score is defined as (CT passed-CT eluted). A high value (5 to 12) indicates good extraction performance. A small positive number means a weak performance. A negative number means that DNA passes directly (failed to capture). If both the pass and elution Cts are high, the DNA remains trapped on the GFF (extraction efficiency score = X).

[0301] In general, it is shown that modified glass fibers capture DNA and RNA from solutions and subsequently release them for PCR testing. GFFs modified with amines, polyamines, imidazoles, and BisTris are shown to capture DNA at low pH and release DNA at high pH via a "charge switch" mechanism. GFFs activated with cyanuric chloride (CC) are attached to GFFs with bisbenzimide (BB) ligands that bind to the minor groove. Although DNA is shown to bind to the BB ligand, at high pH, ​​DNA cannot be effectively released from the GFFs coated with BB, which are fluorescent when bound to dsDNA, and the released DNA can be measured at 360nm or 460nm to show the efficiency of DNA extraction. In order to solve the release problem of the BB ligand, a thermally cleavable aminosilane linker is attached to the GFF, which is then activated with CC and coated with amine-modified BB ligands. Materials and methods.

[0302] Two types of borosilicate GFF disks with different nominal pore sizes and thicknesses were compared. Pall A / E type has a thickness of 0.33 mm and a pore size of 1 μm. Cytiva / Whatman grade GF / F, catalog number 1825-047, GF / F grade has a thickness of 0.43 mm and a pore size of 0.7 μm. The 47 mm disks were stacked on a polypropylene cutting board and punched with a hammer to produce seven 1 / 2 inch disks. The cost was approximately $0.25 / disc. Cytiva disks (Cepheid, Sunnyvale) were also obtained as 0.925 cm laser cut disks. Solvents were obtained from Sigma-Aldrich. Anhydrous solvents were handled under argon. Silanes and other organic reagents were obtained from Sigma-Aldrich, except imidazole silane (BocChem, China). PFP esters and BB-NH2 were prepared using anhydrous techniques and chromatographed over 200 mesh to 400 mesh silica gel using triethylamine in the eluent. Deuterated solvents were used as indicated by 1 H NMR (500 MHz) was used to analyze the intermediate compounds and the final product. Despite literature reports, CDCl3 cannot be used with trityl compounds due to its acidity (the solution turns orange and a hydrolysis peak is visible). UV-vis spectroscopy was performed using a Cary spectrophotometer with a sample cell changer and single beam reading function, with a 1 mL quartz cuvette, and the DMT absorbance was read at 497 nm.

[0303] Silanization (DETA-GFF, method A) of 1 / 2 inch diameter GFF disk in ethanol. Prepare 30 pre-punched 1 / 2 inch diameter disks by stacking several filters (47mm diameter) with a steel punch. Required disks are collected in 50mL polypropylene screw cap centrifuge tubes. 30mL DETA silanization reagents (Sigma Aldrich, catalog number 413348) are added in anhydrous ethanol at 0.25%, 0.5%, 1% and 2% by volume. The disk is placed on a platform shaker and stirred overnight, and multiple tubes can be multiplexed. Excess reagent is discharged, and the disk is repeatedly washed by draining and decantation with fresh volume of methanol in a process of 3 to 6 hours (6 times). Use vacuum desiccator and high vacuum source (<1mm Hg) to drain the disk overnight and remove excess solvent. The disk is stored in the 50mL tube of the same mark for preparation.

[0304] Silanization of 47mm diameter GFF disk in toluene (AP-GFF, method B). The APTMS / toluene method uses a single 47mm diameter GFF disk, crimped into a 20mL borosilicate glass scintillation bottle. Add 2mL of a toluene solution of 0.1M (3-aminopropyl) trimethoxysilane (APTMS) (1.8% by weight). A 20mL vial with a foil-lined lid is tipped over the side to wet the GFF. When wetted with toluene, the filter becomes more flexible and transparent. Several GFF disks can be multiplexed in a rack system. After 2 to 3 hours, the vial is inverted on a 50mL tube to drain (suitably sized to keep them suspended on the tube). 4mL portions of toluene are added with a Pasteur pipette and a 2mL ball. The vial is capped and the filter is simply shaken to wash away excess reagent. The vial is drained and another 4mL portion of toluene is added / shaken / drained. Add a final 4 mL volume of toluene and soak the filter for 30 min / shake / drain. Wash off the toluene in the same manner with 3 x 4 mL of methanol and soak for a final 30 min. Drain the pan and use forceps to move the pan to a clean polypropylene tray. Place labeled bottle caps on the coated pan to prevent curling during vacuum drying. Use a rotary evaporator pump (2 to 5 mmHg) or a high vacuum pump for at least 1 h. Method A Seven punched 1 / 2 inch disks can be obtained from the 47 mm disk and stored in 20 mL glass vials.

[0305] Synthesis of PFP Ester (Pentafluorophenyl 3-[bis(4-methoxyphenyl)phenylmethoxy)propyl Succinate) Figure 2)). Mono-DMT protected 1,3-propanediol was prepared as described (Seela and Kaiser NAR (1987) 3113) or obtained from Cepheid (SO3-OH). A solution of 0.59 g (1.56 mmol) of SO3-OH, 0.44 g (4.4 mmol) of succinic anhydride and 92 mg (0.75 mmol) of 4-dimethylaminopyridine in 12.5 mL of anhydrous pyridine was stirred at room temperature under argon for 4 days. The solution was rotary evaporated to dryness and then co-evaporated with 2×50 mL of toluene. The residue was dissolved in 2.5% methanol, 2.5% triethylamine (TEA) in dichloromethane (DCM) and chromatographed on a 4×13 cm column packed with 175 mL of the same silica gel as above. After 250 mL, the product was eluted with 5% methanol, 5% TEA in DCM. After co-evaporation with 25 mL of toluene, about 200 mL of product was collected and dried on a rotary evaporator to give 0.83 g (92% yield) of succinylated SO3-OH (TEA salt) as a colorless syrup. The NMR spectrum is inconsistent with the spectrum of the present disclosure because they do not report the TEA salt (MW=580). Succinic acid (0.83 g, 1.43 mmol) was converted to PFP ester. 25 mL of DCM and 0.39 mL of TEA were added, and the septum-sealed solution was stirred in ice under argon. A solution of 0.285 mL (0.465 g, 1.66 mmol) of pentafluorophenyl trifluoroacetate (PFPTFA, Aldrich) in 2.5 mL of DCM was added dropwise with a syringe. After 1.5 hours, TLC showed no remaining succinic acid and a single, fast-moving DMT+ spot (Rf=0.6, 1:2 / ethyl acetate: hexane). Rotary evaporation gave 1.37 g of amber slurry, which was dissolved in 2 mL of ethyl acetate and diluted with 3 mL of hexane. The solution was chromatographed in a 4×13 cm column filled with 175 mL of silica gel at a filling ratio of 1:4 / E:H. After 200 mL, about 200 mL of product was collected and dried on a rotary evaporator to give 0.87 g (95% yield) of PFP ester as a colorless slurry. 1H NMR showed product (CD3CN) δ 7.45 (d, 2H), 7.34 (m, 7H), 6.88 (d, 4H), 4.23 (t, 2H), 3.78 (s, 3H), 3.14 (t, 2H), 2.95 (t, 2H), 2.66 (t, 2H), 1.90 (m, 2H). 25 mol% of ethyl acetate was present. The PFP ester was dissolved in 12.5 mL of anhydrous acetonitrile and the solution was divided into two equal parts. One portion of the PFP ester was dried in a tared flask for storage at -20°C under argon (0.41 g). The other portion was dissolved in dry DMF (6.36 mL) to give a 0.1 M solution, and then a 1 mL aliquot was pipetted into a 1.5 mL Eppendorf tube for refrigerated, dry storage.

[0306] DMT determination for measuring the density of alkylamines on GFF. Determine the expected number of GFF discs to be tested and prepare the required volume of solution B (0.3mL / disc). Solution B includes 1mL DMF, 0.4mL TEA and 0.2g DMAP. Each test disc of 1 / 2 inch diameter is placed at the bottom of a labeled 16mL vial (Chemglass, CG-4900-03, 21×70mm, 18-400 thread). 0.300mL solution B and 0.100mL PFP ester are added to each disc. The vial is vortexed briefly to remove any bubbles, and then allowed to stand for at least 1 hour. Use a Pasteur pipette and 2mL ball to remove excess reagent from each vial. Then wash each disc with 3×2mL of DMF, 3×2mL of methanol and 3×2mL of ether. Typically, before removal, allow the third wash to soak for at least 30 minutes. After the last ether wash, the vial is vacuum dried for at least 30 minutes. The dried disks are stable and can be analyzed for DMT content by adding 1.00 mL of 0.1 M p-toluenesulfonic acid in acetonitrile. The orange trityl color is immediately visible, but soak the disks for 30 minutes before measuring the absorbance. If the orange color is intense, dilute 0.100 mL with PTos / ACN at a 1:10 dilution. Use a Cary UV-vis spectrophotometer equipped with a 6-cell converter in single beam mode with a 1 mL cuvette and subtract the pTos / ACN blank baseline. Record the absorbance at 497 nm and calculate the DMT concentration of each disk by dividing by 0.076. Results are given in nmol / disc. Divide by 1.27 cm 2 / plate to obtain nmol / cm 2 The amine density is measured.

[0307] Injection filter DNA extraction and PCR assay.Improved glass fiber filter disc is placed in Cytiva Whatman injection filter holder (Cat. No. 420100).Make the solution of 1mL universal transport medium pass through modified filter disc, and described universal transport medium comprises 500 copies / every milliliter of genomic DNA from Streptococcus pyogenes.To be transferred to CepheidRCC box (with unmodified glass fiber filter with acrylic acid binding agent, 1 micron pore size, 50 mil thickness) by solution, for repurification, polymerase and oligonucleotide introduction, thermal cycle and fluorescence detection.Identical modified filter disc each additionally has the 50mM KOH solution of 1mL through them to elute any remaining DNA from filter disc.Eluent is carried out to identical processing, but in independent Cepheid RCC box, carry out.According to their cycle count (Ct) and endpoint fluorescence (EPF) value, analyze by and elution conditions relative to each other. These values ​​were analyzed relative to a control condition in which 1 mL of a solution containing 500 copies / mL of universal transport medium for genomic DNA was placed directly into one of the Cepheid RCC cassettes.

[0308] Synthesis of CL-53

[0309]

[0310] Step 1. (3-aminopropyl) triethoxysilane (10.76g, 48.61mmol) is weighed into a dry 500mL RB flask, and is placed under argon. Add 100mL anhydrous DCM, and the mixture is cooled to 0 ° C. Add maleic anhydride (4.75g, 48.4mmol), and the reaction is heated to RT, and stirred for 3.5 hours at RT. The mixture is diluted with 250mL toluene, and DCM is removed using a rotary evaporator. Add ZnCl2 (7.29g, 53.5mmol) and hexamethyldisilazane (11.10mL, 53.66mmol), and the mixture is heated at 100 ° C for 14 hours. Filter off solid and remove toluene in a vacuum, then dry overnight under high vacuum. Obtain crude compound 1 (14.22g, 97%), and use it without further purification. 1 H NMR (CDCl3, 500MHz): δ6.67 (s, 2H), 3.80 (q, J = 7.0Hz, 6H), 3.50 (t, J = 7.4Hz, 2H), 1.68 (m, 2H), 1.21 (t, J = 7.0Hz, 9H), 0.58 (m, 2H).

[0311] Step 2. Compound 1 (4.78 g, 15.9 mmol) and 2-(5-methylfuran-2-yl)ethanol (2.00 g, 15.9 mmol) were dissolved in 15 mL of reagent grade ethanol and stirred at RT for 24 h. The ethanol was then removed in vacuo with a water bath set to 30 ° C (without heating the compound). The mixture was chromatographed on silica gel (1.5"×6") using EtOH / hexane (5% to 10% ethanol), and the endo (3.25 g, 48%) isomer and exo (1.81 g, 27%) isomer were cleanly separated. Isomer distribution was performed based on the literature De Bo, G., JACS, 2017, 139, 16768-16771. Endo isomers 1 H NMR (DMSO-d6, 500MHz): δ6.31 (d, J = 5.6 Hz, 1H), 6.17 (d, J = 5.6 Hz, 1H), 4.54 (s, 1H), 3.71 (q, J = 7.0 Hz, 6H), 3.59 (t, J = 7.0 Hz, 2H), 3.41 (d, J = 7.5 Hz, 1H), 3.24 (d, J = 7.4 Hz, 1H), 3.16 (t, J = 7.4 Hz, 2H), 2.21 (m, 1H), 2.11 (m, 1H), 1.63 (s, 3H), 1.38 (m, 2H), 1.11 (t, J = 7.0 Hz, 9H), 0.45 (m, 2H). Exo-isomers 1 H NMR (DMSO-d6, 500MHz): δ6.50(d,J=5.4Hz,1H),6.34(d,J=5.4Hz,1H),4.53(s,1H),3.67(q,J=7.1Hz,6H),3.63(m,2H),3.33(t,J=6.9Hz, 2H), 2.93 (d, J = 6.3Hz, 1H), 2.85 (d, J = 6.3Hz, 1H), 2.11 (m, 1H), 1.96 (m, 1H), 1.52 (s, 3H), 1.48 (m, 2H), 1.11 (t, J = 7.1Hz, 9H), 0.46 (m, 2H).

[0312] Step 3. Under argon, in a dry RB flask, compound 2 (endo isomer, 606 mg, 1.42 mmol) was dissolved in 20 mL of anhydrous DCM. Diisopropylethylamine (500 uL, 2.26 mmol) and carbonyldiimidazole (252 mg, 1.55 mmol) were added and the reaction was stirred at RT for 24 h. Ethylenediamine (190 uL, 2.85 mmol) was added and the reactants were stirred at RT for another 2 hours. The mixture was then filtered through filter paper, diluted with DCM, and extracted with water (3 times) and brine. The filtrate was dried over Na2SO4, and the solvent was removed in vacuo to give 1.45 g (88%) of CL-53. 1 H NMR (DMSO-d6, 500MHz): δ7.09(t,J=5.5Hz,1H),6.34(d,J=5.4Hz,1H),6.22(d,J=5.6H z,1H),4.40(bs,2H),4.09(t,J=6.8Hz,2H),3.69(q,J=7.0Hz,6H),3.44(m,1H),3.25( d, J = 7.4 Hz, 1H), 3.16 (t, J = 7.4 Hz, 2H), 2.96 (q, J = 6.0 Hz, 2H), 2.54 (t, J = 6.5 Hz, 2H), 2.36 (m, 1H), 2.21 (m, 1H), 1.64 (s, 3H), 1.38 (m, 2H), 1.10 (t, J = 7.0 Hz, 9H), 0.46 (m, 2H). LCMS: found m / z 514.2 [M+H], calculated value: 513.7.

[0313] Synthesis of CL-54

[0314]

[0315] The synthesis of CL-54 followed the same procedure as CL-53, however, 2-(5-methylfuran-2-yl)methanol (1 eq) was used instead of 2-(5-methylfuran-2-yl)ethanol in step 2, and bis(3-aminopropyl)amine (3 eq) was used instead of ethylenediamine in step 3. LCMS: found m / z 571.3 [M+H], calculated 570.8.

[0316] Synthesis of CL-56

[0317]

[0318] The synthesis of CL-56 followed the same procedure as CL-53, however spermine (1 eq) was used instead of ethylenediamine in step 3. LCMS: found m / z 656.4 [M+H], calculated 655.9.

[0319] Synthesis of succinylated AP-GFF. A 47 mm diameter aminopropyl coated GFF was prepared using Method B. The disk was rolled into a 20 mL scintillation vial and treated with a solution of 0.06 g succinic anhydride and 10 mg DMAP in 2 mL anhydrous pyridine. After 2 hours, the reagent solution was removed and the disk was washed with 3×3 mL pyridine, 3×3 mL methanol, and 3×3 mL DCM. Vacuum drying for at least 30 minutes gave the succ-AP-GFF disk.

[0320] Synthesis of BisTris-succ-AP-GFF. A 47 mm diameter succinylated aminopropyl glass fiber filter (succ-AP-GFF) was coated with BisTris (MW 209). The disk was inserted into a 20 mL scintillation vial as usual and a solution of 100 mg bis-tris in 1 mL 50 mM imidazole buffer (pH 6.0) was added. A solution containing 100 mg EDC-HCl (MW 191.7) in 1 mL of the same buffer was prepared and immediately added to the succ-AP-GFF disk. The homogeneous solution was allowed to react with the disk overnight, and the filter was then washed as usual with 3×4 mL water and 3×4 mL methanol. The disk was dried under vacuum and tested for DNA binding and release in a PCR assay.

[0321] Tricine-succ-AP-GFF. A 47 mm diameter succinylated aminopropyl glass fiber filter (succ-AP-GFF) was coated with Tricine (MW 179). The procedure was the same as described above for Bis-Tris except that 50 mg of Tricine was used. The disks were dried under vacuum and tested for DNA binding and release in a PCR assay.

[0322] AP-GFF activated with cyanuric chloride (CC-AP-GFF). Aminopropyl-coated glass fiber filter (AP-GFF, 59 nmol / cm 2 ) was reacted with 2 mL of a 0.1 M cyanuric chloride (CC) solution in toluene. 37 mg (0.2 mol) CC was weighed into a 20 mL scintillation vial and 2 mL of toluene was added. The mixture was stirred until dissolved and then the disk was inserted. The vial was sealed and turned upside down to soak the disk at the bottom. After 3 hours, the solution was removed with a Pasteur pipette and washed with 3×4 mL of toluene as usual. The disk was washed with methanol and diethyl ether and then dried under vacuum for storage. A 1 / 2 inch diameter disk was punched out from the 47 mm disk of the CC-AP-GFF disk. One 1 / 2 inch circular disk was tested for residual amine (measured 3 nmol / cm 2). The second 1 / 2 inch disk was tested for amine reactivity. The disk was treated with a 1 M solution of propylenediamine (PDA) in DMF (28 μL of PDA, 0.4 mL of DMF in a 16 mL screw cap vial). After an overnight reaction, the disk was washed with DMF, methanol, and ether. The amine content of the test CC-AP-GFF disks was then measured routinely to produce (27 nmol / disc). The remaining 5 CC-AP-GFF disks were treated with bisbenzimide NH2 (BB-NH2) as described below. Unused CC-AP-GFF was stored in the refrigerator.

[0323] Cyanuric chloride activated CL53-GFF (CC-CL53-GFF). CL53-GFF was prepared using Method B and tested for amine loading (32 nmol / cm 2 ). The material was also tested in a PCR assay with a DNA extraction score of 12. A 47 mm disc of CL53-GFF alone was treated with CC / toluene for 3 hours as described above. The filters were not evaluated for amine content or reacted with PDA. Instead, the discs were used directly to immobilize the bis-benzimidazole amine as described below.

[0324] Synthesis of bis-benzimidohexylamine (BB-NH2). The synthesis of BB-NH2 followed the method of Reed et al. (US 2006 / 0166223) with the following modifications. The synthesis was started with 500 mg of Hoechst 33258, compared to the published 9.5 mg scale. Silica gel purification gave 380 mg of Boc-protected aminohexyl-BB (65% yield). 125 mg of BB-NHBoc was deprotected with 10 mL of trifluoroacetic acid and 10 mL of chloroform. After 18 hours, the solution was dried on a rotary evaporator to give 340 mg of an orange slurry. NMR showed BB-NH2, bis-TEA salt. The material was dissolved in DMF and used for the fixation reaction, using solution B to ensure the free base form of BB-NH2.

[0325] Immobilization of CC-AP-GFF by bis-benzimidyl hexylamine (BB-NH2). A 47 mm diameter disk of CC-AP-GFF was placed in a 20 mL vial. A 0.1 mM BB-NH2 solution (0.123 mmol in 1.23 mL DMF) was mixed with 1 mL of solution B (1 mL DMF, 0.4 mL TEA, 20 mg DMAP) and added to the disk. The disk was soaked overnight and then washed with 5×4 mL portions of DMF and 5×4 mL methanol. The disk was dried under vacuum and tested for DNA binding / release in a PCR assay.

[0326] BB-NH2 was fixed to CC-CL53-GFF. BB-NH2 (0.148 g, about 0.123 mmol) was dissolved in 1 mL of anhydrous DMF and mixed with 1 mL of solution B. The 47 mm disc of CC-CL53-GFF was inserted into a 20 mL scintillation vial and the BB-NH2 solution was added. The filter was soaked overnight and then washed with 5×4 mL of DMF and 5×4 mL of methanol as usual. The disc was dried under vacuum and refrigerated before testing the DNA extraction efficiency in a PCR assay.

[0327] Results and Discussion

[0328] Comparison of silanization methods for amine coated GFFs. Trialkoxysilanes can be used to coat glass surfaces using a variety of solution phase and vapor phase methods. Methods using protic solvents (Method A, ethanol) or aprotic solvents (Method B, toluene) are described herein. Method A was used to prepare GFFs coated with EDA, DETA, PEG-spermine, and imidazole. Method A was also used to coat GFFs with aminosilanes containing thermally cleavable linkers (CL53, CL54, CL55). It was found that method B gave an amine density approximately 2 times higher than method A. For example, the amine density of a Pall disk increased from 17 nmol / cm 2 (Method A) increased to 41 nmol / cm 2 (Method B) Whatman plate from 39nmol / cm 2 (Method A) increased to 88 nmol / cm 2 (Method B). The reaction of the trimethoxysilane groups in APTMS requires displacement by surface silanol groups to form siloxane bonds on the GFF surface. Without wishing to be bound by theory, it is believed that in ethanol, the solvent can reverse the reaction, while in toluene, the displaced ethanol dissolves (low concentrations). However, Method A is more convenient and easy to use with pre-punched discs, but overnight washing increases time. On the other hand, Method B is used to prepare AP-GFF discs with higher loads that require further reaction to attach DNA binding ligands (2-step method), and 0.1M APTMS (2% wt / volume) is used. Ethylenediamine (EDA) silane coating uses Sigma Aldrich, catalog number 104884 and Method A. The epoxysilane coating of GFF uses (3-glycidyloxypropyl) triethoxysilane (Sigma Aldrich, catalog number 50059) and Method B. Imidazole silane coating used N-(trimethoxysilylpropyl)imidazole (Boc Sci, catalog number 70851-51-3) and method A. PEG-spermine silane was prepared from APTES with an average PEG linker length of 45 units and immobilized using method A.

[0329] The structures, amine densities, and DNA extraction performance of some amine-coated GFFs are shown in Table 1. The method was also used to attach aminoalkylsilanes with thermally cleavable linker structures (CL) to GFFs, as described in Table 3.

[0330] Table 1. Structure, amine density, and DNA extraction performance of alkylamine-coated GFFs.

[0331]

[0332] All GFFs except APTMS were silanized using Method A. PEG spermine (average MW 2476) was immobilized at 8.3 mg / mL. Amine density was measured using the DMT assay except for the imidazole-GFFs. DNA was eluted using 50 mM KOH. Imidazole was also eluted using pH 8.5 Tris (not shown). DNA extraction score = Ct passed - Ct eluted.

[0333] Both Methods A and B avoid the 30 min high temperature (100°C) "curing" step commonly used to prepare silanized glass surfaces. The high temperature heating step is presumed to provide a more stable surface by increasing crosslinking of the multilayer silane coating, but heating can be expected to reduce the amine density on the GFF surface. Heating can convert surface ammonium groups to (less stable) free bases that can form irreversible N-oxides. The methods described herein provide good amine density and stability without this destructive heating step. The resulting "vacuum-cured" AP-GFF surface is stable when dried at room temperature. The AP-GFF filter survived a brief treatment with 50 mM KOH (pH 12.5) to elute nucleic acids, but DMT assays showed that soaking in 50 mM KOH for 30 minutes reduced the surface amine density by half (data not shown).

[0334] After validating the DMT assay with AP-GFF, the amine density of other alkylamine coated GFFs was examined. GFFs were prepared using Pall filters and Method A to compare APTMS (monoalkylamine), DETA (trialkylamine), and PEG45-spermine (trialkylamine). The coatings had the amine densities shown in Table 1. Compared to APTMS, the DETA coating had approximately 50% higher amine density (25 nmol / cm 2 to 37 nmol / cm 2 ), rather than threefold. Pentafluorophenyl (PFP) esters react only with sterically accessible amine groups on the GFF surface. Figure 4 As shown, it is believed that the polyamines electrostatically bind to the surface silanols on the GFF. Alternatively, the secondary amines in the aliphatic polyamine linkers may be too sterically hindered for the reaction of the PFP esters. Since the DMT assay showed similar amine loadings to the monoamine APTMS (20 nmol / cm 2), so the long PEG linker does not improve the accessibility of the attached spermine. EDA (dialkylamine) (Whatman GFF) was evaluated and showed an amine density of approximately 50% higher than that of APTES ( Figure 6 ).

[0335] DMT assay of alkylamine groups on GFF. Exposed amino groups on the GFF surface were reacted with pentafluorophenyl (PFP) esters containing a dimethoxytrityl (DMT) reporter group, such as Figure 2 The synthesis of PFP esters from succinic acid is described herein. (Pure, stable) PFP esters are used instead of p-nitrophenyl (PNP) esters or tetrafluorophenyl (TFP) esters.

[0336] DMT PFP ester assays were performed using 1 / 2" diameter AP-GFF disks and rapid acylation reaction conditions. Briefly, AP-GFF disks (laid flat in 16 mL vials) were treated with DMT containing 0.4 mL of PFP ester in a DMF solution containing DMAP and triethylamine. AP-GFF showed complete reaction of surface amines within 20 minutes ( Figure 3 ).

[0337] After washing away the excess PFP ester, the sterically accessible amino groups on the GFF surface are capped with DMT groups. The disk is treated with 1.00 mL of acid (0.1 M p-toluenesulfonic acid in acetonitrile) to release the orange trityl cation. The absorbance at 497 nm is measured using a spectrophotometer (a 1:10 dilution is required). The known extinction coefficient of DMT (ε 497 =76,000M -1 cm -1 ) and Beers Law were used to calculate DMT concentration. Amine density is expressed in nmol / cm 2 (Each 1 / 2 inch disk is 1.27cm 2 ) report. Leaving the PFP ester to react for a longer time did not significantly increase the amine density. Details of the DMT assay for the GFF amine density determination are given herein.

[0338] The formation of PFP ester amide bonds with AP-GFF is concentration dependent. The disclosed method for measuring surface amines in porous supports uses equal volumes of 0.1 M PNP ester and solution B (1 mL DMF, 0.4 mL triethylamine, 40 mg dimethylaminopyridine). For the DMT assay of GFF, the concentration of PFP ester was varied and the resulting DMT concentration was measured. In order to completely wet the GFF disk in a 14 mL vial, a total volume of 0.2 mL was used. Then, the solution was diluted with water and the resulting DMT concentration was measured. Figure 30.2 mL of PFP ester in DMF was added at a concentration of 2.5 μg / mL. After 2 hours of reaction, the disks were washed as usual. Two different GFF types were also compared. As expected, the lower surface area Pall AP-AP-GFF gave lower DMT loading than the thicker Whatman GFF. This experiment also showed that DMT loading increased with increasing PFP ester concentration. For the Pall disks, maximum DMT loading (36 nmol / cm2) was achieved with 0.05 M (25 mM final concentration). 2 For Whatman plates, the DMT loading was increased from 75 nmol / cm with 0.1 M PFP ester. 2 Increased to 88 nmol / cm 2 (Increase of 17%).

[0339] The sterically accessible surface amines must "protrude" from the inner layer and not be bonded to other silanol or alkoxysilane groups ( Figure 4 ). The free base form of the surface propylamine reacts rapidly with the appropriately arranged PFP ester reagent. The triethylamine in "Solution B" acts as a proton scavenger to prevent the formation of ineffective ammonium ions. DMAP (4-dimethylaminopyridine) further acts as a catalyst for amide bond formation. Anhydrous DMF (dimethylformamide) is used to prevent hydrolysis of the PFP ester reagent. In this polar, aprotic solvent, the amide bond is further driven to completion by a huge molar excess of PFP ester. For example, 1.27 cm 2 is soaked with 0.2 mL of 0.1 M PFP ester (20 μmol). 2 AP-GFF Whatman plate. Measured 88 nmol / cm 2 (112 nmol / disc) density of DMT. This is a DMT molar excess of 20,000 / 112 = 180 molar equivalents.

[0340] These chemical factors drive the PFP ester to react with all accessible amines on the GFF surface to a final concentration of 50 mM. To routinely measure the amine density of amine-coated GFFs, it is not necessary to thoroughly cover with DMT. To conserve reagents, the assay described herein uses 100 μL of 0.1 M PFP ester in DMF. 300 μL of Solution B is then added to give a final concentration of 25 mM PFP reagent. We routinely use this approach to characterize the amine density of GFFs, but expect that 17% higher amine loadings can be achieved with a 50 mM PFP ester concentration, as shown in Figure 2. Figure 3 shown.

[0341] After thorough washing and drying as described herein, the PFP ester treated AP-GFF disks are stable and can be stored in 14 mL reaction vials for future DMT assays. After soaking for 30 minutes with 1.00 mL of 0.1 M p-toluenesulfonic acid in acetonitrile (easily dispensed with a pipette), the DMT cationic color is fully formed. The absorbance of the resulting orange solution at 497 nm was measured in a quartz cuvette with a 0.1 M pTos / ACN blank. For 88 nmol / cm 2 AP-GFF plate (A 497 =0.817 au) on-scale measurement, requiring a 1:10 dilution.

[0342] DMT determination of unmodified GFF (negative control). Figure 3 As shown, the DMT level in the blank Pall disk was negligible (1.5 nmol / cm 2 ). However, the Whatman disk showed a significant DMT content (8.7 nmol / cm 2 The increased background may be due to the greater surface area of ​​the Whatman GFF, but there may be differences in the surface chemistry of the Pall GFF. The PFP esters may acylate high pKa silanols on the glass surface. These sites may react with trialkoxyaminosilanes, and thus this "background" is not subtracted from the DMT measurements reported in Table 1. When the AP-GFF disks were succinylated, the background DMT measurement dropped to 1.3 nmol / cm 2 This low background on the Whatman disks shows that the DMT assay can reliably wash out unreacted or hydrolyzed PFP esters.

[0343] DMT assay of AP-GFF disks (positive control). AP-GFF disks (49 punched 1 / 2 inch disks) were prepared using Method B. Unpunched, vacuum dried 47 mm disks were stored in brown glass jars. Punched disks were stored in 20 mL scintillation vials. Amine density was analyzed over time (Table 2). Interestingly, the first few AP-GFF amine density measurements were higher. It could be a change in the DMT assay, a change in the surface area in the disk, or an actual change in the amine density. In any case, AP-GFF was stable for at least 2 months.

[0344] Table 2. AP disk stability over time (shelf life).

[0345]

[0346] AP-GFF (Whatman) was prepared using Method B. Amine density was calculated using the DMT assay using 50 mM PFP ester. AP-GFF discs had an average of 61 (+ / - 3). Unmodified control GFF control discs had an average of 8.4 (+ / - 0.8).

[0347] DMT assay showed higher amine density in Whatman GFF coated with thermally cleavable linker (Table 3). Figure 5 As shown, the CL linker has a rigid linker structure that helps the amine "stick out" of the surface or prevents electrostatic surface binding of the aliphatic polyamine. In Table 1, Pall GFF with 1% DETA silane (Method A) gives 27 nmol / cm 2 amine density, compared to AP-GFF (method B) which gave 41 nmol / cm 2 As shown in Table 3, triamine CL-56 silane on Whatman GFF (Method A) gave a higher amine density (70 nmol / cm 2 , compared with 63 nmol / cm of AP-GFF (method B) 2 quite).

[0348] It is unclear whether the increased amine density of CL54 is due to denser packing during silanization or increased accessibility of PFP esters during the DMT assay. In any case, the density of the measured alkylamine groups is related to the spatial accessibility during DNA capture. Table 3 shows that the amine density of CL did not change much as the amount of silane increased from 0.25% to 1%. Despite the higher amine loading, the DNA extraction performance with CL54-GFF and CL-56 did not improve. These filters effectively bind DNA (high throughput Ct), but cannot fully release DNA with 50mM KOH (high elution Ct). Therefore, the DNA extraction score is lower than the APTMS control. CL53-GFF does show good DNA release at high pH. The DNA extraction performance of the cleavable linker GFF was further studied using thermal cleavage experiments as described below. The structure and synthesis of the CL linker are described herein.

[0349] Aminoalkyl coated GFF disks. The preferred size for the modified GFF material is a strip (1.6 x 11 cm). 1 / 2 inch disks or strips can be coated by soaking a free floating (shaking) portion of GFF in an ethanol silanization solution (Method A). To fit into an extraction box, 9.25 mm diameter disks can be laser cut from the GFF. For assembly lines, GFF can also be used in rolls. A 90 mm disk can be used and assembled into a 100 mm Nalgene Petri dish to produce a 30 x 1 / 2 inch diameter disk.

[0350] Table 3. Structure, amine density, and DNA extraction performance of CL silanes on GFF.

[0351]

[0352] GFF was silanized using Method A. DNA was eluted using pH 8.6 TET (Tris, EDTA, Tween-20). DNA extraction score was calculated for a 2 min elution time at 95°C (Ct passed - Ct eluted).

[0353] Two-step method for attaching DNA binding ligands to GFF. The alkylamine-coated GFF described above can be used as a starting material for attaching DNA binding ligands where silane is not present or is incompatible with the silanization process. Nucleophilic amine-coated surfaces can react with electrophilic carboxylic acid ligands to form amide bonds. This is exemplified by the DMT PFP reagent. Carboxylic acid ligands can also be activated in situ with carbodiimide coupling agents such as EDC. Surface alkylamines are easily converted to succinic acid functionalized GFF (Figure 1, step 2). Succinic acid can be activated to form amide bonds with amine-containing ligands. A method for succinylated GFF is described herein. The method successfully attaches Bis-Tris to EDC coupling chemicals. Amine-containing ligands can also be attached to GFF coated with epoxy silane. Although epoxy silane was successfully attached to GFF using method B, a more effective method for fixing DNA binding ligands was found. The examples describe AP-GFF and CL53-GFF activated with cyanuric chloride. Finally, the immobilization of a bisbenzimide (BB) minor groove binder is described.

[0354] Succinylation of AP-GFF. The conjugation chemistry of the functionalized AP-glass filter fiber (GFF) is similar to the well-known chemistry of modified aminoalkyl-coated CPG (controlled pore glass). CPG beads are commonly used for automated solid phase DNA synthesis. Many surface modifications have been attached to CPG for the introduction of functional groups at the 3'-end of the synthesized oligonucleotides. For example, the reaction conditions developed for CPG (succinic anhydride, pyridine) were used to prepare succinylated AP-GFF (succ-AP-GFF). After extensive washing and drying, the remaining amino groups of succ-AP-GFF were tested using the DMT assay. As previously described, the amine density was almost below the detection limit of the assay (2 nmol / cm 2 ). AP-GFF was also succinylated using succinic anhydride in DMF and triethylamine to drive the reaction. In either case, excess solvent or reagent was removed conventionally and dried under vacuum. These methods for preparing succ-AP-GFF are described herein. The disks were stable and easily stored after vacuum drying.

[0355] Conjugation of DNA-binding ligands to succinylated GFF. Peptide bond formation on glass supports is best performed using anhydrous organic solvents and is generally more efficient than aqueous methods because competing hydrolysis reactions are eliminated. However, some ligands are insoluble in organic solvents and require aqueous solutions. A water-soluble BisTris DNA-binding ligand was successfully conjugated to succ-AP-GFF using EDC at pH 6. Figure 7 As shown, EDC conjugation at pH 6 promotes the reaction of the hydroxyl groups of Bis-Tris to form ester bonds. The resulting BisTris-GFF is shown to capture dsDNA at pH 3 (weak), but is effectively eluted with TET at pH 8.6. Capture (pass) shows Ct 34.1. But elution also shows DNA (Ct 33.8). Both are similar to the control (Ct 32.2). Based on the results, it looks like 25% of the DNA is captured (2Ct delay) and then 25% is completely eluted (2Ct delay). The amine groups in BisTris have a pKa = 6.46, which allows DNA elution at a lower pH.

[0356] Minor Groove Binders (MGBs) on GFFs. Bisbenzimide (BB) molecules are well-known fluorescent DNA-binding molecules. Also known as Hoechst dyes, they are used to stain DNA in cells for fluorescence microscopy. BB dyes have been shown to bind tightly in the minor groove of dsDNA. To explore this novel approach to capture DNA, hexylamine-modified BB dye (BB-NH2) was prepared and an efficient method for its immobilization to AP-GFFs was developed ( Figure 8 ).

[0357] Epoxide coated GFF. Two methods were used to immobilize the amine-containing ligands. First, the previously described epoxide coating was applied to the GFF (EP-GFF). The presence of reactive epoxides on the surface was demonstrated by further treatment with propylenediamine (PDA). The conjugated propylamine groups were measured using the DMT assay and gave only 15 nmol / cm after 22 hours of reaction with PDA. 2 The amine density of the EP-GFF was 1.34 Å (Table 4). The ring was opened with PDA during the reaction of the epoxide group with the primary amine ligand to generate secondary amines and secondary alcohols in the linker structure. Despite the low surface density, the EP-GFF was treated with BB-NH2 overnight and tested for amine density and DNA extraction performance. The secondary amine has a pKa of about 10 and is acylated by PFP esters in the DMT assay (12 nmol / cm 2 ). The poor DNA extraction performance of BB-EP-GFF may be due to this low density.

[0358] Cyanuric chloride coated GFF. Cyanuric chloride is a simple linker that can be used to couple two molecules containing alkylamines. The first alkylamine reacts rapidly to displace the single Cl on the trichlorotriazine ring. The resulting dichlorotriazine (see Figure 8 ) is deactivated but reacts slowly with a second alkylamine to displace the second Cl. The resulting monochlorotriazine is non-reactive. Cyanuric acid-coated GFF (CC-AP-GFF) was prepared by treating AP-GFF with 0.1 M CC in toluene for 3 h. Similar to silanization method B, the toluene solution of CC was rinsed off and the CC-AP-GFF was washed with toluene, methanol, DCM, and dried under vacuum. As described for epoxide-GFF, CC-AP-GFF was further characterized by testing its amine reactivity with propylenediamine. Surprisingly, the cyanuric chloride-activated surface gave good amine density (Table 4, 27 nmol / cm 2 ). Next, CC-AP-GFF was treated with 1 mL of 0.1 M BB-NH2 in DMF and 1 mL of solution B overnight (Method B). CC was allowed to react overnight, and BB-CC-AP-GFF was washed and dried as usual. 1 / 2 inch diameter disks were tested for amine content. As expected, the amine density of BB-coated GFF was close to background levels (8 nmol / cm 2 ), indicating that PFP ester did not react with the immobilized BB.

[0359] The DNA extraction performance of the BB-CC-AP-GFF disks was tested in a PCR assay. The extraction score was X, indicating good DNA binding but no DNA release. The rigid triazine linker structure can provide increased accessibility of the BB ligand to dsDNA, thereby improving DNA binding performance relative to the aliphatic EP-GFF linker. Surprisingly, 50 mM KOH was not sufficient to release the captured dsDNA from the minor groove binding BB surface. It is possible that the unreacted CC groups remain on the surface and covalently capture DNA (compare DNA extraction of freshly prepared CC-AP-GFF, Solution B treated CC-AP-GFF, and BB-CC-AP-GFF). It is also possible that the BB surface density and DNA binding affinity are too high (try more dilute BB-NH2). Capping the unreacted chloro groups on CC-AP-GFF with aminoethanol can also help improve the release of captured DNA from the CC-AP surface. In summary, CC-coated GFFs provide a new method to immobilize amine-containing ligands to aminoalkylated glass supports.

[0360] Table 4. Amine density of coated GFF

[0361]

[0362] All disks are Whatman disks. EP: epoxide silanized using method B. CC: AP-GFF activated with cyanuric chloride. PDA: propylenediamine reaction. Amine density was measured using the DMT assay. Elution for PCR assays used 50 mM KOH. DNA extraction score = (CT passed - CT eluted). * Elution for PCR assays used TET buffer, pH 8.6, heated at 95°C for 10 minutes.

[0363] Thermally cleavable linkers for releasing bisbenzimide captured dsDNA. The thermally cleavable linkers described above (Table 3) allow for the release of strongly bound DNA. Fig. 9 As shown, the linker is cleaved by a reverse Diels-Alder reaction.

[0364] GFF was silanized with a thermally cleavable alkylamine linker (CL53) to yield CL53-GFF (Whatman filter, Method B). The amine density (32 nmol / cm 2 ), and DNA capture performance was measured using PCR assay (extraction score = 12). Figure 7 ), CL53-GFF was further activated with cyanuric chloride and conjugated with BB-NH2 to yield BB-CC-CL53-GFF. As demonstrated by the non-cleavable BB-CC-AP-GFF, the minor groove-binding bisbenzimide ligand efficiently captured double-stranded DNA. However, the cleavable CL53 allowed thermal release of the DNA (extraction score = 10), while the AP linker did not (extraction score = X). Upon heating, the CL53 linker decyclized to cleave the BB ligand and the glass fiber surface. The maleimide-containing fragment remained attached to the glass surface. The cleaved fluorescent Hoechst dye (and the attached furan-containing fragment) remained bound to the minor groove of the released dsDNA, and the complex dissolved into solution. This novel DNA extraction method allows the concentration of the eluted BB-labeled DNA to be determined by liquid phase fluorescence measurements. The fluorescent properties of the released BB dye are similar to those of Hoechst 33342 (ε 343 (MeOH)=47mM -1 cm -1 , Ex = 361 nm, Em = 486 nm), and there are several references using Hoechst 33342 to measure the dsDNA concentration of biological solutions.

[0365] Stoichiometry of amine density and dsDNA binding capacity. The dsDNA (plasmid) binding capacity of unmodified GFF disks (30 mm diameter) from bacterial lysates using 2 M guanidine hydrochloride has been reported to be 30 μg / cm 2GFFs of the same porosity were coated with DNA binding ligands, but DNA and RNA binding capacities were determined without modifying the GFFs. For similar dsDNA binding capacities, 50 nmol / cm 2 The amine density represents the binding ratio of 1 alkylammonium ion / DNA base pair. MGB-type ligands should require lower densities because a single BB molecule binds a 5bp DNA fragment with high affinity. It is believed that the alkylamine, imidazole, Bis-Tris or BB-coated GFFs described herein can separate NAs from complex biological samples. These new GFFs would be valuable if cell lysis could be simplified (no GuHCl), processing time could be shortened, or NA quality could be improved (fewer PCR inhibitors, more specific hybridization).

[0366] Evaluation of hgDNA from plasma on APTES-modified GFF

[0367] The ability of modified glass fiber filters combined with APTES reagent to bind and recover hgDNA from human plasma samples was evaluated. Human plasma was treated with a 1 / 10 dilution of proteinase K (approximately 2 mg / ml) and incubated at room temperature for 15 minutes. The sample was then treated with a CTNG assay bead set containing an oligonucleotide set for a sample adequacy control (SAC) for hgDNA. One milliliter of this plasma and proteinase K sample was processed by the CTNG assay. The assay used 4.5M guanidine thiocyanate buffer in combination with polyethylene glycol for DNA purification and used a glass fiber filter that was not combined with APTES reagent. One milliliter of this sample was also processed using a procedure specifically developed for modified filters without polyethylene glycol. The results are provided in the table below.

[0368] Table 5: GFF modification and hgDNA in human plasma samples (SAC signal from hgDNA in human plasma samples)

[0369]

[0370] Evaluation of hgDNA from plasma on APTES-modified GFF

[0371] Aminopropyltriethoxysilane (APTES) was mixed with a glass fiber filter to chemically modify the glass surface by silanization. The glass fiber was rinsed with 200 proof ethanol, dried, and then built into the modified The modified cartridge was used in the following experiments.

[0372] In the first experiment, a solution of 50mM Tris, 0.1mM EDTA and 0.1% Tween 20 was used in the modified filter box in two chambers. In particular, 250 μL of solution was placed in chamber A together with 1 μg of hgDNA, and the mixture was then passed through a modified filter. In another chamber B, the solution (without hgDNA) was mixed with 20 μL of diluted picogreen dye and filtered hgDNA solution, and then entered into a PCR tube for fluorescence reading. When hgDNA was placed in chamber B instead of chamber A, a standard curve was generated based on fluorescence reading. By performing this conversion, the standard curve conditions of hgDNA were not passed through the modified filter. Therefore, the difference in hgDNA readings was due to the capture of hgDNA on the filter surface by the APTES-modified filter. The results are provided in the table below.

[0373] Table 6: RNA samples evaluated in GeneXpert

[0374] 1 μg hgDNA in 250 μL processed in a modified sample cartridge % of 1μg DNA Unmodified GFF 77％ > 50% GF AP-GFF (volume method) 2％ > 50% GF APTES (hanging strip method) 1％

[0375] In the second experiment, a solution of 50mM Tris, 0.1mM EDTA and 0.1% Tween 20 was used in the modified filter box in two chambers. In particular, 250 μL of solution was placed in chamber A together with 10 μg hgDNA, and the mixture was then passed through the modified filter. In another chamber B, the solution (without hgDNA) was mixed with 20 μL of diluted picogreen dye and filtered hgDNA solution, and then entered the PCR tube for fluorescence reading. When hgDNA was placed in chamber B instead of chamber A, a standard curve was generated based on fluorescence reading. By performing this conversion, the standard curve conditions of hgDNA were not passed through the modified filter. Therefore, the difference in hgDNA readings is due to the capture of hgDNA on the filter surface by the APTES-modified filter. The results are provided in the table below.

[0376] Table 7: RNA samples evaluated in GeneXpert

[0377] 10 μg DNA in 250 μL processed in a modified sample cartridge % of 10 μg DNA Unmodified GFF 63％ > 50% GF AP-GFF (volume method) 2％ > 50% GF APTES (hanging strip method) 2％

[0378] In the third experiment, a solution of 50mM Tris, 0.1mM EDTA and 0.1% Tween 20 was used in the modified filter box in two chambers. In particular, 250 μL of solution was placed in chamber A together with 1 μg of rRNA, and the mixture was then passed through a modified filter. In another chamber B, the solution (rRNA-free) was mixed with 20 μL of diluted picogreen dye and filtered rRNA solution, and then entered into a PCR tube for fluorescence reading. When rRNA was placed in chamber B instead of chamber A, a standard curve was generated based on fluorescence reading. By performing this conversion, the standard curve conditions for rRNA were not passed through the modified filter. Therefore, the difference in rRNA readings was due to the capture of rRNA on the filter surface by the APTES-modified filter. The results are provided in the table below.

[0379] Table 8: RNA samples evaluated in GeneXpert

[0380]

[0381] Chemical vapor deposition (CVD) method is used to make modified glass fiber. GFF sheet is suspended in vacuum oven. The whole cleaning, dehydration and deposition process is carried out at 150 ℃. In short, the glass fiber surface is first plasma cleaned. This surface cleaning is followed by dehydration removal to remove the remaining water from the surface. Then aminosilane (e.g., APTES) is introduced into a sealed chamber, and the pressure of the deposition chamber is increased to 2 to 3 Torr. The reaction time of the surface with the gas phase adsorbate is 5min. After deposition, three cleaning cycles are carried out, which consist of adding N2 gas and then evacuating. These cleaning cycles are carried out for safety reasons and to improve the quality of deposition. They are used to remove the remaining silane from the chamber before the chamber is opened, and they help to remove any unreacted silane from the surface of the substrate. Fig.12 CVD modified sheets (CVD 1 to 4) are shown to have better or equal performance than the ethanol soak method (85A) or the control.

[0382] Functional testing of amine-modified cassettes using Covid Plus and CTNG assays. The ability of modified glass fiber filters combined with APTES reagents to process and evaluate Covid-19 targets in artificial clinical nasopharyngeal swab matrix assays and CTNG targets in Zeptometrix matrix was evaluated. Boxes with modified glass fiber filters were constructed: Box 116A had 2% AP-GFF pre-cut strips; Box 116B had 2% AP-GFF pre-cut strips; Box 116C had 2% AP-GFF pre-cut pieces + separated with a glass stirring rod; and Box 116D had 2% AP-GFF pre-cut pieces. In these Covid-19 and CTNG assays, 300μl of artificial samples were treated with 4.5MGTC lysis buffer, 50mM tris, 0.1mM EDTA, 0.1% Tween 20 wash buffer reagent, and 40mM KOH elution reagent at pH 12. The elution buffer was neutralized with 400mM Tris HCl beads. The results are provided in the table below.

[0383] Table 9: GFF Modified and Covid Plus Samples in Clinical Nasopharyngeal Swab Matrix

[0384]

[0385] Table 10: GFF-modified and CTNG samples in clinical matrices

[0386]

[0387]

[0388] Functional testing of modified glass fiber filters and removal of chaotropic lysis buffer. The ability of the modified glass fiber filters to purify DNA and RNA is advantageous with respect to the lysis solution used. The lysis solution does not need to contain high salt content, nor does it need to contain polyethylene glycol or ethanol. The data below show successful purification of viral RNA using modified glass fiber filters without the addition of chaotropic salts or polyethylene glycol. In particular, when the viral particles were in a solution containing 2% Brij58 detergent, the viral particles were easily lysed by the detergent solubilization, and the RNA was freely bound to the modified glass fiber.

[0389] Table 11: GFF modified and Covid Plus samples in the absence and presence of chaotropic agents

[0390]

[0391] The use of modified glass fibers allows for the processing of higher volumes as well as the reduction of chaotropes and binding agents as shown above in Tables 9 to 11. Overall, these advantages provide sample preparation time (SP time) advantages for DNA / RNA purification due to the use of fewer reagents.

[0392] The results shown above indicate that the amine-modified glass fiber filter cartridge performs comparable or better than another cartridge type (e.g., RCC-corrected cartridge C, see, e.g., WO2021263101A1 and WO2015013676A1, which utilize RCC cartridges to process SARS-CoV2 and CTNG samples, respectively). The simplified reagent chemistry provided by the amine-modified surface allows for faster time to results, elimination of several types of chemicals in sample preparation, and RT-PCR assays with higher sensitivity due to reduced reagent carryover.

[0393] Evaluation of hgDNA and CT / NG from human urine on APTES-modified GFF.

[0394] The ability of modified glass fiber filters combined with APTES reagent to bind and recover hgDNA and CT / NG from human urine samples was evaluated. The samples were treated with a CTNG assay bead set containing an oligonucleotide set for hgDNA, Chlamydia trachomatis, and Neisseria gonorrhoeae sample adequacy controls (SACs). One milliliter of the sample was treated using a 4.5M guanidine thiocyanate buffer in combination with polyethylene glycol for DNA purification and an unmodified glass fiber filter (not combined with APTES reagent). One milliliter of the sample was also treated with a procedure specifically formulated for modified filters without polyethylene glycol. The results are provided in Tables 12 and 13 below.

[0395] Table 12: GFF modified and CTNG samples

[0396]

[0397] Table 13: GFF modified and CTNG samples

[0398]

[0399] Capture efficiency of modified glass fiber filters for large plasma samples.

[0400] Further experiments were carried out to extract cell-free DNA from EDTA plasma. For the revised box C scheme, 1 mL of plasma was mixed with 100 μl of proteinase K (about 20 mg / ml, Roche). The mixture was vortexed for 10 to 15 seconds and incubated for 1 min at RT. 2 ml of lysis reagent was added, further vortexed for 10 to 15 seconds and incubated for 10 min at RT. The resulting 4.5 ml lysate was loaded onto the GX instrument, and the remaining steps of DNA purification were carried out using standard reagents from Cepheid commercial assays. For the modified glass fiber filter box, 2 ml of plasma was mixed with 100 μl of proteinase K (about 20 mg / ml, Roche). The mixture was vortexed for 10 to 15 seconds and incubated for 10 min at RT. 2 ml of lysis reagent (5 M GuHCl, 1% Tween-20 and 0.0075% SE-15 defoamer) was added, further vortexed for 10 to 15 seconds and incubated for 10 min at RT. The resulting approximately 4 ml lysate was loaded onto the GX instrument, and the remaining steps of DNA purification were performed using standard reagents from Cepheid commercial assays. The DNA extracted from 1 ml plasma (RCC) or 2 ml plasma (ModGFF) was measured using digital PCR ESR1_E380Q mutation detection assay. As shown below, compared to using correction box C to process 1 ml plasma, when ModGFF was used to process 2 ml plasma, the amount of WT and mutant alleles representing cell-free DNA was detected to be about 2 times higher. An exception is the ModGFF-1 box, which is due to changes in processing efficiency.

[0401] Table 14: GFF Modification and Cell-free DNA Samples

[0402] Sample Box target ddPCR readings AVG (cpn / ul) Dilution factor Elution volume (ul) Number of copies per cassette ModGFF-1 E380Q 53.4 2.2 65.0 7710 ModGFF-2 E380Q 83.5 2.2 65.0 12065 ModGFF-4 E380Q 80.8 2.2 65.0 11669 ModGFF-5 E380Q 72.4 2.2 65.0 10454 ESR1 RCC-1 E380Q 22.0 2.2 70.0 3429 ESR1 RCC-2 E380Q 28.5 2.2 70.0 4436 ESR1 RCC-3 E380Q 15.9 2.2 70.0 2478 ModGFF-1 WT 26.7 2.2 65.0 3856 ModGFF-2 WT 40.6 2.2 65.0 5869 ModGFF-4 WT 39.7 2.2 65.0 5741 ModGFF-5 WT 35.3 2.2 65.0 5103 ESR1 RCC-1 WT 14.8 2.2 70.0 2305 ESR1 RCC-1 WT 17.9 2.2 70.0 2778 ESR1 RCC-1 WT 12.4 2.2 70.0 1929

[0403] Capture efficiency of modified GFF for targets from more than 3 mL of whole blood.

[0404] Genomic streptococcal DNA was spiked into fresh blood to a final concentration of 500 copies / mL. The blood was then mixed in a ratio of 1 part blood: 0.5 part enzymatic reagent (proteinase K): 0.91 part chaotropic agent (5M GuHCl), and allowed to stand at room temperature for 20 minutes to form a sample lysate. The sample lysate was then passed through a 10 micron polypropylene disk of 25 mm diameter using a 12 mL syringe. The sample lysate was then loaded into the two chambers of the box (with a total volume of 6.32 mL) and then filtered through the 2%-APTES GFF in the cartridge. By processing the sample lysate of over 6 ml (6.32 mL), over 3 mL (3.225 mL) of blood was processed through a single filter. The discarded sample was then collected and loaded into the two chambers of the second box, which was then run again through a modified GFF. The filters of the two boxes were rinsed with TET, and eluted using high pH KOH, and then PCR was performed. For the Ct results of the two boxes, see Table 15.

[0405] Table 15: Capture efficiency of modified glass fiber filters for large volume blood samples

[0406] Streptococcus A Ct Streptococcus A EPF Box Sample 28.425 573.25 By Sample 33.9 371.25 Delta: +5.475 -202

[0407] The Ct obtained from each box can be compared. The Ct from the first box corresponds to the efficiency of capturing the target DNA from the blood and then releasing it into the PCR by the modified GFF. The Ct from the second box provides details about how many copies of the target DNA can bypass the capture on the initial filter and can be considered as a "pass" result. Using the approximate value between the Streptococcus A copy number and the measured Ct based on the LOD study from the commercial Streptococcus A assay, the delay between the initial modified GFF and the second modified GFF>5Ct, indicating that compared with the PCR run of the eluate from the first filter, only about 2% of the Streptococcus DNA copies are present in the PCR (or, compared with the second modified GFF, 40x of Streptococcus DNA is effectively captured on the initial modified GFF). This shows that the modified GFF is efficient when extracting pathogen DNA from large volumes of human genomic DNA.

[0408] All publications, patents, patent applications, and other references cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was individually indicated to be incorporated by reference for all purposes.

[0409] While various specific embodiments have been shown and described, it will be appreciated that changes can be made without departing from the spirit and scope of the invention.

Claims

1. A separation material for nucleic acid separation, comprising: A glass fiber solid support comprising borosilicate glass and a compound bonded to the glass fiber solid support, the compound being derived from a structure represented by the following formula: Y-(L) y -SiX3 in, Y is a DNA binding ligand selected from an alkylamine, a cycloalkylamine, an alkoxyamine, a polyamine moiety, an arylamine, an intercalator, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof, L is a linker selected from an alkyl group, a heteroalkyl group, an olefin group, a heteroolefin group, a polyacrylic acid, a Diels-Alder adduct, or a combination thereof, Each X is independently selected at each occurrence from a hydrolyzable group, an alkyl group, a heteroalkyl group, an alkenyl group, or two or three Xs are combined to form one or more cyclic groups, or one X is combined with Y to form a cyclic azasilane, and y is either 0 or 1.

2. A separation material for nucleic acid separation, comprising: a glass fiber solid support comprising a Diels-Alder adduct with a DNA binding ligand, cyanuric chloride, or a combination thereof, wherein the adduct or cyanuric chloride is chemically bonded to the glass fiber solid support, optionally via a linker.

3. The separation material of claim 2, wherein the DNA binding ligand comprises an amine group, an intercalator, a minor groove binder, a peptide, an amino acid, a protein or a combination thereof, preferably comprises an alkylamine, a cycloalkylamine, an alkoxyamine, a polyamine moiety, an arylamine, an intercalator, a DNA groove binder, a peptide, an amino acid, a protein or a combination thereof.

4. The separation material of any one of claims 1 to 3, wherein the DNA binding ligand comprises an alkylamine group, an imidazole group, a bisbenzimide minor groove binder, or a combination thereof.

5. The separation material of any one of claims 1 to 4, wherein the DNA binding ligand is selected from spermine, methylamine, ethylamine, propylamine, ethylenediamine, diethylenetriamine, 1,3-dimethyldipropyldiamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium hydrochloride, tris(2-aminoethyl)amine or a combination thereof.

6. The separation material of any one of claims 1 to 5, wherein the Diels-Alder adduct is derived from an unsaturated cyclic imine group.

7. The separation material according to any one of claims 1 to 6, wherein the Diels-Alder adduct is derived from a structure represented by the following general formula, an isomer, a salt, a tautomer or a combination thereof, wherein Y' is the DNA binding ligand, and L, X and y are as defined in claim 1.

8. The separation material of any one of claims 1 to 7, wherein the linker, when present, is selected from an alkylene oxide group, an alkylene group, cyanuric chloride, an alkylamine, or a combination thereof.

9. The separation material according to any one of claims 1 to 8, wherein at least two X are independently selected from halogen, alkoxy, dialkylamino, triflate, or are combined with the Si atom to which they are attached to form a silatricycle, a cyclic siloxane, a polysilsesquioxane or a silazane, preferably wherein at least two X are independently selected from an alkoxy group (e.g. ethoxy or methoxy).

10. The separation material according to any one of claims 1 to 9, wherein the compound or the Diels-Alder adduct is derived from one of the following structures: 3-aminopropyltrimethoxysilane, aminoalkylsilazotricyclo, 3-(2-aminoethyl)aminopropyltriethoxysilane, 3-(2-aminoethyl)aminopropyltrimethoxysilane, or a combination thereof, and wherein n is an integer from 0 to 10, from 1 to 10, or from 1 to 5.

11. The separation material of any one of claims 1 to 10, wherein the compound is derived from 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, or a combination thereof.

12. The separation material according to any one of claims 1 to 11, wherein the glass fiber solid support has a molecular weight of 10 nmol / cm 2 or greater, 20nmol / cm 2 or greater, 35nmol / cm 2 or greater, or 30 nmol / cm 2 Up to 100nmol / cm 2 The surface density of the compound or Diels Alder adduct.

13. The separation material of any one of claims 1 to 12, wherein the glass fiber solid support has a pH of at least 10 μg / cm 2 , 20μg / cm 2 or greater, 35 μg / cm 2 or greater, or 30 μg / cm 2 Up to 100 μg / cm 2 DNA binding capacity.

14. The separation material of any one of claims 1 to 13, wherein the glass fiber solid support has a pore size of 0.2 μm to 3 μm, 0.2 μm to 2 μm, 0.5 μm to 1.0 μm, or 0.6 μm to 0.8 μm.

15. The separation material of any one of claims 1 to 14, wherein the glass fiber solid support comprises beads that facilitate mechanical lysis, wherein the beads are selected from glass beads, silica beads, or a combination thereof.

16. The separation material according to any one of claims 1 to 15, wherein the glass fiber solid support has a thickness of 35 g / m 2 Up to 100g / m 2 , preferably 50g / m 2 Up to 85g / m 2 , or 70g / m 2 Up to 85g / m 2 The basis weight.

17. The separation material of any one of claims 1 to 16, wherein the glass fiber solid support has a fiber diameter of 1 μm to 100 μm, preferably 1 μm to 50 μm, or 1 μm to 25 μm.

18. The separation material of any one of claims 1 to 17, wherein the glass fiber solid support has a thickness of 250 μm to 2,000 μm, 300 μm to 1,500 μm, 300 μm to 1,000 μm, 300 μm to 750 μm, or 350 μm to 500 μm.

19. A method for isolating nucleic acid from a biological sample, the method comprising: (a) contacting the nucleic acid with the separation material of any one of claims 1 to 16; and (b) eluting the nucleic acid from the separation material.

20. A method for isolating nucleic acid from a biological sample, the method comprising: (a) contacting the nucleic acid with a composition comprising a Diels-Alder adduct, the Diels-Alder adduct comprising a DNA binding ligand, and (b) concentrating the nucleic acid onto a solid support, wherein the Diels-Alder adduct is optionally chemically bonded to the solid support.

21. The method of claim 20, wherein the solid support comprises a material selected from the group consisting of silica, glass, vinyl backbone polymers, mica, polycarbonate, zeolite, titanium dioxide, magnetic beads, glass beads, cellulose filters, polycarbonate filters, polytetrafluoroethylene filters, polyvinyl pyrrolidone filters, polyethersulfone filters, glass fiber filters, or combinations thereof, preferably wherein the solid support is a glass fiber filter.

22. The method of any one of claims 19 to 21, wherein the biological sample is blood, plasma, serum, semen, cerebrospinal fluid, tissue, tears, urine, feces, saliva, a smear specimen, a respiratory sample, a nasopharyngeal sample, a vaginal swab, a vaginal mucus sample, a vaginal tissue sample, a vaginal cell sample, a bacterial culture, a mammalian cell culture, a viral culture, a human cell, bacteria, extracellular fluid, pancreatic juice, a cell lysate, a PCR reaction mixture or an in vitro nucleic acid modification reaction mixture, preferably wherein the biological sample is blood, plasma, a respiratory sample or a vaginal swab.

23. The method of any one of claims 19 to 22, wherein the biological sample comprises a nucleic acid selected from genomic DNA, total RNA, short DNA, small DNA, tumor-derived nucleic acid, methylated DNA, microbial nucleic acid, bacterial nucleic acid, viral nucleic acid, cell-free nucleic acid, or a combination thereof.

24. The method of any one of claims 19 to 23, wherein before step a) or simultaneously with step a), the biological sample is contacted with a lysis buffer comprising one or more of a chaotropic agent, a salt, a buffer, a surfactant, a defoaming agent, a binding agent, or a combination thereof.

25. The method of claim 24, wherein in the method the chaotropic agent is used at a concentration of less than 4.5M, less than 2M or less than 1M in the lysis buffer.

26. The method of any one of claims 19 to 24, wherein the method does not use a chaotropic agent or a lysis buffer.

27. The method of any one of claims 19 to 26, wherein the nucleic acid is contacted with a binding agent, a filtering agent, a washing agent, or a combination thereof while concentrating or before eluting the nucleic acid.

28. The method of claim 27, wherein the filtering and / or washing reagents comprise a binding agent selected from polyalkylene oxides (eg PEG 200) or salts.

29. The method of any one of claims 27 to 28, wherein the binding agent is used in a concentration of less than 40% v / v, less than 30% v / v, less than 20% v / v or less than 10% v / v in the filter and / or the detergent.

30. The method of any one of claims 19 to 26, wherein the method does not use a binding agent.

31. The method of any one of claims 19 to 30, wherein the method further comprises eluting the nucleic acid with an eluent, preferably wherein the eluent has a pH greater than about 7, greater than about 8, greater than about 9, or wherein elution comprises heating the nucleic acid to a temperature of 100°C or less, 95°C or less, 85°C or less, 75°C or less, 65°C or less, 55°C or less in the presence of an eluent; sonicating the nucleic acid; photochemically cleaving the compound / composition; or a combination thereof.

32. The method of any one of claims 19 to 31, wherein the method further comprises eluting the nucleic acid with an eluent having a pH greater than about 10.

33. The method of any one of claims 19 to 32, wherein the method comprises eluting the nucleic acid with an eluent having a pH of about 10 to about 13.

34. The method of any one of claims 19 to 33, wherein the method further comprises neutralization with a buffer.

35. The method of any one of claims 19 to 34, wherein the method comprises neutralization with Tris HCl.

36. The method of any one of claims 19 to 35, wherein the method is performed in a cassette, preferably in an automated cassette.

37. A method for detecting nucleic acid in a biological sample, comprising: (a) isolating the nucleic acid from the biological sample using the method of any one of claims 19 to 36; (b) eluting the nucleic acid from the solid support with an eluent; and (c) detecting the nucleic acid.

38. A sample kit for separating and detecting nucleic acids from a biological sample, comprising: a cartridge body having a plurality of chambers defined therein, wherein the plurality of chambers are in fluid communication via a fluid path of the cartridge, and wherein at least one chamber is configured to receive the biological sample, a reaction vessel configured to amplify the nucleic acid by thermal cycling, and a filter disposed in the fluid path between the plurality of chambers and the reaction vessel, wherein the filter comprises a separation material according to any one of claims 1 to 18, The plurality of chambers and the reaction container independently include reagents for releasing nucleic acids from the biological sample and primers and probes for detecting the nucleic acids.

39. A sample kit for separating and detecting nucleic acids from a biological sample, comprising: A cartridge having a plurality of chambers therein, wherein the plurality of chambers comprises: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality of chambers; a lysis chamber in fluid communication with the sample chamber, the lysis chamber comprising reagents for releasing nucleic acids, optionally wherein the sample chamber and the lysis chamber are one and the same; a reaction vessel fluidly connected to the plurality of chambers of the cartridge and configured to ii) amplify nucleic acid and detect a plurality of amplification products; a filter disposed in a fluid path between the lysis chamber and the reaction vessel, wherein the filter comprises a solid support modified with a DNA binding ligand selected from the group consisting of an alkylamine, a cycloalkylamine, an alkoxyamine, a polyamine moiety, an arylamine, an intercalator, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof, and A plurality of primers and / or probes are disposed in one or more chambers or reaction vessels among the plurality of chambers for detecting the nucleic acid.

40. A sample box as described in claim 39, wherein the solid support comprises a material selected from silica, glass, vinyl skeleton polymer, mica, polycarbonate, zeolite, titanium dioxide, magnetic beads, glass beads, cellulose filters, polycarbonate filters, polytetrafluoroethylene filters, polyvinyl pyrrolidone filters, polyether sulfone filters, glass fiber filters or combinations thereof.

41. The sample cartridge of any one of claims 39 to 40, wherein the lysis chamber comprises: Valve body and bonnet, wherein the valve body is connected to the valve cap to define the cracking chamber therebetween, and The filter is held in a cracking chamber fixed between the valve body and the valve cap.

42. The cartridge of any one of claims 38 to 41, wherein the biological sample has a volume of up to 10 mL, preferably 300 μL to 10 mL, 500 μL to 5 mL or 1 mL to 5 mL.

43. A sample box as described in any one of claims 39 to 42, wherein the lysis chamber has a fluid flow path between the inlet in the cap and the outlet in the valve body, and the fluid flow path is fluidly connected to the fluid displacement area of ​​the valve body, wherein the fluid displacement area can be depressurized by movement of the syringe to introduce the fluid into the fluid displacement area, and can be pressurized by movement of the syringe to discharge the fluid from the fluid displacement area.

44. The sample cartridge of any one of claims 38 to 43, wherein the sample cartridge together with the reagents allows a flow rate of up to about 100 μL / sec, such as about 10 μL / sec to about 100 μL / sec.

45. The sample cartridge of any one of claims 38 to 44, wherein the sample cartridge together with the reagents allows pressures below 100 psi, below 80 psi, or below 60 psi.

46. ​​A sample box as described in any one of claims 38 to 45, wherein the box is an automated box.

47. A method for detecting nucleic acid in a biological sample obtained from a subject, the method comprising: a) contacting nucleic acid from the biological sample with a primer set and optional probe in a sample kit according to any one of claims 38 to 46; b) subjecting the nucleic acid, primer pair, and optional probe to amplification conditions; c) detecting the presence of the amplification product, optionally by real-time PCR, melting curve analysis, or a combination thereof, and d) detecting the presence of the nucleic acid in the biological sample based on detecting the amplification product.

48. The method of claim 47, wherein: a) contacting nucleic acid from the sample with a primer set and optionally a probe in a sample kit comprises: placing the biological sample in the cartridge, the cartridge comprising a cartridge body having a plurality of chambers in fluid communication, a reaction vessel having one or more reaction chambers and configured to amplify the nucleic acid, a fluid path between the plurality of chambers and the reaction vessel, and a filter in the fluid path; and If the biological sample comprises cells, lysing cells in the biological sample with one or more lysing reagents present in at least one chamber of the plurality of chambers; b) said subjecting the nucleic acid, primer pair, and optional probe to amplification conditions comprises amplifying said nucleic acid with primers and probe present in solution in at least one of said plurality of chambers; and c) said subjecting the nucleic acid, primer pair, and optional probe to amplification conditions comprises amplifying said nucleic acid with primers and probe present in solution in at least one of said plurality of chambers.

49. The method of any one of claims 47 to 48, wherein the biological sample is contacted with a lysis reagent comprising one or more of a chaotropic agent, a salt, a buffer, a surfactant, a defoaming agent, a binding agent, a precipitating agent, or a combination thereof.

50. The method of claim 49, wherein the chaotropic agent is used at a concentration of less than 2.0 mol / mL in the lysis buffer.

51. The method of any one of claims 47 to 49, wherein the method does not comprise the use of a chaotropic agent or a lysis buffer.

52. The method of any one of claims 47 to 51, wherein the method further comprises contacting the nucleic acid with a binding agent, a filtering agent and / or washing to promote binding of the nucleic acid to the filter while removing non-target material.

53. The method of claim 52, wherein the binding agent comprises PEG and is used at a concentration of less than 30% v / v in the filter and / or the detergent.

54. The method of any one of claims 47 to 52, wherein the method does not use a binding agent.

55. The method of any one of claims 47 to 54, wherein amplification is performed by nested PCR, gradient PCR, isothermal PCR, qPCR or RT-PCR.

56. The method of any one of claims 47 to 55, wherein amplification is performed by a real-time PCR multiplex assay.

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