Inactivated virus preservation solution, preparation method and purification device

Abstract

The application discloses an inactivated virus storage solution, a preparation method and a purification device, wherein the raw materials of the inactivated virus storage solution comprise a pH buffer, a non-ionic surfactant, a reducing agent, a free metal ion chelating agent, a solid-supported chelating agent and water; the solid-supported chelating agent contains DTPA functionalized resin; and the concentration of each raw material in water is as follows: the pH buffer is 0.5 mM to 2.0 mM; the non-ionic surfactant is 0.002% (v / v) to 0.02% (v / v); the reducing agent is 0.2 mM to 1.0 mM; the free metal ion chelating agent is 0.2 mM to 1.0 mM; and the DTPA functionalized resin is 5% (w / v) to 40% (w / v); and the DTPA functionalized resin is prepared by maleimide monoamide-DTPA and activated thiol resin. The application can completely inactivate viruses at normal temperature to make the viruses lose infectivity and ensure biological safety; meanwhile, the application can maintain the complete structure of virus particles and does not release nucleic acid; and the inactivated virus storage solution removes metal ions and inhibits nucleases by adding the functionalized resin as the solid-supported chelating agent.

Description

Technical Field

[0001] This invention relates to the fields of virus detection and molecular diagnostics, and in particular to an inactivated virus preservation solution, its preparation method, and its purification apparatus. Background Technology

[0002] In fields such as virus detection and molecular diagnostics, post-collection sample preservation and pretreatment are crucial. The main function of sample preservation solutions is to maintain sample stability and prevent nucleic acid degradation during the period from sample collection to testing, while ensuring biosafety during transportation and handling. Nucleic acid extraction and purification are key steps in releasing nucleic acids from preserved samples and removing PCR inhibitors. Serum and plasma are the most common sample types in molecular diagnostics, widely used for nucleic acid detection of pathogens such as viral hepatitis (HBV, HCV), HIV, and dengue fever. However, serum / plasma samples contain high concentrations of proteins (albumin, immunoglobulins) and metal ions (Ni²⁺). + Co² + Cu² + Zn² + Pb² + Cr³ + Zn² + Ca² + Fe³ +Viruses are major inhibitors of PCR reactions, and effectively removing these inhibitors while simplifying procedures is a key technical challenge in this field. Currently, sample preservation methods are mainly divided into two categories based on the method of virus treatment: non-inactivated and inactivated. 1. Non-inactivated preservation solutions are typically based on cell culture media (such as those with added serum, antibiotics, BSA, etc.) to maintain viral activity and structural integrity, supporting subsequent virus isolation and culture, vaccine development, and sequencing research. Its biggest advantage is the preservation of the virus's infectivity, making it suitable for live virus-related research. However, its drawbacks are also significant: the samples are infectious, and in locations such as primary clinics and customs ports where strict biosafety laboratories (such as P2+ laboratories) are lacking, opening the sample can generate aerosols, easily leading to operator infection and laboratory contamination, which is strictly prohibited in on-site testing. Furthermore, non-inactivated samples require cold chain transportation (4℃ or -20℃), which is costly and cumbersome, limiting its application in resource-constrained scenarios. 2. Existing inactivated preservation solutions inactivate viruses under strongly alkaline conditions (pH>11) or with high concentrations of chemical lysis agents (such as high concentrations of guanidine salts) to ensure biosafety during transportation and handling. However, their core drawback is that inactivation also destroys the intact structure of the virus particles, causing nucleic acids to be prematurely exposed to nucleases in the preservation solution, affecting the long-term stability of the nucleic acids. Although some products attempt to alleviate this problem by adding nuclease inhibitors or stabilizers, these components are costly, require stringent storage conditions, have limited inhibitory effects on certain resistant nucleases, and still pose a risk of degradation under high temperature or long-term storage conditions.

[0003] Currently, mainstream nucleic acid extraction methods include column extraction, magnetic bead extraction, direct amplification / extraction-free methods, and functionalized resin-assisted extraction. Column extraction utilizes the principle of binding nucleic acids under high-salt conditions and eluting under low-salt conditions using a silica membrane. It requires multiple centrifugation operations, is time-consuming, and relies on instruments such as centrifuges. While this method yields high purity and good nucleic acid quality, it is complex (typically requiring 6-10 steps including lysis, binding, washing, and elution), takes 30-60 minutes, and requires equipment such as centrifuges and vortex mixers, making it unsuitable for molecular point-of-care testing (POCT) and basic field testing. Magnetic bead extraction utilizes surface-modified magnetic beads to bind nucleic acids under high-salt conditions, achieving purification through magnetic separation. It requires a nucleic acid extractor or magnetic rack. This method can be automated, but the instruments are expensive and the equipment is bulky, making it difficult to meet the needs of home self-testing and field testing. Direct amplification / extraction-free methods involve directly lysing samples with chemical lysis buffers (such as those containing surfactants, chelating agents, and release salts), and then using the supernatant for amplification. While simple, these methods lack purification capabilities, as PCR inhibitors (such as proteins, heme, and humic acids) from the sample enter the reaction system, affecting detection sensitivity and accuracy. For serum / plasma samples, high concentrations of albumin and immunoglobulins significantly inhibit PCR. Functionalized resin-assisted extraction, such as Chelex-100 resin, inhibits nuclease activity by chelating metal ions. However, Chelex-100 consists of free resin particles, requiring sedimentation after use, which is cumbersome and cannot specifically remove immunoglobulin-based PCR inhibitors. Furthermore, the functional group IDA in Chelex-100 resin is a tridentate ligand, which has limited chelation stability with metal ions, potentially leading to the re-release of metal ions under high-temperature conditions.

[0004] Current preservation solutions have the following shortcomings: 1. Biosafety risks of non-inactivated preservation solutions: Non-inactivated preservation solutions maintain viral activity, making samples infectious. In locations such as primary clinics and customs ports that lack strict biosafety laboratories (such as P2+ laboratories), opening the sample can generate aerosols, easily leading to operator infection and laboratory contamination, which is strictly prohibited in on-site testing. Furthermore, non-inactivated samples require cold chain transportation (4℃ or -20℃), which is costly and cumbersome. 2. Structural damage issues with inactivated preservation solutions: Most existing inactivated preservation solutions use strongly alkaline or high-concentration chemical lysis agents. While these can inactivate the virus and ensure transportation safety, they also damage the integrity of the viral particle structure, causing premature exposure of nucleic acids to nucleases in the preservation solution, affecting long-term stability.

[0005] Current nucleic acid extraction technologies suffer from the following shortcomings: 1. Complex operation and instrument dependence: While column-based and magnetic bead methods offer high purity, they involve numerous steps (lysis, binding, washing, elution) requiring equipment such as centrifuges and extraction instruments, making them unsuitable for molecular point-of-care testing (POCT) and scenarios like primary care clinics and border crossings. 2. Weak anti-interference capability of direct amplification: Existing extraction-free release reagents simplify the operation, but their ability to remove PCR inhibitors in complex samples (such as serum, plasma, and feces) is limited, leading to decreased detection sensitivity and increased false negative rates. 3. Instability of nucleic acids after lysis: Existing direct amplification / extraction-free reagents directly and irreversibly lyse viral envelopes and protein coats using high-concentration surfactants or dissociative salts, releasing both nucleic acids and endogenous nucleases from the sample. Although nuclease inhibitors or stabilizers can be added subsequently, these components are costly, require stringent storage conditions, and have limited inhibitory effects on certain resistant nucleases, resulting in gradual degradation of nucleic acids at room temperature. 4. Limitations of metal ion inhibitor treatment: Some release agents use free chelating agents (such as EDTA and DTPA) to remove metal ions to inhibit nucleases, but these free chelating agents will enter the PCR system along with the template, competingly chelating the Mg²⁺ required for Taq enzyme. + Cofactors affect amplification efficiency. Serum / plasma samples themselves contain high concentrations of Ca²⁺. + and Fe³ + This problem is particularly prominent. 5. Lack of in-situ removal methods for protein inhibitors: Existing direct amplification release agents have poor removal capabilities for protein inhibitors (such as albumin, hemoglobin, and immunoglobulins). Proteins not only inhibit PCR themselves, but can also interfere with amplification by binding to nucleic acids or adsorbing Taq enzymes. 6. Insufficient integrated application of functionalized resins in the field of nucleic acid extraction. Immobilized metal affinity chromatography (IMAC) is a commonly used technique for protein purification, with IDA (iminodiacetic acid) and NTA (nitrotriacetic acid) as the mainstream ligands. By immobilizing Ni² + Co² + Histidine-tag (His-tag) fusion protein was purified by metal ion purification.

[0006] Therefore, in view of the shortcomings of existing technologies, there is an urgent need for an inactivated virus preservation solution, preparation method and purification device that can completely inactivate the virus at room temperature and maintain the integrity of the virus particle structure to make up for the deficiencies of existing technologies. Summary of the Invention

[0007] The first objective of this invention is to overcome at least one of the shortcomings of the prior art and provide an inactivated virus preservation solution. This inactivated virus preservation solution can completely inactivate viruses at room temperature, eliminate the risk of biotransmission, and ensure biosafety, while simultaneously preserving the complete structure of the virus particles and preventing premature release of nucleic acids. It also maintains the integrity of the virus particle structure, preventing the release of nucleic acids, which are tightly encapsulated within a protein shell or envelope, physically isolating nucleases in the preservation solution. Furthermore, this inactivated virus preservation solution incorporates functionalized resin as a chelating agent to remove metal ions that inhibit nucleases.

[0008] The above-mentioned objectives of the present invention are achieved through the following technical measures: A virus inactivation preservation solution is provided, the raw materials of which include a pH buffer, a nonionic surfactant, a reducing agent, a free metal ion chelating agent, a loading chelating agent, and water; the loading chelating agent contains DTPA functionalized resin.

[0009] The concentrations of each raw material in water are as follows: pH buffer: 0.5mM~2.0mM; Nonionic surfactants: 0.002% (v / v) to 0.02% (v / v); Reducing agent: 0.2mM~1.0mM; Free metal ion chelating agent: 0.2mM~1.0mM; DTPA functionalized resin: 5% (w / v) to 40% (w / v).

[0010] The DTPA functionalized resin was prepared by reacting maleimide monoamide-DTPA with an activated thiol resin.

[0011] The immobilized chelating agent also contains at least one of EGTA functionalized resin and bifunctional phosphonic acid functionalized resin.

[0012] When the raw material contains EGTA functionalized resin, the concentration of EGTA functionalized resin is 1% (w / v) to 20% (w / v).

[0013] When the raw material contains difunctional phosphonic acid functionalized resin, the concentration of the difunctional phosphonic acid functionalized resin is 1% (w / v) to 20% (w / v).

[0014] The EGTA functionalized resin was prepared by ring-opening reaction of epoxy-activated agarose resin with ethylene glycol diethyl ether diaminetetraacetic acid (EGTA).

[0015] The preparation method of the bifunctional phosphonic acid functionalized resin is as follows: Step C1: Sulfonate polystyrene-divinylbenzene macroporous resin beads with sulfonating reagent to obtain sulfonated resin; Step C2: The sulfonated resin obtained in step C1 is subjected to a chloromethylation reaction using a chloromethylation reagent to obtain a chloromethylated sulfonated resin; Step C3: The chloromethylated sulfonated resin obtained in step C2 is reacted with trialkyl phosphite via an Arbuzov reaction to obtain a resin intermediate containing methyl phosphonate groups. Step C4: Hydrolyze the resin intermediate containing methyl phosphonate groups obtained in step C3 to obtain a bifunctional phosphonic acid functionalized resin containing sulfonic acid groups and phosphonic acid groups.

[0016] Furthermore, the concentrations of each raw material in water are as follows: pH buffer: 1.0 mM; Nonionic surfactants: 0.01% (v / v) to 0.013% (v / v); Reducing agent: 0.5mM; Free metal ion chelating agent: 0.5mM; DTPA functionalized resin: 20% (w / v) to 30% (w / v).

[0017] When the raw material contains EGTA functionalized resin, the concentration of EGTA functionalized resin is 5% (w / v) to 15% (w / v).

[0018] When the raw material contains a difunctional phosphonic acid functionalized resin, the concentration of the difunctional phosphonic acid functionalized resin is 5% (w / v) to 15% (w / v).

[0019] Preferably, the preparation method of the above-mentioned DTPA functionalized resin is carried out by the following steps: Step A1: Dissolve maleimide monoamide-DTPA in coupling buffer to obtain DTPA ligand solution, wherein the coupling buffer is PBS buffer containing Tween-20 and EDTA. Step A2: React the activated thiol resin with the DTPA ligand solution obtained in step A1 to obtain reactant A; Step A3: Add blocking solution A to reactant A from step A2 to react and obtain the DTPA functionalized resin. The blocking solution A contains L-cysteine ​​in PBS buffer.

[0020] Preferably, the preparation method of the above-mentioned DTPA functionalized resin is carried out by the following steps: Step A1: Dissolve maleimide monoamide-DTPA in a coupling buffer to obtain a DTPA ligand solution. The concentration of the DTPA ligand solution is 2 mg / mL to 10 mg / mL. The coupling buffer is 50 mM PBS buffer with 0.05% Tween-20 and 0.05 mM EDTA added, and the pH of the coupling buffer is 7.2. Step A2: React the activated thiol resin with 5 times the volume of the ligand solution obtained in step A1 at room temperature for 3 hours. After the reaction is completed, reactant A is obtained. Step A3: Add blocking solution A to reactant A in step A2 and react for 1 hour to obtain the DTPA functionalized resin. The blocking solution A is prepared by adding 50 mM L-cysteine ​​solid dissolved in 50 mM PBS buffer, and the pH value of the PBS buffer is 7.2. The weight ratio of reactant A to blocking solution A is 1:1.

[0021] Preferably, the above-mentioned EGTA functionalized resin is prepared by the following steps: Step B1: Dissolve EGTA in ethylene glycol diethyl ether diaminetetraacetic acid in an alkaline coupling buffer to obtain an EGTA ligand solution; the alkaline coupling buffer is a Na2CO3-NaHCO3 buffer containing NaCl. Step B2: React the epoxy-activated agarose resin with the EGTA ligand solution obtained in step B1 to obtain reactant B.

[0022] Step B3: React reactant B obtained in step B2 with blocking solution B to obtain EGTA functionalized resin; the blocking solution is a Na2CO3-NaHCO3 buffer solution containing ethanolamine.

[0023] Preferably, the above-mentioned EGTA functionalized resin is prepared by the following steps: Step B1: Dissolve EGTA (ethylene glycol diethyl ether diaminetetraacetic acid) in an alkaline coupling buffer to obtain an EGTA ligand solution; the concentration of the EGTA ligand solution is 20 mg / mL to 50 mg / mL; the alkaline coupling buffer is 0.5 M NaCl added to a 0.1 M Na2CO3-NaHCO3 buffer, and the pH value of the alkaline coupling buffer is 9.5. Step B2: React epoxy-activated agarose resin with 5 times the volume of the EGTA ligand solution obtained in step B1 in a 40°C water bath shaker for 20 hours to obtain reactant B; Step B3: The reactant B obtained in step B2 is sealed with blocking solution B at 40°C for 4 hours to obtain EGTA functionalized resin; the blocking solution B is 1M ethanolamine dissolved in 0.1M Na2CO3-NaHCO3 buffer solution, and the pH value of the blocking solution B is 9.5, and the weight ratio of reactant B to blocking solution B is 1:1.

[0024] Preferably, the preparation method of the above-mentioned bifunctional phosphonic acid functionalized resin is as follows: Step C1: Sulfonate polystyrene-divinylbenzene macroporous resin beads with sulfonating reagent, wash and dry to obtain sulfonated resin; Step C2: Swelling agent and chloromethylating agent are added to the sulfonated resin obtained in step C1 to swell it, and then a catalyst is added to carry out the chloromethylation reaction of the sulfonated resin obtained in step C1 to obtain chloromethylated sulfonated resin. Step C3: The chloromethylated sulfonated resin obtained in step C2 is reacted with trialkyl phosphite under heating in an inert solvent to carry out the Arbuzov reaction, thereby obtaining a resin intermediate containing methyl phosphonate groups. Step C4: The resin intermediate containing methyl phosphonate groups obtained in step C3 is subjected to acidic or alkaline hydrolysis to obtain a bifunctional phosphonic acid functionalized resin containing sulfonic acid groups and phosphonic acid groups.

[0025] Preferably, the preparation method of the above-mentioned bifunctional phosphonic acid functionalized resin is as follows: Step C1: Sulfonate polystyrene-divinylbenzene macroporous resin beads at 80℃~150℃ for 2h~8h using sulfonating reagent, then wash and dry to obtain sulfonated resin. Step C2: Swelling agent and chloromethylating agent are added to the sulfonated resin obtained in step C1 to swell it, and then a catalyst is added to carry out chloromethylation reaction of the sulfonated resin obtained in step C1 to obtain chloromethylated sulfonated resin. The swelling agent is dichloromethane or tetrachloroethane. Step C3: The chloromethylated sulfonated resin obtained in step C2 is reacted with trialkyl phosphite at 100℃~160℃ in an inert solvent for 8h~48h to obtain a resin intermediate containing methyl phosphonate groups. Step C4: The resin intermediate containing methyl phosphonate groups obtained in step C3 is subjected to acidic or alkaline hydrolysis to obtain a bifunctional phosphonic acid functionalized resin containing sulfonic acid groups and phosphonic acid groups.

[0026] Preferably, the pH buffer is a Tris-HCl buffer; Preferably, the nonionic surfactant is a mixture of Tween 80 and Triton X-100, and the volume ratio of Tween 80 to Triton X-100 is 1:(0.3~1).

[0027] Preferably, the reducing agent is at least one of dithiothreitol (DTT), β-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP).

[0028] Preferably, the free metal ion chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA), diethyl ether diaminetetraacetic acid (EGTA), and diethylenetriaminepentaacetic acid (DTPA).

[0029] Preferably, the sulfonating agent is at least one of concentrated sulfuric acid, fuming sulfuric acid, chlorosulfonic acid, or sulfur trioxide.

[0030] Preferably, the chloromethylating agent is at least one of chloromethyl methyl ether or 1,4-dichloromethoxybutane.

[0031] Preferably, the catalyst is at least one of anhydrous zinc chloride, anhydrous tin tetrachloride, or anhydrous ferric chloride.

[0032] Preferably, the above-mentioned trialkyl phosphite is at least one of trimethyl phosphite, triethyl phosphite, or tripropyl phosphite.

[0033] Preferably, the inert solvent is at least one of N,N-dimethylformamide, toluene, xylene, or dioxane.

[0034] A second objective of this invention is to overcome at least one of the shortcomings of the prior art by providing a method for preparing an inactivated virus preservation solution. This method involves mixing the raw materials to obtain the inactivated virus preservation solution.

[0035] The above-mentioned objectives of the present invention are achieved through the following technical measures: A method for preparing the inactivated virus preservation solution as described above is provided, comprising mixing a pH buffer, a nonionic surfactant, a reducing agent, a free metal ion chelating agent and a functionalized resin, and then adding purified water to the target volume to obtain the inactivated virus preservation solution.

[0036] A third objective of this invention is to overcome at least one of the shortcomings of the prior art by providing a purification apparatus. This purification apparatus can effectively filter and remove functionalized resin particles from the inactivated virus preservation solution and immunoglobulins from the sample.

[0037] The above-mentioned objectives of the present invention are achieved through the following technical measures: A purification apparatus is provided, comprising: The bottle contains the inactivated virus preservation solution described above; A bottle cap, which is detachably fitted to the open end of the bottle body; A composite filter element is used to trap cell debris and blood clots in the sample, trap the immobilized chelating agent in the inactivated virus preservation solution, and adsorb immunoglobulins in the sample. The composite filter element is sealed and assembled at the outlet end of the bottle cap. A dust cover, which is detachably fitted onto the outside of the bottle cap.

[0038] The bottle cap includes a cap body and a dropper. The cap body and the dropper are integrally connected. The cap body is detachably assembled to the outlet end of the bottle body. The dropper is provided with a liquid flow channel. The dust cover is detachably fitted onto the outside of the dropper.

[0039] The composite filter element is disposed between the cover body and the drip tip. The composite filter element includes a pre-filtration layer, an immunoglobulin adsorption layer and a support layer. The pre-filtration layer, the immunoglobulin adsorption layer and the support layer are stacked in sequence, and the support layer is close to the liquid flow channel.

[0040] The pre-filter layer is made of polyethylene, polypropylene, or ultra-high molecular weight polyethylene (UHMWPE); the immunoglobulin adsorption layer is made of at least one of Protein A resin, Protein G resin, or Protein L resin; and the support layer is made of polyethylene, polypropylene, or ultra-high molecular weight polyethylene (UHMWPE).

[0041] The beneficial effects of this invention are: 1. This inactivated virus preservation solution utilizes the synergistic effect of nonionic surfactants and reducing agents to completely inactivate viruses at room temperature, rendering them non-infectious and ensuring biosafety, while maintaining the integrity of the virus particle structure and preventing the release of nucleic acids. The viral nucleic acid is tightly encapsulated within a protein coat, physically isolating nucleases in the preservation solution, achieving long-term stable preservation without the need for expensive nuclease inhibitors. This resolves the technical contradiction of existing preservation solutions where "inactivation and structural integrity are mutually exclusive." The nonionic surfactants act gently on the virus, achieving inactivation without disintegrating the particles, while the reducing agent disrupts disulfide bonds in the viral structural proteins. The synergistic effect of both enhances the inactivation effect, but because the concentration is controlled at a low level, it does not cause complete disintegration of the virus particles. 2. This inactivated virus preservation solution uses functionalized resin as an immobilization chelating agent to remove metal ions (calcium, magnesium, iron, lead, copper, manganese, cadmium, mercury), inhibiting nucleases. Furthermore, the chelates are retained by the filter cartridge and do not enter the PCR system, thus not competing with the Mg²⁺ chelate for the Taq polymerase active site. 2+ ; 3. The inactivated virus preservation solution contains a low concentration of free metal ion chelating agents, which are used to rapidly inhibit nuclease activity and prevent nucleic acid degradation in the early stages of sample mixing. This concentration is much lower than the amount of EDTA used in conventional lysis buffer (usually greater than 1 mM), and its inhibitory effect on Taq enzyme is negligible and does not affect amplification efficiency; 4. Applicable to processing various sample types such as serum, plasma, urine, feces (pre-processing), cervical secretions, and sputum (liquefaction); 5. This purification device is equipped with a composite filter element, specifically consisting of a pre-filtration layer, an immunoglobulin adsorption layer, and a support layer. The immunoglobulin adsorption layer can efficiently adsorb immunoglobulins such as IgG, IgA, and IgM in serum and plasma, addressing the core issues of protein binding to nucleic acids and Taq enzyme inhibition. Combined with the pre-filtration layer and support layer, it physically traps blood clots, cell debris, and functionalized resin particles, achieving simultaneous removal of large particles, metal ions, and protein inhibitors. 6. The sample undergoes pretreatment steps such as inactivation, metal ion removal, immunoglobulin adsorption, and resin retention in this purification device. The detection equipment only needs to retain heating, quantitative sample addition, and amplification detection functions, eliminating the need for a complex nucleic acid extraction module. This purification device relies on dropper squeezing for filtration, eliminating the need for pipettes, centrifuges, and magnetic bead extractors. Only the lysis step requires a simple metal bath, completely eliminating the limitations of large equipment required by traditional column and magnetic bead methods. This purification device eliminates the need for repeated opening and closing of the cap for pipetting, and when paired with a matching automated PCR device, it enables a fully enclosed sample input and result output process, with no open nucleic acid transfer operations. It is suitable for use in primary healthcare institutions, customs, ports, entry-exit quarantine, field emergency disease control testing, and on-site disease control testing. Attached Figure Description

[0042] The invention will be further described with reference to the accompanying drawings, but the contents of the drawings do not constitute any limitation on the invention.

[0043] Figure 1 This is a schematic diagram of the purification device.

[0044] Figure 2 for Figure 1 The cross-sectional view along the "AA" direction.

[0045] Figure 3 The amplification curve is shown in Example 5.

[0046] exist Figures 1 to 3 Including: Bottle body 100 Bottle cap 200, cap body 210, dropper 220 Composite filter element 300, pre-filtration layer 310, immunoglobulin adsorption layer 320, support layer 330 Dust cover 400. Detailed Implementation

[0047] The technical solution of the present invention will be further described with reference to the following embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the raw materials and reagents used in the following embodiments can be purchased from conventional biochemical reagent stores or pharmaceutical companies.

[0048] Example 1

[0049] An inactivated virus preservation solution comprises a pH buffer, a nonionic surfactant, a reducing agent, a free metal ion chelating agent, a loading chelating agent, and water; wherein the loading chelating agent contains at least one of DTPA functionalized resin, EGTA functionalized resin, and bifunctional phosphonic acid functionalized resin.

[0050] The nonionic surfactant is a mixture of Tween 80 and Triton X-100, with a volume ratio of 1:(0.3~1). The concentration of the nonionic surfactant in the virus inactivation preservation solution is the sum of the volume fraction concentrations of Tween 80 and Triton X-100. The pH buffer is Tris-HCl buffer; the reducing agent is at least one of dithiothreitol (DTT), β-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP). The free metal ion chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA), glycol diethyl ether diaminetetraacetic acid (EGTA), and diethylenetriaminepentaacetic acid (DTPA).

[0051] The nonionic surfactant inactivates the virus at room temperature (making it non-infectious) while maintaining the integrity of the virus particle structure, without damaging the viral envelope or protein coat, ensuring that nucleic acid is not released. The pH buffer is used to maintain the pH stability of the preservation solution, providing a suitable alkaline environment for virus inactivation. The reducing agent of this invention is used to assist in virus inactivation by disrupting disulfide bonds in the viral envelope, thereby enhancing the inactivation effect.

[0052] It should be noted that functionalized resins, acting as immobilized metal chelating agents, involve immobilizing DTPA, EGTA, and bifunctional phosphonic acids onto the resin. These immobilized metal chelating agents are used for both specific and non-specific chelation of metal ions (such as Ca²⁺) in samples. + Fe³ + Pb² + Cu² + Mn² + Cd² + Hg² +(etc.) to inhibit nuclease activity. Unlike free metal ion chelators, the immobilized chelator of this invention is covalently linked to the resin particles. The complex formed after chelating metal ions can be retained by the external filter and will not enter the final PCR reaction system, thus avoiding competition between these metal ions and the enzyme active site, thereby inhibiting the PCR reaction. The inactivated virus preservation solution of this invention contains both a free metal ion chelator and an immobilized metal chelator (at least one of DTPA functionalized resin, EGTA functionalized resin, or bifunctional phosphonic acid functionalized resin). These two are not redundant or contradictory, but rather have a clear division of labor and synergistic effect.

[0053] The role of the free metal ion chelating agent is immediate protection. In the initial stage of sample and preservation solution mixing (0-30 seconds), the free metal ion chelating agent can rapidly bind with free metal ions (such as Fe³⁺) in the sample. + Al³ + Cr³ + Co³ + Ca² + Mg² + The free metal ion chelating agent binds to the resin particles, immediately inhibiting nuclease activity and preventing nucleic acid degradation before extraction and purification. This rapid reaction, completing chelation within seconds, is a significant advantage over immobilized metal chelating agents. The immobilized chelating resin, on the other hand, provides deep removal and retention. In subsequent adsorption stages (e.g., after sample settling for 1-10 minutes), the immobilized chelating resin further removes any remaining metal ions from the sample. Because the immobilized chelating agent is covalently linked to the resin particles, the chelated complex is effectively retained by the composite filter element (especially the pre-filter layer) of this invention and will not enter the final PCR reaction system.

[0054] The concentrations of each raw material in water are as follows: pH buffer: 0.5mM~2.0mM; Nonionic surfactants: 0.002% (v / v) to 0.02% (v / v); Reducing agent: 0.2mM~1.0mM; Free metal ion chelating agent: 0.2mM~1.0mM; DTPA functionalized resin: 5% (w / v) to 40% (w / v); When the raw material contains EGTA-functionalized resin, the concentration of EGTA-functionalized resin is 1% (w / v) to 20% (w / v). When the raw material contains difunctional phosphonic acid-functionalized resin, the concentration of difunctional phosphonic acid-functionalized resin is 1% (w / v) to 20% (w / v).

[0055] It should be noted that the amount of free metal ion chelating agent in the inactivated virus preservation solution of this invention is low, far lower than the EDTA concentration in conventional lysis buffers (typically 2-5 mM). At this concentration, the inhibitory effect on Taq enzyme is negligible and does not affect amplification efficiency.

[0056] The DTPA functionalized resin is prepared by reacting maleimide monoamide-DTPA with an activated thiol resin. The specific preparation method of the DTPA functionalized resin comprises the following steps: Step A1: Dissolve maleimide monoamide-DTPA in coupling buffer to obtain DTPA ligand solution, wherein the coupling buffer is PBS buffer containing Tween-20 and EDTA. Step A2: React the activated thiol resin with the DTPA ligand solution obtained in step A1 to obtain reactant A; Step A3: Add blocking solution A to reactant A from step A2 to react and obtain the DTPA functionalized resin. The blocking solution A contains L-cysteine ​​in PBS buffer.

[0057] It should be noted that DTPA, or diethylenetriaminepentaacetic acid, is a potent aminopolycarboxylic acid chelating agent. Its molecular structure contains multiple coordinating atoms (nitrogen and oxygen atoms), enabling it to form stable water-soluble complexes with various metal ions. DTPA rapidly forms water-soluble complexes with ions such as calcium, magnesium, iron, lead, copper, manganese, cadmium, and mercury. Unlike existing technologies such as IDA (iminodiacetic acid) and NTA (nitrotriacetic acid), DTPA is an octadentate ligand, which is too stable when binding to metal ions, hindering competitive elution of His-tag proteins. Therefore, DTPA is not a mainstream choice for immobilized metal affinity chromatography (IMAC). However, this invention covalently links DTPA to resin particles, allowing the chelated complex to be effectively retained by the composite filter element of this invention, preventing it from entering the final PCR reaction system. This enables DTPA to be well-suited for use in immobilized metal affinity chromatography (IMAC).

[0058] The activated thiol resin of this invention refers to a solid-phase support on which reactive thiol groups (-SH) are introduced into the surface or matrix through chemical modification. The substrate can be agarose (such as Sepharose), polystyrene, cellulose, polyacrylamide, etc. These thiol groups can undergo efficient and specific addition reactions with maleimide groups under mild conditions to form stable thioether bonds. This invention does not limit the specific type of activated thiol resin used, as long as its surface contains reactive thiol groups. This invention uses Activated Thiol Sepharose 4B as the activated thiol resin, and polystyrene, agarose, or cellulose resins with surface-modified thiol groups. Specifically, the activated thiol resin used in this invention is Activated Thiol Sepharose 4B, purchased from Beijing Weishi Bohui Chromatography Technology Co., Ltd. Maleimide monoamide-DTPA was purchased from Shanghai Jinpan Biotechnology Co., Ltd., with CAS number 2032239-75-9.

[0059] The coupling buffer of this invention is defined according to the type of coupling reaction. For maleimide-thiol reactions, the coupling buffer is a neutral or weakly acidic buffer, including but not limited to: 10 mM-100 mM phosphate buffer (PBS buffer), 10 mM-100 mM Tris-HCl buffer, or 10 mM-100 mM acetate buffer, with a pH range of 6.0-7.5. Preferably, the coupling buffer is a 50 mM phosphate buffer (pH 7.2) containing 0.05% Tween-20 and 0.05 mM EDTA. The addition of EDTA is used to chelate any trace metal ions that may be present, preventing them from catalyzing the oxidation of thiol groups.

[0060] EGTA-functionalized resins are prepared by a ring-opening reaction between epoxy-activated agarose resin and ethylene glycol diethyl ether diaminetetraacetic acid (EGTA). Specifically, the EGTA-functionalized resins are prepared through the following steps: Step B1: Dissolve EGTA in ethylene glycol diethyl ether diaminetetraacetic acid in an alkaline coupling buffer to obtain an EGTA ligand solution; the alkaline coupling buffer is a Na2CO3-NaHCO3 buffer containing NaCl. Step B2: React the epoxy-activated agarose resin with the EGTA ligand solution obtained in step B1 to obtain reactant B; Step B3: React reactant B obtained in step B2 with blocking solution B to obtain EGTA functionalized resin; the blocking solution is a Na2CO3-NaHCO3 buffer solution containing ethanolamine.

[0061] The alkaline coupling buffer includes 0.05-0.2M Na2CO3-NaHCO3 buffer or phosphate buffer, with a pH range of 9.0-11.0, and optionally contains 0.5M-1.0M NaCl to improve coupling efficiency.

[0062] The epoxy-activated agarose resin of this invention refers to a solid-phase support on which epoxy groups (-CH(O)CH2) are introduced onto the surface of agarose gel through chemical methods. Under alkaline conditions, the epoxy groups can undergo ring-opening reactions with various nucleophilic groups (such as amino, hydroxyl, and thiol groups) to form stable covalent bonds. The "resin" used to prepare the difunctional phosphonic acid functionalized resin refers to a polymer resin as the matrix material, specifically a polystyrene-divinylbenzene (PS-DVB) macroporous resin. This resin has a porous structure, high specific surface area, and good mechanical stability, providing sufficient sites for subsequent functionalization modification. This invention does not limit the specific type, degree of crosslinking (DVB content), and particle size of the PS-DVB macroporous resin used, but preferably a macroporous PS-DVB resin with a crosslinking degree of 6%-8%. Specifically, this invention uses epoxy-activated Sepharose 6B as the epoxy-activated agarose resin, as well as similar epoxy-activated agarose and polyacrylamide resins. Specifically, the epoxy-activated agarose resin used in this invention is Epoxy-activated Sepharose 6B, which was purchased from Beijing Weishi Bohui Chromatography Technology Co., Ltd.

[0063] For the ring-opening reaction of EGTA's epoxy groups, the alkaline coupling buffer comprises (0.05-0.2) M Na₂CO₃-NaHCO₃ buffer or (0.05-0.2) M phosphate buffer, with a pH range of 9.0-11.0. Preferably, the alkaline coupling buffer is a 0.1 M Na₂CO₃-NaHCO₃ buffer (pH 9.5) containing 0.5 M NaCl. A high concentration of NaCl helps reduce nonspecific adsorption and improves coupling efficiency.

[0064] The preparation method of the bifunctional phosphonic acid functionalized resin is as follows: Step C1: Sulfonate polystyrene-divinylbenzene macroporous resin beads with sulfonating reagent to obtain sulfonated resin; Step C2: The sulfonated resin obtained in step C1 is subjected to a chloromethylation reaction using a chloromethylation reagent to obtain a chloromethylated sulfonated resin; Step C3: The chloromethylated sulfonated resin obtained in step C2 is reacted with trialkyl phosphite via an Arbuzov reaction to obtain a resin intermediate containing methyl phosphonate groups. Step C4: Hydrolyze the resin intermediate containing methyl phosphonate groups obtained in step C3 to obtain a bifunctional phosphonic acid functionalized resin containing sulfonic acid groups and phosphonic acid groups.

[0065] The polystyrene-divinylbenzene macroporous resin beads were purchased from Tianjin Nankai Hecheng Technology Co., Ltd., model D101.

[0066] It should be noted that the sulfonation reaction of this invention refers to the chemical reaction of introducing a sulfonic acid group (-SO3H) onto the aromatic ring of the polymer. The sulfonating agents used in this invention include, but are not limited to, at least one of concentrated sulfuric acid (H2SO4, concentration ≥98%), fuming sulfuric acid (sulfuric acid containing free SO3), chlorosulfonic acid (HSO3Cl), or sulfur trioxide (SO3). The sulfonation reaction can be carried out under solvent-free conditions, or inert solvents such as dichloroethane or tetrachloroethane can be used as swelling agents. The degree of sulfonation can be adjusted by controlling the reaction temperature, time, and amount of sulfonating agent. The sulfonated resin needs to be repeatedly washed with deionized water until neutral to remove free sulfonating agents.

[0067] The chloromethylation reaction of this invention refers to the chemical reaction of introducing a chloromethyl group (-CH2Cl) onto an aromatic ring. This reaction is typically carried out under Lewis acid catalysis. To avoid using highly toxic chloromethyl methyl ether, this invention preferably uses the safer 1,4-dichloromethoxybutane or hydrochloric acid / paraformaldehyde system as the chloromethylation reagent. The specific operation of the chloromethylation reaction is as follows: the sulfonated resin is fully swollen in a solvent with the chloromethylation reagent and catalyst, and reacted at 40°C to 70°C for 4 to 12 hours. After the reaction, the resin is repeatedly washed with solvents such as methanol and acetone to remove residual reagents and catalyst, yielding the chloromethylated sulfonated resin. The amount of chloromethyl group introduced can be adjusted by changing the amount of catalyst and the reaction time.

[0068] The Arbuzov reaction of this invention refers to the reaction of a haloalkane with a trialkyl phosphite to generate a dialkyl alkyl phosphonate. In this invention, a chloromethylated sulfonated resin is used as the haloalkane component and reacted with an excess of a trialkyl phosphite (such as at least one of trimethyl phosphite, triethyl phosphite, or tripropyl phosphite) in an inert solvent (such as at least one of N,N-dimethylformamide, toluene, xylene, or dioxane) under heating, converting the chloromethyl group to a phosphonate group. In this reaction, the mass ratio of the chloromethylated sulfonated resin to the trialkyl phosphite is preferably 1:5 to 1:15. After the reaction is complete, the resin is repeatedly washed with an organic solvent (such as anhydrous ethanol or acetone) to remove unreacted trialkyl phosphite and byproducts, yielding a resin intermediate containing a phosphonate group.

[0069] The hydrolysis reaction of this invention is a step of converting phosphonate groups into phosphonic acid groups. For methyl or ethyl phosphonate, acidic or alkaline hydrolysis is typically used. Acidic hydrolysis: The phosphonate resin is immersed in 6M hydrochloric acid and refluxed at 80°C to 100°C for 12 to 24 hours. Alkaline hydrolysis: The phosphonate resin is immersed in 2M sodium hydroxide solution and refluxed at 80°C to 100°C for 12 to 24 hours. After the reaction, the resin is converted to the acidic form (-PO3H2) with hydrochloric acid. After hydrolysis, the resin is repeatedly washed with deionized water until neutral to obtain the final bifunctional phosphonic acid-functionalized resin.

[0070] The blocking solution of this invention is used to block unreacted active groups on a solid support to reduce nonspecific adsorption. For the preparation of DTPA functionalized resins, to block the residual thiol groups after the thiol-maleimide reaction, blocking solution A can be selected from: a buffer solution of (10-100) mM L-cysteine, mercaptoethanol, or 2-mercaptoethylamine. For EGTA functionalized resins, to block the residual epoxy groups after the epoxy group reaction, blocking solution B can be selected from: (0.5-1.0) M ethanolamine dissolved in (0.5-2.0) M Na2CO3-NaHCO3 buffer (pH 9.0-9.5) or dissolved in (0.5-2.0) M Tris-HCl buffer (pH 8.0-9.0).

[0071] The method for preparing the inactivated virus preservation solution involves mixing a pH buffer, a nonionic surfactant, a reducing agent, a free metal ion chelating agent, and a functionalized resin, and then adding purified water to the target volume to obtain the inactivated virus preservation solution.

[0072] This inactivated virus preservation solution uses a low-concentration nonionic surfactant combined with a reducing agent for synergistic effect. It can completely inactivate the virus and eliminate the risk of infection at room temperature without completely disintegrating the virus particles. The nucleic acid is encapsulated in a protein shell and physically isolated from the nuclease in the preservation solution, allowing for long-term stable preservation without the need for nuclease inhibitors. This solves the technical challenge of traditional reagent inactivation and the inability to maintain the integrity of the virus structure. This inactivated virus preservation solution also contains a free metal chelating agent and a functional resin for immobilization. The low-concentration free chelating agent can quickly bind metal ions and block nucleic acid degradation in the early stage of sample mixing, and the amount used is much lower than that of conventional lysis buffer. It hardly interferes with the magnesium ions required by Taq enzyme and does not affect PCR amplification. The immobilization resin can deeply remove various metal ions such as calcium, iron, and copper to form chelates. With the matching filter, the chelates can be completely retained to prevent them from entering the amplification system and competing for cofactors. This preservation solution is highly versatile and can be used for various clinical samples such as serum, plasma, urine, feces, cervical secretions, and sputum. Only feces and sputum require simple pretreatment and liquefaction beforehand.

[0073] Preparation Example 1

[0074] 1. Preparation method of DTPA functionalized resin: Step A1: Dissolve maleimide monoamide-DTPA in coupling buffer to prepare a DTPA ligand solution with a concentration of 2 mg / mL. The coupling buffer is prepared by adding 0.05% Tween-20 and 0.05 mM EDTA to 50 mM PBS buffer, and the pH of the coupling buffer is 7.2. Step A2: Take 1 mL of activated thiol resin, specifically Activated Thiol Sepharose 4B, and wash it three times with 10 times its volume of coupling buffer. Then, mix the washed activated thiol resin with 5 times its volume of DTPA ligand solution and react it on a rotary mixer at room temperature for 3 hours. Stable thioether bonds are formed through the selective reaction of maleimide groups and thiol groups. After the reaction is complete, wash the resin five times with coupling buffer to remove uncoupled ligands, and obtain reactant A. Step A3: Blocking solution A is added to reactant A from step A2. Blocking solution A is prepared by dissolving 50 mM L-cysteine ​​solid in 50 mM PBS buffer, and the pH of the PBS buffer is 7.2. The mixture is rotated and mixed at room temperature for 1 hour to block the residual thiol active sites on the resin. The resin is then washed alternately with 10 mL of PBS buffer and deionized water to obtain DTPA functionalized resin. The weight ratio of reactant A to blocking solution A is 1:1.

[0075] 2. Preparation of EGTA functionalized resin

[0076] Step B1: Dissolve EGTA (ethylene glycol diethyl ether diaminetetraacetic acid) in alkaline coupling buffer to prepare an EGTA ligand solution with a concentration of 20 mg / mL to 50 mg / mL; wherein the alkaline coupling buffer is 0.5 M NaCl added to 0.1 M Na2CO3-NaHCO3 buffer, and the pH value of the alkaline coupling buffer is 9.5. Step B2: Take 1 mL of epoxy-activated agarose resin (such as Epoxy-activated Sepharose 6B), wash it three times with 10 times its volume of deionized water, and then wash it twice with 10 times its volume of alkaline coupling buffer. Mix the washed resin with 5 times its volume of the EGTA ligand solution obtained in Step B1, and react it in a 40°C water bath shaker for 20 hours at 150 rpm. Covalent linkage is formed through the ring-opening reaction of the amino and epoxy groups of EGTA. After the reaction is completed, wash reactant B with 10 mL to 100 mL of deionized water to obtain reactant B. Step B3: Reactant B obtained in step B2 and blocking solution B are blocked at 40°C for 4 hours to block unreacted epoxy groups. Blocking solution B is 1M ethanolamine dissolved in 0.1M Na2CO3-NaHCO3 buffer, and the pH of blocking solution B is 9.5. Finally, the mixture is washed three times alternately with 10mL deionized water, 0.1M acetate buffer (containing 0.5M NaCl, pH 4.0), and 0.1M Tris-HCl buffer (containing 0.5M NaCl, pH 8.0) to obtain EGTA functionalized resin. The weight ratio of reactant B to blocking solution B is 1:1. 3. Preparation of bifunctional phosphonic acid functionalized resins Step C1, sulfonation reaction Take 100g of polystyrene-divinylbenzene macroporous resin (PS-DVB, crosslinking degree 6%, particle size 0.3-1.0mm) and place it in a 2L three-necked flask. Add 500mL of concentrated sulfuric acid (98%). Place the flask in an oil bath and slowly heat it to 120℃. Stir the mixture at this constant temperature for 8 hours. After the reaction is complete, cool it to room temperature and slowly pour the reaction mixture into 5L of ice water. Filter the mixture and wash the resin repeatedly with deionized water until the washing solution is neutral. The sulfonated resin is then dried in a vacuum drying oven at 60℃ until constant weight.

[0077] Step C2, chloromethylation reaction

[0078] The resin was placed in a 1L three-necked flask, and 400mL of 1,4-dichloromethoxybutane was added to 50g of the sulfonated resin obtained in step C1. Then, 200mL of dichloroethane was added, and the mixture was allowed to swell at room temperature for 2 hours. 1,4-dichloromethoxybutane served as the chloromethylating agent, and dichloroethane as the swelling agent. Then, 15g of anhydrous zinc chloride was added as a catalyst, and the mixture was heated to 50℃ and stirred at this temperature for 8 hours. After the reaction was complete, the resin was cooled, filtered, and washed repeatedly with anhydrous ethanol and acetone, followed by washing with deionized water until neutral. Finally, the resin was dried in a vacuum drying oven at 60℃ to constant weight to obtain the chloromethylated sulfonated resin.

[0079] Step C3, Arbuzov reaction

[0080] Take 30g of the chloromethylated sulfonated resin obtained in step C2 and place it in a 500mL three-necked flask. Add 250mL of trimethyl phosphite and 100mL of N,N-dimethylformamide (DMF) as inert solvents. Heat the flask to 140℃ and stir under nitrogen protection for 24 hours. After the reaction is complete, cool and filter. Wash the resin repeatedly with anhydrous ethanol to remove unreacted trimethyl phosphite and byproducts to obtain a resin intermediate containing phosphonate methyl groups.

[0081] Step C4, hydrolysis reaction

[0082] The resin intermediate containing phosphonate methyl ester groups obtained in step C3 was placed in a 1L three-necked flask, and 600mL of 6M hydrochloric acid solution was added. The flask was heated to 90°C and stirred under reflux for 24 hours. After the reaction was completed, the mixture was cooled, filtered, and the resin was repeatedly washed with deionized water until the washing solution was neutral. A bifunctional phosphonate functionalized resin containing both sulfonic acid groups and phosphonate groups was obtained.

[0083] Preparation Example 2

[0084] 1. Preparation method of DTPA functionalized resin: Step A1: Dissolve maleimide monoamide-DTPA in coupling buffer to prepare a DTPA ligand solution with a concentration of 10 mg / mL. The coupling buffer is prepared by adding 0.05% Tween-20 and 0.05 mM EDTA to 50 mM PBS buffer, and the pH of the coupling buffer is 7.2. Step A2: Take 1 mL of activated thiol resin, specifically Activated Thiol Sepharose 4B, and wash it twice with 8 volumes of coupling buffer. Then, mix the washed activated thiol resin with 4 volumes of DTPA ligand solution and react it on a rotary mixer at room temperature for 4 hours. Stable thioether bonds are formed through the selective reaction of maleimide groups and thiol groups. After the reaction is complete, wash the resin 5 times with coupling buffer to remove uncoupled ligands, and obtain reactant A. Step A3: Blocking solution A is added to reactant A from step A2. Blocking solution A is prepared by dissolving 50 mM L-cysteine ​​solid in 50 mM PBS buffer, and the pH of the PBS buffer is 7.2. The mixture is rotated and mixed at room temperature for 1 hour to block the residual thiol active sites on the resin. The resin is then washed with 100 mL of large amount of PBS buffer and deionized water alternately to obtain DTPA functionalized resin. The weight ratio of reactant A to blocking solution A is 1:1.

[0085] 2. Preparation of EGTA functionalized resin

[0086] Step B1: Dissolve EGTA (ethylene glycol diethyl ether diaminetetraacetic acid) in alkaline coupling buffer to prepare an EGTA ligand solution with a concentration of 50 mg / mL; wherein the alkaline coupling buffer is 0.5 M NaCl added to 0.1 M Na2CO3-NaHCO3 buffer, and the pH value of the alkaline coupling buffer is 9.5. Step B2: Take 1 mL of epoxy-activated agarose resin (e.g., Epoxy-activated Sepharose 6B), wash it three times with 10 times its volume of deionized water, and then wash it twice with 10 times its volume of alkaline coupling buffer. Mix the washed resin with 5 times its volume of the EGTA ligand solution obtained in Step B1, and react it in a 40°C water bath shaker for 20 hours at 150 rpm. Covalent linkage is formed through the ring-opening reaction of the amino and epoxy groups of EGTA. After the reaction is complete, wash reactant B with 100 mL of deionized water to obtain reactant B. Step B3: Reactant B obtained in step B2 and blocking solution B are blocked at 40°C for 4 hours to block unreacted epoxy groups. Blocking solution B is 1M ethanolamine dissolved in 0.1M Na2CO3-NaHCO3 buffer, and the pH of blocking solution B is 9.5. Finally, the mixture is washed three times alternately with 10mL deionized water, 0.1M acetate buffer (containing 0.5M NaCl, pH 4.0), and 0.1M Tris-HCl buffer (containing 0.5M NaCl, pH 8.0) to obtain EGTA functionalized resin. The weight ratio of reactant B to blocking solution B is 1:1. 3. Preparation of bifunctional phosphonic acid functionalized resins Step C1, sulfonation reaction Take 100g of polystyrene-divinylbenzene macroporous resin (PS-DVB, crosslinking degree 6%, particle size 0.3-1.0mm) and place it in a 2L three-necked flask. Add 500mL of fuming sulfuric acid (25%). Place the flask in an oil bath and slowly heat it to 120℃. Stir the mixture at this constant temperature for 8 hours. After the reaction is complete, cool it to room temperature and slowly pour the reaction mixture into 5L of ice water. Filter the mixture and wash the resin repeatedly with deionized water until the washing solution is neutral. The sulfonated resin is obtained and dried in a vacuum drying oven at 60℃ until constant weight.

[0087] Step C2, chloromethylation reaction

[0088] In a 1L three-necked flask, 400mL of chloromethyl methyl ether was added to 50g of the sulfonated resin obtained in step C1, followed by 200mL of dichloroethane. The mixture was allowed to swell at room temperature for 2 hours, where chloromethyl methyl ether served as the chloromethylating agent and dichloroethane as the swelling agent. Then, 15g of anhydrous tin tetrachloride was added as a catalyst, and the mixture was heated to 50℃ and stirred at this temperature for 8 hours. After the reaction was complete, the mixture was cooled, filtered, and washed repeatedly with anhydrous ethanol and acetone, followed by washing with deionized water until neutral. Finally, the resin was dried in a vacuum drying oven at 60℃ until constant weight to obtain the chloromethylated sulfonated resin.

[0089] Step C3, Arbuzov reaction

[0090] Take 30g of the chloromethylated sulfonated resin obtained in step C2 and place it in a 500mL three-necked flask. Add 250mL of triethyl phosphite and 100mL of toluene, where toluene is used as an inert solvent. Heat the flask to 140℃ and stir under nitrogen protection for 24 hours. After the reaction is complete, cool and filter. Wash the resin repeatedly with anhydrous ethanol to remove unreacted triethyl phosphite and byproducts to obtain a resin intermediate containing methyl phosphonate groups.

[0091] Step C4, hydrolysis reaction

[0092] The resin intermediate containing phosphonate methyl ester groups obtained in step C3 was placed in a 1L three-necked flask, and 600mL of 6M hydrochloric acid solution was added. The flask was heated to 90°C and stirred under reflux for 24 hours. After the reaction was completed, the mixture was cooled, filtered, and the resin was repeatedly washed with deionized water until the washing solution was neutral. A bifunctional phosphonate functionalized resin containing both sulfonic acid groups and phosphonate groups was obtained.

[0093] Preparation Example 3

[0094] 1. Preparation method of DTPA functionalized resin: Step A1: Dissolve maleimide monoamide-DTPA in coupling buffer to prepare a DTPA ligand solution with a concentration of 8 mg / mL. The coupling buffer is prepared by adding 0.05% Tween-20 and 0.05 mM EDTA to 50 mM PBS buffer, and the pH of the coupling buffer is 7.2. Step A2: Take 1 mL of activated thiol resin, specifically Activated Thiol Sepharose 4B, and wash it twice with 12 volumes of coupling buffer. Then, mix the washed activated thiol resin with 6 volumes of DTPA ligand solution and react it on a rotary mixer at room temperature for 5 hours. Stable thioether bonds are formed through the selective reaction of maleimide groups and thiol groups. After the reaction is complete, wash the resin 5 times with coupling buffer to remove uncoupled ligands, and obtain reactant A. Step A3: Blocking solution A is added to reactant A from step A2. Blocking solution A is prepared by dissolving 50 mM L-cysteine ​​solid in 50 mM PBS buffer, and the pH of the PBS buffer is 7.2. The mixture is rotated and mixed at room temperature for 1 hour to block the residual thiol active sites on the resin. The resin is then washed alternately with 80 mL of PBS buffer and deionized water to obtain DTPA functionalized resin. The weight ratio of reactant A to blocking solution A is 1:1.

[0095] 2. Preparation of EGTA functionalized resin

[0096] Step B1: Dissolve EGTA (ethylene glycol diethyl ether diaminetetraacetic acid) in alkaline coupling buffer to prepare an EGTA ligand solution with a concentration of 30 mg / mL; wherein the alkaline coupling buffer is 0.5 M NaCl added to 0.1 M Na2CO3-NaHCO3 buffer, and the pH value of the alkaline coupling buffer is 9.5. Step B2: Take 1 mL of epoxy-activated agarose resin (e.g., Epoxy-activated Sepharose 6B), wash it three times with 8 times its volume of deionized water, and then wash it twice with 8 times its volume of alkaline coupling buffer. Mix the washed resin with 4 times its volume of the EGTA ligand solution obtained in Step B1, and react it in a 40°C water bath at 150 rpm for 20 hours. Covalent linkage is formed through the ring-opening reaction of the amino and epoxy groups of EGTA. After the reaction is complete, wash reactant B with 80 mL of deionized water to obtain reactant B. Step B3: Reactant B obtained in step B2 and blocking solution B are blocked at 40°C for 4 hours to block unreacted epoxy groups. Blocking solution B is 1M ethanolamine dissolved in 0.1M Na2CO3-NaHCO3 buffer, and the pH of blocking solution B is 9.5. Finally, the mixture is washed three times alternately with 10mL deionized water, 0.1M acetate buffer (containing 0.5M NaCl, pH 4.0), and 0.1M Tris-HCl buffer (containing 0.5M NaCl, pH 8.0) to obtain EGTA functionalized resin. The weight ratio of reactant B to blocking solution B is 1:1. 3. Preparation of bifunctional phosphonic acid functionalized resins Step C1, sulfonation reaction Take 100g of polystyrene-divinylbenzene macroporous resin (PS-DVB, crosslinking degree 6%, particle size 0.3-1.0mm) and place it in a 2L three-necked flask. Add 500mL of chlorosulfonic acid. Place the flask in an oil bath and slowly heat it to 120℃. Stir the mixture at this constant temperature for 8 hours. After the reaction is complete, cool it to room temperature and slowly pour the reaction mixture into 5L of ice water. Filter the mixture and wash the resin repeatedly with deionized water until the washing solution is neutral. The sulfonated resin is then dried in a vacuum drying oven at 60℃ until constant weight.

[0097] Step C2, chloromethylation reaction

[0098] The resin was placed in a 1L three-necked flask, and 400mL of 1,4-dichloromethoxybutane was added to 50g of the sulfonated resin obtained in step C1. Then, 200mL of tetrachloroethane was added, and the mixture was allowed to swell at room temperature for 2 hours. 1,4-dichloromethoxybutane served as the chloromethylating agent, and tetrachloroethane as the swelling agent. Then, 15g of anhydrous ferric chloride was added as a catalyst, and the mixture was heated to 50℃ and stirred at this temperature for 8 hours. After the reaction was complete, the resin was cooled, filtered, and washed repeatedly with anhydrous ethanol and acetone, followed by washing with deionized water until neutral. Finally, the resin was dried in a vacuum drying oven at 60℃ to constant weight to obtain the chloromethylated sulfonated resin.

[0099] Step C3, Arbuzov reaction

[0100] Take 30g of the chloromethylated sulfonated resin obtained in step C2 and place it in a 500mL three-necked flask. Add 250mL of tripropyl phosphite and 100mL of xylene, where xylene is used as an inert solvent. Heat the flask to 140℃ and stir under nitrogen protection for 24 hours. After the reaction is complete, cool and filter. Wash the resin repeatedly with anhydrous ethanol to remove unreacted tripropyl phosphite and byproducts to obtain a resin intermediate containing methyl phosphonate groups.

[0101] Step C4, hydrolysis reaction

[0102] The resin intermediate containing phosphonate methyl ester groups obtained in step C3 was placed in a 1L three-necked flask, and 600mL of 6M hydrochloric acid solution was added. The flask was heated to 90°C and stirred under reflux for 24 hours. After the reaction was completed, the mixture was cooled, filtered, and the resin was repeatedly washed with deionized water until the washing solution was neutral. A bifunctional phosphonate functionalized resin containing both sulfonic acid groups and phosphonate groups was obtained.

[0103] Sample Example

[0104] Each inactivated virus preservation solution was prepared according to the preparation method of the inactivated virus preservation solution in Example 1, and the formulation of the inactivated virus preservation solution is shown in Table 1: Table 1. Composition and concentration of various inactivated virus preservation solutions

[0105] The DTPA-functionalized resin, EGTA-functionalized resin, and bifunctional phosphonic acid-functionalized resin in combination I were prepared in preparation example 2; the DTPA-functionalized resin, EGTA-functionalized resin, and bifunctional phosphonic acid-functionalized resin in combination J were prepared in preparation example 3; and the DTPA-functionalized resin, EGTA-functionalized resin, and bifunctional phosphonic acid-functionalized resin in the other combinations were prepared in preparation example 1.

[0106] Example 1

[0107] The virus inactivation experiment verifies whether the virus inactivation preservation solution of the present invention can completely inactivate the virus at room temperature, thereby rendering it non-infectious and ensuring the biosafety of staff.

[0108] Sample preparation: H1N1 virus culture medium, titer 1×10⁻⁶ 6 TCID50 / mL; adenovirus culture medium, titer 1×10⁻⁶ 6 TCID50 / mL; Mycoplasma pneumoniae culture medium, concentration 1×10⁻⁶ 6 All CFU / mL samples were preserved using a non-inactivating preservation solution.

[0109] Experimental Groups: The present invention group: H1N1 virus culture medium, adenovirus culture medium, mycoplasma pneumoniae culture medium and inactivated virus preservation solution of sample example are mixed at a volume ratio of 1:5, allowed to stand at room temperature for 1 minute, and then desalted using a desalting column.

[0110] Positive control group: H1N1 virus culture medium and adenovirus culture medium were mixed with an equal volume of DMEM, and Mycoplasma pneumoniae culture medium was mixed with an equal volume of PBS. The mixture was then left to stand at room temperature for 1 minute (without inactivation).

[0111] Procedure: Take samples from each group of H1N1 virus culture medium and perform 10-fold serial dilutions using DMEM (10... -1 Up to 10 -8 MDCK cells were seeded in 96-well plates, and viral TCID50 was measured; samples from each group of adenovirus culture medium were serially diluted 10-fold using DMEM (10... -1 Up to 10 -8 H1 HeLa cells were inoculated into 96-well plates for viral TCID50 assay; samples from each group of Mycoplasma pneumoniae culture medium were serially diluted 10-fold with PBS (10... -1 Up to 10 -8 Take 100 μL and spread it on a Mycoplasma pneumoniae solid culture medium plate to count CFU, as shown in Table 2.

[0112] Table 2. Inactivation data of different viruses in different inactivated virus preservation solutions

[0113] As shown in the table above, the virus inactivation preservation solution of this invention can completely inactivate H1N1 influenza virus, adenovirus, and mycoplasma pneumoniae after standing at room temperature for 1 minute, rendering them inactive. Operators can safely operate on ordinary laboratory benches without the need for a biosafety cabinet, significantly reducing the biosafety risks of on-site testing.

[0114] Example 2

[0115] Temperature-controlled lysis and release experiments were conducted to verify the "temperature-controlled switch" mechanism of the inactivated virus preservation solution of this invention, which maintains the integrity of virus particles (without releasing nucleic acid) at room temperature and effectively releases nucleic acid after high-temperature heating. Sample preparation: Samples from each group in Effect Example 1 after inactivation were collected.

[0116] Experimental Groups: Heating group: Take 1 mL of inactivated H1N1 virus sample, heat at 98℃ for 30 seconds, then perform PEG precipitation (PEG8000, final concentration 10%, precipitate at 4℃ for 2 hours, centrifuge at 12,000 rpm for 30 minutes), and collect the supernatant.

[0117] Unheated group: Take 1 mL of inactivated H1N1 virus sample, do not heat it, directly perform PEG precipitation, and collect the supernatant.

[0118] Detection method: Take the supernatant of each group and use RT-qPCR to detect and compare the Ct values, as shown in Table 3.

[0119] Explanation of principle: PEG precipitation can precipitate intact viral particles and mycoplasma, but it cannot precipitate free nucleic acids. If the viral particles or mycoplasma are intact, they are precipitated by PEG, and there is no nucleic acid in the supernatant (high Ct value or no amplification); if the viral particles or mycoplasma are lysed, the nucleic acid is released into the supernatant, and the nucleic acid remains in the supernatant after PEG precipitation (low Ct value).

[0120] Table 3. Temperature-controlled lysis and release data for different inactivated virus preservation solutions

[0121] Note: Undet is the Ct value without amplification.

[0122] As shown in Table 3, the unheated group showed no amplification signal, indicating that the inactivated virus preservation solution of this invention maintained the complete structure of the virus particles at room temperature, was precipitated by PEG, and contained no free nucleic acid in the supernatant. The heated group showed obvious amplification signals, indicating that high-temperature heating effectively destroyed the viral envelope and protein coat, releasing nucleic acid, which remained in the supernatant.

[0123] Example 2

[0124] A purification device, such as Figure 1 and Figure 2 As shown, it includes: Bottle 100 contains the inactivated virus preservation solution of the present invention; Bottle cap 200, which is detachably mounted on the open end of the bottle body 100.

[0125] The composite filter element 300 is used to trap cell debris and blood clots in the sample, trap viruses, immobilized chelating agents in the inactivated virus preservation solution of the present invention, and adsorb immunoglobulins in the sample. The composite filter element 300 is sealed and assembled at the outlet end of the bottle cap 200.

[0126] The bottle cap 200 includes a cap body 210 and a dropper 220. The cap body 210 and the dropper 220 are integrally connected. The cap body 210 is detachably mounted on the upper end of the bottle body 100. The dropper 220 is provided with a liquid flow channel. The dust cover is detachably fitted onto the outside of the dropper 220.

[0127] The composite filter element 300 is disposed between the cover body 210 and the drip tip 220. The composite filter element 300 includes a pre-filtration layer 310, an immunoglobulin adsorption layer 320, and a support layer 330, which are stacked sequentially, with the support layer 330 close to the liquid flow channel. The pre-filtration layer 310 is made of polyethylene, polypropylene, or ultra-high molecular weight polyethylene (UHMWPE). The immunoglobulin adsorption layer 320 is made of at least one of Protein A resin, Protein G resin, or Protein L resin. The support layer 330 is made of polyethylene, polypropylene, or UHMWPE.

[0128] A dust cover, which is detachably fitted onto the outside of the dropper cap 200.

[0129] In this embodiment, the pre-filter layer 310 is specifically made of polyethylene, with a diameter matching the diameter of the bottle cap 200, a thickness of 2 mm, and a pore size of 50 μm. The pre-filter layer 310 is used to trap large particles such as cell debris, resin particles, and blood clots, while maintaining a non-specific adsorption rate of less than 5% for nucleic acids. The immunoglobulin adsorption layer 320 is at least one of Protein A resin, Protein G resin, or Protein L resin, which is purchased from Beijing Weishi Bohui Chromatography Technology Co., Ltd., and is used to adsorb immunoglobulins in samples (especially serum and blood samples). In this embodiment, Protein A resin, Protein G resin, and Protein L resin are mixed in a weight ratio of 1:1:1, and the thickness of the immunoglobulin adsorption layer 320 is 1 mm.

[0130] In this embodiment, the support layer 330 is specifically made of polyethylene, with a diameter matching the diameter of the bottle cap 200, a thickness of 1 mm, and a pore size of 50 μm. The function of the polyethylene support layer 330 is to support the immunoglobulin adsorption layer 320 and prevent resin leakage.

[0131] The capacity of the purification device can be determined according to actual needs. In this embodiment, the purification device is described using 1.5 mL as an example, and the diameters of the pre-filtration layer 310, the immunoglobulin adsorption layer 320, and the support layer 330 are specifically 4 mm. When using the purification device, it is inverted, and the bottle 100 is squeezed. The liquid flows sequentially through the pre-filtration layer 310, the immunoglobulin adsorption layer 320, and the support layer 330, and the sample flows out from the dropper 220 along with the liquid.

[0132] The purification device features a composite filter element 300 composed of a pre-filter layer 310, an immunoglobulin adsorption layer 320, and a support layer 330. The immunoglobulin adsorption layer 320 efficiently adsorbs immunoglobulins from serum and plasma, solving the core challenges of protein binding to nucleic acids and inhibiting Taq enzymes. Simultaneously, the pre-filter layer 310 and support layer 330 physically trap blood clots, cell debris, and functionalized resin particles from the inactivated virus preservation solution, removing large particulate impurities and protein-based PCR inhibitors. Samples can undergo all pretreatment processes, including immunoglobulin adsorption and impurity trapping, within the device, allowing for subsequent analysis. The testing equipment only needs to have heating, quantitative sample addition, and amplification detection functions. It does not need to be equipped with a complex nucleic acid extraction module. The device relies on the squeezing of the dropper bottle to achieve the filtration operation. There is no need for pipettes, centrifuges, or magnetic bead extractors. Only the lysis step can use a simple metal bath, eliminating the dependence on large equipment for traditional column extraction and magnetic bead extraction methods. The entire process does not require repeated opening and closing of the cap for pipetting. When used with the matching automatic PCR equipment, it can achieve a fully closed operation of "sample in, result out", avoiding the contamination risk of open nucleic acid transfer. It is suitable for applications in primary medical institutions, customs ports of entry and exit quarantine, field emergency and on-site disease control testing and other scenarios.

[0133] Example 3

[0134] Comparison Table 1: Effects of different inactivated virus preservation solutions on serum Ca²⁺ + Fe³ + The removal efficiency and its impact on subsequent PCR were investigated. The samples used were serum from healthy individuals (with H1N1 virus culture and exogenous Ca2+ added). 2+ Up to 2.5 mM, Fe 3+ Up to 0.05mM).

[0135] Procedure: Each combination was performed using the same procedure (sample:inactivated virus preservation solution = 1:10, with 100 μL of sample and 1 mL of inactivated virus preservation solution, incubated at room temperature for 5 minutes, purified using the purification device of Example 2 and filtered through a composite filter), the flow-through was collected, heated at 98°C for 30 seconds, and the flow-through was used as a template for qPCR detection of the H1N1 M gene. (PCR inhibition was assessed), and the results are shown in Table 4.

[0136] Table 4. Ct values ​​of various inactivated virus preservation solutions

[0137] As shown in Table 4, DTPA-functionalized resins possess broad-spectrum metal ion chelating capabilities. EGTA-functionalized resins also exhibit strong chelating ability for Ca²⁺. + It exhibits high selectivity. Bifunctional phosphonic acid-functionalized resins exhibit high selectivity for Fe³⁺. + It exhibits high selectivity, with combination F achieving the optimal broad-spectrum metal ion removal effect; the metal ion spectra differ among different samples, and the combined use of multiple functionalized resins can cover a wider range of inhibitor types.

[0138] Example of effect 4

[0139] The composite filter element 300 of the purification device in Example 2 was compared with a single-layer filter element. Under the same conditions, control group 1 consisted of a single-layer filter element, with only a pre-filtration layer 310 made of polyethylene, polypropylene, or ultra-high molecular weight polyethylene (UHMWPE) with a thickness of 2 mm and a pore size of 50 μm, and no immunoglobulin adsorption layer 320 or support layer 330. Control group 2 consisted of no filter element, meaning the supernatant after inactivation was taken directly without filtration.

[0140] In this example, the inactivated virus preservation solution used in the sample was formulated using combination F. The sample was a hepatitis B virus positive serum sample.

[0141] Operating steps: Serum samples and preservation solutions were mixed at a volume ratio of 1:10, and 100 μL of serum sample and 1 mL of inactivated virus preservation solution were incubated at room temperature for 2 minutes. The mixture was then filtered through different filter cartridges, and the flow-through was collected. The flow-through was heated at 98°C for 30 seconds before hepatitis B virus qPCR detection, as shown in Table 5.

[0142] Table 5. qPCR detection data of hepatitis B virus using different filtration methods

[0143] As shown in Table 5, the purification device of this invention exhibits the lowest Ct value, indicating that the pre-filter layer 310 effectively traps resin particles, and the Protein A / G / L layer effectively adsorbs immunoglobulins from the serum sample. The control group 1, lacking the immunoglobulin adsorption layer 320, shows a significantly higher Ct value, indicating that immunoglobulins in the serum significantly inhibit PCR. The composite filter element 300 in the purification device of this invention achieves a synergistic extraction effect of large particle trapping, immunoglobulin adsorption, and resin barrier, significantly improving detection sensitivity.

[0144] Example 5

[0145] The purification device of the present invention extracts and detects hepatitis B virus DNA in serum samples at high temperature, verifying the complete processing procedure for hepatitis B virus (HBV, DNA virus) in serum samples.

[0146] The sample was a hepatitis B virus positive serum sample.

[0147] The specific steps of the invention group's operation are as follows: In the purification apparatus of Example 2, add 100 μL of serum sample, followed by 1 mL of inactivated virus preservation solution (using the formula of Combination F). Gently mix and let stand at room temperature for 1 minute (virus inactivation, structural integrity). Invert the purification apparatus of Example 2 and squeeze the bottle 100°C to allow the liquid to pass through the composite filter cartridge 300, collecting the flow-through. Take 100 μL of the flow-through, heat at 98°C for 30 seconds (high-temperature lysis to release nucleic acid), and then perform hepatitis B virus qPCR detection.

[0148] Comparison group settings: Control group A (lytic direct amplification method): Commercially available lytic release agent (containing high concentration of NP-40+EDTA), sample and release agent mixed at 1:10, incubated at room temperature for 10 minutes, without heating, and directly performed qPCR detection.

[0149] Control group B (column extraction method): commercially available silica membrane centrifuge column method, operated according to the instructions (lysis, binding, washing, elution, about 40 minutes).

[0150] Control group C (unextracted control): The sample was mixed with PBS at a ratio of 1:10 and directly subjected to qPCR detection without any treatment, as shown in Table 6.

[0151] Table 6. qPCR detection data of hepatitis B virus using different filtration methods

[0152] Note: Undet is the Ct value without amplification.

[0153] like Figure 3 As can be seen from the amplification curves, both the amplification curves of the present invention group and control group B, as recorded in real time by the qPCR instrument, exhibit a typical S-shape with a high plateau phase. The corresponding amplification curve of control group A shows a lower plateau phase, while control group C shows no amplification curve. Therefore, the Ct value of the present invention group is close to that of the column extraction method, and the extraction effect is close to the standard of the column method. Compared with the lysis-type direct amplification method, the sensitivity of the present invention group is significantly improved. The operation time is only 2 minutes, which is 95% shorter than the column extraction method, and no centrifuge is required. The sample is inactivated at room temperature, making the operation safe and suitable for field use at the grassroots level.

[0154] Example 6

[0155] The purification device of the present invention is used to extract and detect chikungunya virus RNA in serum samples at high temperature, verifying the effectiveness of the purification device in extracting and detecting RNA viruses at high temperature.

[0156] The sample was a positive serum sample for Chikungunya fever.

[0157] The specific steps of the invention group's operation are as follows: In the purification apparatus of Example 2, add 100 μL of serum sample, followed by 1 mL of inactivated virus preservation solution (using the formula of Combination F). Gently mix and let stand at room temperature for 1 minute (virus inactivation, structural integrity). Invert the purification apparatus of Example 2 and squeeze the bottle 100°C to allow the liquid to pass through the composite filter cartridge 300, collecting the flow-through. Take 100 μL of the flow-through, heat at 98°C for 30 seconds (high-temperature lysis to release nucleic acid), and then perform hepatitis B virus qPCR detection.

[0158] Comparison group settings: Control group A (lytic direct amplification method): Commercially available lytic release agent, sample and release agent mixed at a ratio of 1:10, incubated at room temperature for 10 minutes, without heating, and directly performed qPCR detection.

[0159] Control group B (column extraction method): commercially available silica membrane centrifugation column method (containing RNase inhibitor), operated according to the instructions, as shown in Table 7.

[0160] Table 7. qPCR detection data of Kenyan fever with different filtration methods

[0161] As shown in Table 7, the detection performance of this invention for RNA viruses is close to that of column extraction, and significantly superior to the lysis-based direct amplification method. In the lysis-based direct amplification method, the viral structure is destroyed, exposing the RNA to nucleases, resulting in a significantly increased Ct value, indicating that the RNA has been partially degraded or not extracted, and that there are many inhibitors present. This invention achieves a complete process for room-temperature inactivation and high-temperature lysis and release of RNA viruses, making it particularly suitable for on-site detection of RNA viruses.

[0162] Example 7

[0163] Applicability verification for different sample types: Verify the applicability of this purification device to different sample types (urine, feces, cervical secretions, sputum).

[0164] Sample preparation: Urine: Urine samples spiked with UU (Ureaplasma urealyticum) Feces: Fecal samples spiked with rotavirus (pre-treatment method: 0.1g feces + 1mL water, centrifuge and collect the supernatant). Cervical secretions: Cervical brush samples spiked with HPV Sputum: Sputum samples spiked with Mycobacterium tuberculosis (pre-treatment method: equal volume of 2% N-acetylcysteine, liquefy at 37℃ for 20 minutes, centrifuge and mix the precipitate with the preservation solution). Operating steps: After each sample is processed according to the corresponding pretreatment method, the sample is added to the purification device of Example 2 and mixed with the inactivated virus preservation solution of the sample example using the formula of combination F at a ratio of 1:10. After standing at room temperature for 1 minute, the sample is filtered by squeezing through the composite filter cartridge 300. After the flow-through solution is heated at 98°C for 30 seconds, the corresponding pathogen is detected by qPCR. The results are shown in Table 8.

[0165] Table 8. qPCR detection data for different sample types

[0166] Note: Undet is the Ct value without amplification.

[0167] As shown in Table 8, the purification device of this invention is suitable for various complex samples such as urine, feces, cervical secretions, and sputum. Its detection effect is equivalent to column extraction and significantly superior to direct amplification. For samples with high levels of inhibitors, such as feces, direct amplification cannot detect them, while this invention can still effectively detect them.

[0168] Example 8

[0169] By combining the purification device of the present invention with a PCR instrument with automatic heating function, an integrated POCT detection system is obtained.

[0170] Experimental objective: To verify the compatibility of the present invention with a PCR instrument with automatic heating function (specifically, a molecular detection system and device with sample heating and quantitative sample addition function as described in Chinese Patent Publication No. CN119464045A), and to achieve fully enclosed POCT detection from sample input to result output.

[0171] This integrated POCT testing system consists of: 1. The purification apparatus of Embodiment 2 of the present invention, wherein the inactivated virus preservation solution used in the sample example uses the formulation of combination F.

[0172] 2. PCR instrument with automatic heating function: It has a heating module, a quantitative sample loading module, an amplification and fluorescence detection module (for its heating function, see the publication number CN119464045A, a molecular detection system and device with sample heating and quantitative sample loading function).

[0173] Sample: Chikungunya virus positive serum sample.

[0174] Operating procedures: Step 1, Sample Collection and Room Temperature Inactivation: Take 100 μL of serum sample and add it to 1 mL of inactivated virus preservation solution from the purification device in Example 2. Tightly cap the bottle and invert it 10 times (approximately 1 minute). In this step, the virus is completely inactivated (non-infectious), but the virus particle structure remains intact, and the nucleic acid is not released.

[0175] Step 2, Sample Addition: Invert the purification device and squeeze the liquid to allow it to pass sequentially through the composite filter cartridge 300. The flow-through solution then enters the amplification reaction container compatible with the PCR instrument. This step automatically removes functionalized resin particles and immunoglobulins from the sample.

[0176] Step 3: Automatic high-temperature lysis to release nucleic acid: The equipment's heating module heats the flow-through liquid to 98°C and maintains it for 30 seconds to destroy the viral envelope and protein coat, releasing nucleic acid.

[0177] Step 4, Automatic quantitative sample addition: The equipment quantitatively extracts an appropriate amount of lysis solution and automatically adds it to the reaction chamber pre-loaded with lyophilized amplification reagents.

[0178] Step 5, Automatic Amplification and Detection: The device starts the amplification program and collects fluorescence signals in real time, automatically interprets the results (positive / negative), and displays them on the device screen.

[0179] Control group setup: Traditional magnetic bead method + PCR (control group): Nucleic acid was extracted using commercially available magnetic beads and then detected by real-time fluorescence PCR, as shown in Table 9.

[0180] Table 9. Data Comparison between Integrated POCT Testing System and Control Group

[0181] As shown in Table 9, the purification device of this invention, when used in conjunction with the automated heating quantitative detection equipment, achieves a fully enclosed POCT detection process from sample input to result output. Operators only need to add the sample; all other steps are completed automatically by the equipment, reducing manual operation time to approximately one minute. Compared to traditional magnetic bead extraction, the operation time of this invention is reduced by more than 95%, and it eliminates the need for specialized equipment such as magnetic bead extractors and biosafety cabinets, making it more suitable for on-site testing at grassroots levels.

[0182] In summary, the purification device of this invention is highly compatible with PCR instruments with automatic heating function, forming an integrated POCT-PCR detection system with full functions of sample inactivation, inhibitor removal, high-temperature lysis, quantitative sample addition, and amplification detection. It has significant application value and market prospects in the field of point-of-care molecular testing.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A virus inactivation preservation solution, characterized in that: The raw materials include a pH buffer, a nonionic surfactant, a reducing agent, a free metal ion chelating agent, a supported chelating agent, and water; the supported chelating agent contains DTPA functionalized resin. The concentrations of each raw material in water are as follows: pH buffer: 0.5mM~2.0mM; Nonionic surfactants: 0.002% (v / v) to 0.02% (v / v); Reducing agent: 0.2mM~1.0mM; Free metal ion chelating agent: 0.2mM~1.0mM; DTPA functionalized resin: 5% (w / v) to 40% (w / v); The DTPA functionalized resin was prepared by reacting maleimide monoamide-DTPA with activated thiol resin.

2. The inactivated virus preservation solution according to claim 1, characterized in that: The immobilized chelating agent also contains at least one of EGTA functionalized resin and bifunctional phosphonic acid functionalized resin. When the raw material contains EGTA functionalized resin, the concentration of EGTA functionalized resin is 1% (w / v) to 20% (w / v); When the raw material contains bifunctional phosphonic acid functionalized resin, the concentration of the bifunctional phosphonic acid functionalized resin is 1% (w / v) to 20% (w / v); Among them, EGTA functionalized resin is prepared by ring-opening reaction of epoxy activated agarose resin and ethylene glycol diethyl ether diaminetetraacetic acid EGTA; The preparation method of the bifunctional phosphonic acid functionalized resin is as follows: Step C1: Sulfonate polystyrene-divinylbenzene macroporous resin beads with sulfonating reagent to obtain sulfonated resin; Step C2: The sulfonated resin obtained in step C1 is subjected to a chloromethylation reaction using a chloromethylation reagent to obtain a chloromethylated sulfonated resin; Step C3: The chloromethylated sulfonated resin obtained in step C2 is reacted with trialkyl phosphite via an Arbuzov reaction to obtain a resin intermediate containing methyl phosphonate groups. Step C4: Hydrolyze the resin intermediate containing methyl phosphonate groups obtained in step C3 to obtain a bifunctional phosphonic acid functionalized resin containing sulfonic acid groups and phosphonic acid groups.

3. The inactivated virus preservation solution according to claim 2, characterized in that: The concentrations of each raw material in water are as follows: pH buffer: 1.0 mM; Nonionic surfactants: 0.01% (v / v) to 0.013% (v / v); Reducing agent: 0.5mM; Free metal ion chelating agent: 0.5mM; DTPA functionalized resin: 20% (w / v) to 30% (w / v).

4. The inactivated virus preservation solution according to claim 2, characterized in that: When the raw material contains EGTA functionalized resin, the concentration of EGTA functionalized resin is 5% (w / v) to 15% (w / v); When the raw material contains a difunctional phosphonic acid functionalized resin, the concentration of the difunctional phosphonic acid functionalized resin is 5% (w / v) to 15% (w / v).

5. The inactivated virus preservation solution according to claim 1, characterized in that: The preparation method of the DTPA functionalized resin is carried out by the following steps: Step A1: Dissolve maleimide monoamide-DTPA in coupling buffer to obtain DTPA ligand solution, wherein the coupling buffer is PBS buffer containing Tween-20 and EDTA. Step A2: React the activated thiol resin with the DTPA ligand solution obtained in step A1 to obtain reactant A; Step A3: Add blocking solution A to reactant A from step A2 to react and obtain the DTPA functionalized resin. The blocking solution A contains L-cysteine ​​in PBS buffer.

6. The inactivated virus preservation solution according to claim 2, characterized in that, The EGTA functionalized resin was prepared by the following steps: Step B1: Dissolve EGTA (ethylene glycol diethyl ether diaminetetraacetic acid) in an alkaline coupling buffer to obtain an EGTA ligand solution; the alkaline coupling buffer is a Na2CO3-NaHCO3 buffer containing NaCl. Step B2: React the epoxy-activated agarose resin with the EGTA ligand solution obtained in step B1 to obtain reactant B; Step B3: React reactant B obtained in step B2 with blocking solution B to obtain EGTA functionalized resin; the blocking solution B is a Na2CO3-NaHCO3 buffer solution containing ethanolamine.

7. The inactivated virus preservation solution according to claim 2, characterized in that: The preparation method of the bifunctional phosphonic acid functionalized resin: Step C1: Sulfonate polystyrene-divinylbenzene macroporous resin beads with sulfonating reagent, wash and dry to obtain sulfonated resin; Step C2: Swelling agent and chloromethylating agent are added to the sulfonated resin obtained in step C1 to swell it, and then a catalyst is added to carry out the chloromethylation reaction of the sulfonated resin obtained in step C1 to obtain chloromethylated sulfonated resin. Step C3: The chloromethylated sulfonated resin obtained in step C2 is reacted with trialkyl phosphite under heating in an inert solvent to carry out the Arbuzov reaction, thereby obtaining a resin intermediate containing methyl phosphonate groups. Step C4: The resin intermediate containing methyl phosphonate groups obtained in step C3 is subjected to acidic or alkaline hydrolysis to obtain a bifunctional phosphonic acid functionalized resin containing sulfonic acid groups and phosphonic acid groups. The pH buffer is a Tris-HCl buffer. The nonionic surfactant is a mixture of Tween 80 and Triton X-100; The reducing agent is at least one of dithiothreitol (DTT), β-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP). The free metal ion chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA), diethyl ether diaminetetraacetic acid (EGTA), and diethylenetriaminepentaacetic acid (DTPA). The sulfonating agent is at least one of concentrated sulfuric acid, fuming sulfuric acid, chlorosulfonic acid, or sulfur trioxide. The chloromethylating agent is at least one of chloromethyl methyl ether or 1,4-dichloromethoxybutane; The catalyst is at least one of anhydrous zinc chloride, anhydrous tin tetrachloride, or anhydrous ferric chloride. The trialkyl phosphite is at least one of trimethyl phosphite, triethyl phosphite, or tripropyl phosphite; The inert solvent is at least one of N,N-dimethylformamide, toluene, xylene, or dioxane.

8. A method for preparing an inactivated virus preservation solution as described in any one of claims 1 to 7, characterized in that: A pH buffer, nonionic surfactant, reducing agent, free metal ion chelating agent and functionalized resin are mixed, and then purified water is added to the target volume to obtain the inactivated virus preservation solution.

9. A purification apparatus, characterized in that, include: The bottle (100) contains the inactivated virus preservation solution as described in any one of claims 1 to 7; A bottle cap (200) is detachably fitted to the open end of the bottle body (100); The composite filter element (300) is used to trap cell debris and blood clots in the sample, trap the immobilized chelating agent in the inactivated virus preservation solution, and adsorb immunoglobulins in the sample. The composite filter element (300) is sealed and assembled at the outlet end of the bottle cap (200). A dust cover, which is removably fitted onto the outside of the bottle cap (200).

10. The purification apparatus according to claim 9, characterized in that: The bottle cap (200) includes a cap body (210) and a dropper (220). The cap body (210) and the dropper (220) are integrally connected. The cap body (210) is detachably assembled to the outlet end of the bottle body (100). The dropper (220) is provided with a liquid flow channel. The dust cover is detachably fitted onto the outside of the dropper (220). The composite filter element (300) is disposed between the cover body (210) and the dripper (220). The composite filter element (300) includes a pre-filtration layer (310), an immunoglobulin adsorption layer (320), and a support layer (330). The pre-filtration layer (310), the immunoglobulin adsorption layer (320), and the support layer (330) are stacked in sequence, and the support layer (330) is close to the liquid flow channel. The pre-filter layer (310) is polyethylene, polypropylene or ultra-high molecular weight polyethylene (UHMWPE); the immunoglobulin adsorption layer (320) is at least one of Protein A resin, Protein G resin or Protein L resin; the support layer (330) is polyethylene, polypropylene or ultra-high molecular weight polyethylene (UHMWPE).

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