Nucleic acid releasing agent for isothermal amplification and use method thereof

Abstract

The invention relates to the field of molecular biology, and discloses a nucleic acid releasing agent for isothermal amplification and a use method of the nucleic acid releasing agent. Comprising the following steps: step 1, preparing a polyoxypropylene polyoxyethylene copolymer diluent, a defoaming agent SE-15 diluent, an ethyl phenyl polyethylene glycol NP-40 diluent, a sodium polyethylene sulfonate diluent and a Proclin300 diluent in sequence through RNA enzyme-free water; and 2, sequentially preparing an ethylenediamine tetraacetic acid disodium salt mother solution, a sodium dodecyl sulfate mother solution and a glycine mother solution through RNA enzyme-free water. Sodium polyethylene sulfonate can be specifically combined with protein amplification inhibitors in a sample to remove mucoprotein, hemoglobin, polysaccharide and other inhibitors in the swab sample, and disodium ethylene diamine tetraacetate can chelate metal ions to reduce the influence of nuclease on target nucleic acid.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a nucleic acid release agent for isothermal amplification and its method of use. Background Technology

[0002] In the field of molecular biology, nucleic acid detection is a core technology for pathogen diagnosis, genotyping, and molecular epidemiological investigation. It determines the presence and load of a target pathogen by extracting, amplifying, and detecting the target nucleic acid in a sample. With the rapid growth in demand for point-of-care testing (POCT), isothermal amplification technology has become the preferred method for nucleic acid testing in non-laboratory settings such as primary hospitals, customs ports, and field sites because it does not rely on the temperature cycling function of a PCR instrument, can rapidly complete amplification at a constant temperature, and has a simple operating procedure.

[0003] Traditional nucleic acid release agents often use a combination of guanidine salt denaturants, a single surfactant, and a basic chelating agent, resulting in weak resistance to amplification inhibitors. Substances such as mucosal mucin, hemoglobin, and polysaccharides in swab samples cannot be effectively removed. These substances can non-specifically bind to key enzymes in isothermal amplification, leading to decreased enzyme activity and affecting amplification efficiency and stability. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a nucleic acid release agent for isothermal amplification and its usage method, solving the problem that traditional nucleic acid release agents have weak resistance to amplification inhibitors, affecting amplification efficiency and stability.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a nucleic acid releasing agent for isothermal amplification, comprising the following steps: Step 1: Prepare diluents for polyoxypropylene-polyoxyethylene copolymer, defoamer SE-15, ethyl phenyl polyethylene glycol NP-40, sodium polyethylene sulfonate, and Proclin 300 sequentially using RNase-free water. Step 2: Prepare disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, and glycine mother liquor sequentially using RNase-free water; Step 3: Mix the polyoxypropylene-polyoxyethylene copolymer diluent, defoamer SE-15 diluent, ethyl phenyl polyethylene glycol NP-40 diluent, sodium polyethylene sulfonate diluent, Proclin 300 diluent, disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, glycine mother liquor, and RNase-free water to obtain a mixture. Step 4: After sterile filtration of the mixture, dispense it into sterile containers to obtain the nucleic acid release agent.

[0006] By employing the above technical solution, sodium polyvinyl sulfonate can specifically bind to protein amplification inhibitors in the sample; ethylphenyl polyethylene glycol NP-40 can deeply denature impurities and remove inhibitors such as mucosal mucin, hemoglobin, and polysaccharides in swab samples; disodium ethylenediaminetetraacetate can chelate metal ions to reduce the impact of nucleases on target nucleic acids; and glycine can maintain the pH stability of the system to meet the activity requirements of key enzymes for isothermal amplification. Simultaneously, the standardized preparation process ensures consistent concentrations of each functional component in each batch of the release agent, avoiding fluctuations in anti-inhibition effects due to component inhomogeneity. This prevents non-specific binding of inhibitors to key enzymes for isothermal amplification, achieving stable enzyme activity and efficient amplification of target nucleic acids during isothermal amplification. This solves the problem of weak anti-amplification inhibitor capabilities in traditional nucleic acid release agents, which affects amplification efficiency and stability.

[0007] Preferably, in step one, the dilution ratios of the polyoxypropylene-polyoxyethylene copolymer diluent, the defoamer SE-15 diluent, the ethyl phenyl polyethylene glycol NP-40 diluent, and the Proclin 300 diluent are all 5 times. The preparation method involves mixing the polyoxypropylene-polyoxyethylene copolymer stock solution, the defoamer SE-15 stock solution, the ethyl phenyl polyethylene glycol NP-40 stock solution, and the Proclin 300 stock solution with RNase-free water at a volume ratio of 1:4. The dilution ratio of the sodium polyethylene sulfonate diluent is 1000 times, and the preparation method involves mixing the sodium polyethylene sulfonate stock solution with RNase-free water at a volume ratio of 1:999.

[0008] Preferably, in step two, the concentration of the disodium ethylenediaminetetraacetate mother liquor is 50 mM, and it is prepared by dissolving disodium ethylenediaminetetraacetate in RNase-free water, adjusting the pH to 8.0, and then making up to a final volume to obtain the disodium ethylenediaminetetraacetate mother liquor. The concentration of the sodium dodecyl sulfonate mother liquor is 10 mM, and it is prepared by dissolving sodium dodecyl sulfonate in RNase-free water at 50°C, cooling to room temperature, and then making up to a final volume to obtain the sodium dodecyl sulfonate mother liquor. The concentration of the glycine mother liquor is 50 mM, and it is prepared by weighing glycine, dissolving it in RNase-free water, adjusting the pH to 7.4, and then making up to a final volume to obtain the glycine mother liquor.

[0009] Preferably, in step three, the mixing involves adding RNase-free water to a mixing container, followed by the sequential addition of polyoxypropylene-polyoxyethylene copolymer diluent, defoamer SE-15 diluent, ethyl phenyl polyethylene glycol NP-40 diluent, sodium polyethylene sulfonate diluent, disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, glycine mother liquor, and Proclin 300 diluent, and then mixing them uniformly using a vortex mixing method. The vortex speed of the vortex mixing method is 2000-2500 rpm, and the mixing time is 30 seconds to 1 minute.

[0010] Preferably, in step three, the mixture is prepared in the following amounts per 1 mL: 10 μL of polyoxypropylene-polyoxyethylene copolymer diluent, 2.5 μL of defoamer SE-15 diluent, 5 μL of ethyl phenyl polyethylene glycol NP-40 diluent, 30 μL of sodium polyethylene sulfonate diluent, 20 μL of disodium ethylenediaminetetraacetate mother liquor, 60 μL of sodium dodecyl sulfonate mother liquor, 50 μL of glycine mother liquor, 2.5 μL of Proclin 300 diluent, and the remainder is RNase-free water.

[0011] Preferably, in step four, the aseptic filtration process involves using a sterile syringe to push the mixture through a cellulose acetate membrane for filtration.

[0012] Preferably, in step four, the sterile container is a sterile centrifuge tube made of polypropylene.

[0013] A nucleic acid release agent for isothermal amplification is prepared by the above-described preparation method.

[0014] A method for using a nucleic acid releasing agent for isothermal amplification, comprising the above-mentioned nucleic acid releasing agent for isothermal amplification, including the following steps: Insert the swab sample into the container containing the nucleic acid release agent, rotate the swab to elute the sample into the nucleic acid release agent, remove the swab, and obtain a mixture; After incubating the container containing the mixture, if there is residue in the mixture, the mixture is centrifuged and the supernatant is taken as a template for isothermal amplification.

[0015] Preferably, the incubation temperature is 37°C and the time is 15 minutes. The isothermal amplification is recombinase nuclease isothermal amplification, the amplification process temperature is 39-42°C, and the amplification incubation time is 10-15 minutes.

[0016] This invention provides a nucleic acid releasing agent for isothermal amplification and its method of use. It has the following beneficial effects: 1. This invention utilizes sodium polyvinyl sulfonate to specifically bind to protein amplification inhibitors in samples, ethylphenyl polyethylene glycol NP-40 to deeply denature contaminating proteins, and to remove inhibitors such as mucosal mucin, hemoglobin, and polysaccharides from swab samples. Disodium EDTA can chelate metal ions to reduce the impact of nucleases on target nucleic acids, while glycine can maintain pH stability to meet the activity requirements of key enzymes in isothermal amplification. Simultaneously, standardized preparation processes ensure consistent concentrations of each functional component in each batch of the release agent, avoiding fluctuations in anti-inhibition effects due to component inhomogeneity. This prevents non-specific binding of inhibitors to key enzymes in isothermal amplification, achieving stable enzyme activity and efficient amplification of target nucleic acids during isothermal amplification. This solves the problem of weak anti-amplification inhibitor capabilities in traditional nucleic acid release agents, which affect amplification efficiency and stability.

[0017] 2. This invention utilizes disodium EDTA to inhibit nuclease activity, reducing the risk of RNA degradation. Glycine maintains the pH of the system within a suitable range for RNA stability. Combined with an RNase-free preparation environment and the preservative effect of Proclin 300, a multi-layered protection mechanism is formed. Compared to the limitations of traditional release agents, which are prone to RNA degradation and have short shelf life at room temperature, RNA samples treated with this release agent can maintain amplification activity for a longer period at room temperature without relying on cold chain transportation and storage. It is not only suitable for routine laboratory testing but also meets the needs of delayed amplification after sample processing in POCT scenarios such as primary hospitals, customs ports, and field sites, thus broadening the application scope of nucleic acid testing.

[0018] 3. This invention establishes a unified standard through a process of pre-dilution preparation, mother liquor preparation, component mixing, and aseptic filtration and dispensing. This avoids performance differences between different batches due to uneven component concentrations, ensuring that the lysis capacity, anti-inhibition effect, and nucleic acid protection performance of each batch of products remain stable, providing a solid guarantee for the reliability and repeatability of subsequent nucleic acid testing results. Attached Figure Description

[0019] Figure 1 This is an LFD detection band image after treating a throat swab with 1 mL of the first release agent in Example 1; Figure 2 The LFD detection band diagram is shown after treating a throat swab with 1.5 mL of the first release agent in Example 1. Figure 3 This is an LFD detection band image after treating a throat swab with 2 mL of the first release agent in Example 1; Figure 4 This is an image of the LFD detection bands after treating a pharyngeal swab with 1 mL of the nucleic acid release agent in Example 1; Figure 5 The image shows the LFD detection bands after treating a throat swab with 1.5 mL of the nucleic acid release agent from Example 1. Figure 6 The image shows the LFD detection bands after treating a pharyngeal swab with 2 mL of the nucleic acid release agent in Example 1. Figure 7 This is an LFD detection band diagram of the sample treated with the first-release agent for 15 minutes in Example 2; Figure 8 This is an LFD detection band diagram of the sample treated with the first-release agent for 30 minutes in Example 2; Figure 9 This is an LFD detection band diagram of the sample treated with the first-release agent for 60 minutes in Example 2; Figure 10This is an LFD detection band diagram of the sample treated with release agent S for 15 minutes in Example 2; Figure 11 This is an LFD detection band diagram of the sample treated with release agent S for 30 minutes in Example 2; Figure 12 This is an LFD detection band diagram of the sample treated with release agent S for 60 min in Example 2; Figure 13 This is an LFD detection band diagram of the sample treated with Wang Fang release agent for 15 minutes in Example 2; Figure 14 This is an LFD detection band image of the sample treated with Wang Fang release agent for 30 minutes in Example 2; Figure 15 This is an LFD detection band diagram of the sample treated with Wang Fang release agent for 60 minutes in Example 2; Figure 16 This is a flowchart of a method for preparing a nucleic acid releasing agent for isothermal amplification proposed in this invention; Figure 17 This is a flowchart illustrating a method for using a nucleic acid releasing agent for isothermal amplification, as proposed in this invention. Detailed Implementation

[0020] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see the appendix Figure 1 -Appendix Figure 17 This invention provides a method for preparing a nucleic acid releasing agent for isothermal amplification, comprising the following steps: Step 1: Prepare diluents for polyoxypropylene-polyoxyethylene copolymer, defoamer SE-15, ethyl phenyl polyethylene glycol NP-40, sodium polyethylene sulfonate, and Proclin 300 sequentially using RNase-free water. Furthermore, in step one, the dilution ratios of the polyoxypropylene-polyoxyethylene copolymer diluent, the defoamer SE-15 diluent, the ethyl phenyl polyethylene glycol NP-40 diluent, and the Proclin 300 diluent are all 5 times. The preparation method involves mixing the polyoxypropylene-polyoxyethylene copolymer stock solution, the defoamer SE-15 stock solution, the ethyl phenyl polyethylene glycol NP-40 stock solution, and the Proclin 300 stock solution with RNase-free water at a volume ratio of 1:4. The dilution ratio of the sodium polyethylene sulfonate diluent is 1000 times, and the preparation method involves mixing the sodium polyethylene sulfonate stock solution with RNase-free water at a volume ratio of 1:999.

[0022] Specifically, in step one, the preparation of the diluent requires selecting an appropriate dilution factor based on the concentration characteristics of each raw material stock solution to ensure that each component reaches the target effective concentration during subsequent mixing. As an option, the dilution factor for the polyoxypropylene / polyoxyethylene copolymer diluent, defoamer SE-15 diluent, ethylphenyl polyethylene glycol NP-40 diluent, and Proclin 300 diluent is set to 5 times. The preparation method involves separately measuring the polyoxypropylene / polyoxyethylene copolymer stock solution, defoamer SE-15 stock solution, ethylphenyl polyethylene glycol NP-40 stock solution, and Proclin 300 stock solution, and mixing each stock solution with RNase-free water at a volume ratio of 1:4. During mixing, a vortex mixing method is required to ensure thorough integration of the stock solution and RNase-free water. The vortex speed can be controlled at 1500-2000 rpm, and the mixing time is 20-30 seconds.

[0023] The preparation of sodium polyvinyl sulfonate diluent needs to be different from the other four diluents. Because the concentration of sodium polyvinyl sulfonate stock solution is extremely high, and it needs to play a role in binding proteins in the sample and reducing the residual amplification inhibitors in the subsequent release agent, its dilution factor is set to 1000 times. The preparation method is to measure the sodium polyvinyl sulfonate stock solution and mix it with RNase-free water at a volume ratio of 1:999. When mixing, the RNase-free water should be added slowly while gently inverting the container to avoid generating air bubbles that may affect subsequent mixing. After mixing, it can be dispensed into 1.5mL sterile centrifuge tubes, labeled, and stored in a refrigerator at 4℃ for no more than 7 days.

[0024] Step 2: Prepare disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, and glycine mother liquor sequentially using RNase-free water; Furthermore, in step two, the concentration of the disodium ethylenediaminetetraacetate mother liquor is 50 mM. It is prepared by dissolving disodium ethylenediaminetetraacetate in RNase-free water, adjusting the pH to 8.0, and then bringing the volume to a final volume. The concentration of the sodium dodecyl sulfate mother liquor is 10 mM. It is prepared by dissolving sodium dodecyl sulfate in RNase-free water at 50°C, cooling to room temperature, and then bringing the volume to a final volume. The concentration of the glycine mother liquor is 50 mM. It is prepared by weighing glycine, dissolving it in RNase-free water, adjusting the pH to 7.4, and then bringing the volume to a final volume.

[0025] Specifically, in step two, each mother liquor needs to be prepared as a fixed-volume solution of a specific concentration so that it can be added to the mixture in a fixed volume to ensure that the concentration of each component in the final solution is controllable. Specifically, disodium ethylenediaminetetraacetate, as a chelating agent, needs to inhibit nuclease activity by chelating metal ions in the sample; its mother liquor concentration needs to be sufficient to effectively exert a chelating effect in the final solution. Sodium dodecyl sulfate, as a surfactant, needs to assist in sample lysis by denaturing proteins; too high a concentration may affect the subsequent isothermal amplification enzyme activity, while too low a concentration will result in insufficient lysis. Glycine is used to maintain the pH stability of the release agent system, providing a suitable stable environment for nucleic acids; its concentration needs to be adapted to other components to avoid pH fluctuations.

[0026] The concentration of the disodium ethylenediaminetetraacetate (EDTA) stock solution was set at 50 mM. The preparation method involved weighing an appropriate amount of EDTA, adding it to RNase-free water, and stirring to dissolve. Since EDTA has low solubility in a neutral environment, the pH of the solution was adjusted to 8.0 using 1 mol / L NaOH solution. After complete dissolution, the solution was transferred to a volumetric flask and brought to the target volume with RNase-free water. The volumetric flask was then inverted 5-10 times to ensure uniform concentration.

[0027] The concentration of sodium dodecyl sulfonate mother liquor was set at 10 mM. The preparation method was to weigh the corresponding mass of sodium dodecyl sulfonate, add RNase-free water, and place it in a 50°C water bath. During this time, gently stir with a glass rod to assist dissolution, avoiding high temperature from damaging the structure of sodium dodecyl sulfonate. After complete dissolution, remove it, cool it to room temperature, and then transfer it to a volumetric flask and make up to volume with RNase-free water.

[0028] The concentration of the glycine stock solution was set at 50 mM. The preparation method was as follows: weigh an appropriate amount of glycine, add RNase-free water and stir until completely dissolved, adjust the pH to 7.4 with 1 mol / L HCl solution, and then transfer to a volumetric flask and make up to volume with RNase-free water.

[0029] Step 3: Mix the polyoxypropylene-polyoxyethylene copolymer diluent, defoamer SE-15 diluent, ethyl phenyl polyethylene glycol NP-40 diluent, sodium polyethylene sulfonate diluent, Proclin 300 diluent, disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, glycine mother liquor, and RNase-free water to obtain a mixture. Further, in step three, the mixing involves adding RNase-free water to the mixing container, followed by the sequential addition of polyoxypropylene-polyoxyethylene copolymer diluent, defoamer SE-15 diluent, ethyl phenyl polyethylene glycol NP-40 diluent, sodium polyethylene sulfonate diluent, disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, glycine mother liquor, and Proclin 300 diluent. The mixture is then vortexed for uniform mixing at a rotation speed of 2000-2500 rpm for 30 seconds to 1 minute.

[0030] Furthermore, in step three, the amount of each component in the mixture per 1 mL is as follows: 10 μL of polyoxypropylene-polyoxyethylene copolymer diluent, 2.5 μL of defoamer SE-15 diluent, 5 μL of ethyl phenyl polyethylene glycol NP-40 diluent, 30 μL of sodium polyethylene sulfonate diluent, 20 μL of disodium ethylenediaminetetraacetate mother liquor, 60 μL of sodium dodecyl sulfonate mother liquor, 50 μL of glycine mother liquor, 2.5 μL of Proclin 300 diluent, and the remainder is RNase-free water.

[0031] Specifically, in step three, when mixing, RNase-free water should be added to the mixing container first. RNase-free water serves as a solvent base, reducing the local concentration of each concentrated component when added later and minimizing adverse interactions between components. Then, the following components are added sequentially: polyoxypropylene-polyoxyethylene copolymer diluent, defoamer SE-15 diluent, ethyl phenyl polyethylene glycol NP-40 diluent, sodium polyethylene sulfonate diluent, disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, glycine mother liquor, and Proclin 300 diluent. Each component must be pre-mixed after addition to ensure complete dispersion before adding the next component.

[0032] Step three involves vortex mixing, with the vortex speed set at 2000-2500 rpm and the mixing time at 30 seconds to 1 minute. In some embodiments, if the volume of the mixture is large, vortex mixing can be performed in stages: after adding 3-4 components, vortex for 15-20 seconds, then add the remaining components and continue vortexing to further ensure the mixing effect. The amount of each component in the mixture must be strictly controlled to ensure that each component in 1 mL of the mixture can work synergistically. Specifically, the amounts of each component per 1 mL of the mixture are set as follows: 10 μL of polyoxypropylene / polyoxyethylene copolymer diluent, 2.5 μL of defoamer SE-15 diluent, 5 μL of ethyl phenyl polyethylene glycol NP-40 diluent, 30 μL of sodium polyvinyl sulfonate diluent, 20 μL of disodium ethylenediaminetetraacetate stock solution, 60 μL of sodium dodecyl sulfonate stock solution, 50 μL of glycine stock solution, and 2.5 μL of Proclin 300 diluent. The remaining volume is made up to 1 mL with RNase-free water. The 10 μL of polyoxypropylene / polyoxyethylene copolymer diluent ensures that its concentration in the final solution is sufficient to help destroy the viral capsid. The 30 μL of sodium polyvinyl sulfonate diluent effectively binds to protein inhibitors in the sample, and the 20 μL of disodium ethylenediaminetetraacetate stock solution ensures that its concentration in the final solution reaches 1 mM, meeting the requirements for chelating metal ions.

[0033] Step 4: After sterile filtration of the mixture, dispense it into sterile containers to obtain the nucleic acid release agent.

[0034] Furthermore, in step four, the aseptic filtration process involves using a sterile syringe to push the mixture through a cellulose acetate membrane for filtration.

[0035] Furthermore, in step four, the sterile container is a sterile centrifuge tube made of polypropylene.

[0036] Specifically, the purpose of aseptic filtration in step four is to remove any particulate impurities and microorganisms that may be present in the mixture, preventing contamination during subsequent sample processing. Dispensing facilitates single-use and reduces contamination and concentration changes caused by repeated opening. Specifically, aseptic filtration uses a sterile syringe to push the mixture through a cellulose acetate membrane. The cellulose acetate membrane has a pore size of 0.22 μm, effectively trapping bacteria, fungi, and other microorganisms while allowing small molecules to pass through. Sterile containers are selected from polypropylene centrifuge tubes. Storage conditions are set as follows: no more than 2 hours at room temperature (20-25°C), no more than 7 days at 4°C, and no more than 3 months at -20°C, with no more than 3 freeze-thaw cycles to avoid component degradation due to repeated freeze-thaw cycles.

[0037] This invention utilizes a multifunctional combination of sodium polyvinyl sulfonate (a polyvinyl sulfonate) as an anti-inhibition component, ethylphenyl polyethylene glycol NP-40 as a denaturing component, and disodium ethylenediaminetetraacetate (EDTA) and glycine as a synergistic agent. A standardized preparation process is employed, including dilution ratio, stock solution concentration preparation, sequential mixing, and aseptic filtration and dispensing. Sodium polyvinyl sulfonate specifically binds to protein amplification inhibitors in the sample; ethylphenyl polyethylene glycol NP-40 deeply denatures impurities, clearing mucosal mucin, hemoglobin, polysaccharides, and other inhibitors from swab samples; disodium EDTA chelates metal ions to reduce the impact of nucleases on target nucleic acids; and glycine maintains pH stability to match the activity requirements of key enzymes in isothermal amplification. The standardized preparation process ensures consistent concentrations of all functional components in each batch of the release agent, avoiding fluctuations in anti-inhibition effects due to component inhomogeneity. This prevents non-specific binding of inhibitors to key enzymes in isothermal amplification, achieving stable enzyme activity and efficient amplification of target nucleic acids during isothermal amplification. This solves the problem of weak anti-amplification inhibitor capabilities in traditional nucleic acid release agents, which affects amplification efficiency and stability.

[0038] A nucleic acid release agent for isothermal amplification is prepared by the above-described preparation method.

[0039] Specifically, the polyoxypropylene-polyoxyethylene copolymer, along with ethylphenyl polyethylene glycol NP-40 and sodium dodecyl sulfonate, works together to release nucleic acids by disrupting the cell membranes of cells or the capsid structure of viruses in the sample. Disodium ethylenediaminetetraacetate (EDTA) chelates magnesium and calcium ions in the sample, inhibiting nuclease activity and preventing nucleic acid degradation. Glycine maintains the pH of the release agent system at 7.2-7.6, thus matching the requirements of nucleic acids. Sodium polyvinyl sulfonate binds to amplification inhibitors such as proteins and hemoglobin in the sample, reducing their interference with subsequent isothermal amplification. Defoamer SE-15 eliminates bubbles generated during sample processing, preventing them from affecting sample elution and subsequent sample loading. Proclin 300 acts as a preservative, inhibiting microbial growth and extending the shelf life of the release agent. RNase-free water serves as a solvent, ensuring uniform dispersion of all components. A method for using a nucleic acid releasing agent for isothermal amplification, comprising the above-mentioned nucleic acid releasing agent for isothermal amplification, including the following steps: Insert the swab sample into the container containing the nucleic acid release agent, rotate the swab to elute the sample into the nucleic acid release agent, remove the swab, and obtain the mixture; After incubating the container containing the mixture, if there is residue in the mixture, the mixture is centrifuged and the supernatant is taken as a template for isothermal amplification.

[0040] Furthermore, the incubation temperature is 37℃ for 15 minutes, and the isothermal amplification is recombinase nuclease isothermal amplification, with the amplification temperature at 39-42℃ and the amplification incubation time at 10-15 minutes.

[0041] Specifically, swab samples must be processed within one hour of collection to avoid nucleic acid degradation due to prolonged storage. The swab sample is inserted into a container containing the aforementioned nucleic acid release agent, and the swab is rotated to elute the sample into the agent. The swab is then removed, yielding a mixture. The container containing the mixture is then incubated, and subsequent processing is selected based on the clarity of the mixture. Generally, incubation allows the lysis components in the release agent to fully function, ensuring complete nucleic acid release. Specifically, the incubation temperature is set at 37°C to promote the activity of the lysis components while avoiding nucleic acid degradation due to high temperatures; the incubation time is set at 15 minutes. After incubation, if the mixture contains mucosal residue or other impurities, it needs to be centrifuged, and the supernatant used as a template for isothermal amplification. The centrifugation speed is set at 3000 rpm, and the centrifugation time is set at 1 minute. If the mixture is clear and free of obvious residue, the supernatant can be directly used as a template for isothermal amplification.

[0042] Isothermal amplification, specifically recombinase nuclease isothermal amplification (RPA), requires no temperature cycling and allows for rapid nucleic acid amplification at a constant temperature, exhibiting good compatibility with nucleic acid release agents. Specifically, the amplification temperature is set at 39-42℃, which is the optimal activity temperature range for key RPA enzymes such as recombinases and single-stranded binding proteins; the incubation time is set at 10-15 minutes. Alternatively, the amplification system also includes enzyme beads, magnesium ion beads, upstream and downstream primers, and a probe. The concentrations of the upstream and downstream primers are set at 10 μmol / L, and the probe concentration is also set at 10 μmol / L to ensure specific binding of the primers and probe to the target nucleic acid, while avoiding false positives caused by primer dimers or non-specific probe binding.

[0043] The following is a further description with reference to specific embodiments: Example 1 Effect of release agent volume on sensitivity: The effect of different release agents at different volumes on the detection sensitivity was tested, using SARS-CoV-2 as the sample. 3, 4, and 5 refer to the viral sample concentration after release agent treatment, and 10, 20, and 30 refer to further dilutions of 0, 2, and 3 times at different viral concentrations.

[0044] Experimental steps: Add pharyngeal swabs and virus samples at volumes of 1, 1.5, and 2 ml of release agent (3, 4, and 5 refer to the original virus solution diluted 10³, 10⁴, and 10⁵ times, respectively), process for 10 min, and use as templates for RPA amplification.

[0045] To perform the RPA experiment: Add one enzyme ball and one magnesium ion ball to each reaction in sequence, add 0.5u of each upstream and downstream primer, add 0.25u of probe, and add template to reach a total volume of 25u.

[0046] After mixing, incubate at 42 degrees Celsius for 10 minutes.

[0047] To perform the LFD experiment, dilute the reaction solution with 200 μL of water and transfer it to the test strip using a pipette. Observe the bands after ten minutes.

[0048] Please refer to the experimental results. Figures 1-6 : 1. The effect of the release agent was improved at 1 ml and 1.5 ml. A band appeared in sample 4 (i.e., the original sample diluted 10*4), and disappeared as the sample concentration decreased. However, this was not observed at 2 ml, and no band appeared in sample 4 at any concentration.

[0049] 2. The self-prepared release agent could detect sample 4 at volumes of 1, 1.5, and 2 ml, and showed a decreasing trend in sensitivity as the sample concentration decreased. Overall, the 1 and 1.5 ml volumes performed better. The self-prepared release agent was a nucleic acid release agent prepared using a method for preparing nucleic acid release agents for isothermal amplification.

[0050] Example 2 Template degradation rate experiment of release agent: The ability of different release agents to preserve RNA after treating pharyngeal swabs was detected. Virus-containing pharyngeal swabs treated with release agents were left to stand for different times as templates and used for amplification to determine the impact on detection sensitivity.

[0051] Experimental steps: 1. Add 200u of the release agent (three types of release agents) to 10u of the SARS-CoV-2 sample, mix well, and let stand for 15 / 30 / 60 min respectively. Use as a template.

[0052] 2. Perform RPA experiments: Add one enzyme ball and one magnesium ion ball to each reaction in sequence, add 0.5u of each upstream and downstream primer, add 0.25u of probe, and add template to reach a total volume of 25u.

[0053] 3. After mixing, incubate at 42 degrees Celsius for 15 minutes.

[0054] 4. Perform the LFD experiment. Dilute the reaction solution with 200 μL of water and transfer it to the test strip using a pipette. Observe the band after ten minutes.

[0055] Please refer to the experimental results. Figures 7-15All three release agents were detectable in sample 5 within the first 15 minutes. Even after 60 minutes of treatment, the other two release agents were still detectable in sample 5. The band for sample 5 became faint or undetectable due to degradation in the other two release agents.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a nucleic acid releasing agent for isothermal amplification, characterized in that, Includes the following steps: Step 1: Prepare diluents for polyoxypropylene-polyoxyethylene copolymer, defoamer SE-15, ethyl phenyl polyethylene glycol NP-40, sodium polyethylene sulfonate, and Proclin 300 sequentially using RNase-free water. Step 2: Prepare disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, and glycine mother liquor sequentially using RNase-free water; Step 3: Mix the polyoxypropylene-polyoxyethylene copolymer diluent, defoamer SE-15 diluent, ethyl phenyl polyethylene glycol NP-40 diluent, sodium polyethylene sulfonate diluent, Proclin 300 diluent, disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, glycine mother liquor, and RNase-free water to obtain a mixture. Step 4: After sterile filtration of the mixture, dispense it into sterile containers to obtain the nucleic acid release agent.

2. The method for preparing a nucleic acid releasing agent for isothermal amplification according to claim 1, characterized in that: In step one, the dilution ratios of the polyoxypropylene-polyoxyethylene copolymer diluent, the defoamer SE-15 diluent, the ethyl phenyl polyethylene glycol NP-40 diluent, and the Proclin 300 diluent are all 5 times. The preparation method involves mixing the polyoxypropylene-polyoxyethylene copolymer stock solution, the defoamer SE-15 stock solution, the ethyl phenyl polyethylene glycol NP-40 stock solution, and the Proclin 300 stock solution with RNase-free water at a volume ratio of 1:

4. The dilution ratio of the sodium polyethylene sulfonate diluent is 1000 times, and the preparation method involves mixing the sodium polyethylene sulfonate stock solution with RNase-free water at a volume ratio of 1:

999.

3. The method for preparing a nucleic acid releasing agent for isothermal amplification according to claim 1, characterized in that: In step two, the concentration of the disodium ethylenediaminetetraacetate mother liquor is 50 mM. It is prepared by dissolving disodium ethylenediaminetetraacetate in RNase-free water, adjusting the pH to 8.0, and then bringing the volume to a final volume. The concentration of the sodium dodecyl sulfonate mother liquor is 10 mM. It is prepared by dissolving sodium dodecyl sulfonate in RNase-free water at 50°C, cooling to room temperature, and then bringing the volume to a final volume. The concentration of the glycine mother liquor is 50 mM. It is prepared by weighing glycine, dissolving it in RNase-free water, adjusting the pH to 7.4, and then bringing the volume to a final volume.

4. The method for preparing a nucleic acid releasing agent for isothermal amplification according to claim 1, characterized in that: In step three, the mixing process involves adding RNase-free water to a mixing container, followed by the sequential addition of polyoxypropylene-polyoxyethylene copolymer diluent, defoamer SE-15 diluent, ethyl phenyl polyethylene glycol NP-40 diluent, sodium polyethylene sulfonate diluent, disodium ethylenediaminetetraacetate mother liquor, sodium dodecyl sulfonate mother liquor, glycine mother liquor, and Proclin 300 diluent. The mixture is then vortexed to ensure uniform mixing. The vortex speed is 2000-2500 rpm, and the mixing time is 30 seconds to 1 minute.

5. The method for preparing a nucleic acid releasing agent for isothermal amplification according to claim 1, characterized in that: In step three, the mixture is prepared in the following amounts per 1 mL: 10 μL of polyoxypropylene-polyoxyethylene copolymer diluent, 2.5 μL of defoamer SE-15 diluent, 5 μL of ethyl phenyl polyethylene glycol NP-40 diluent, 30 μL of sodium polyethylene sulfonate diluent, 20 μL of disodium ethylenediaminetetraacetate mother liquor, 60 μL of sodium dodecyl sulfonate mother liquor, 50 μL of glycine mother liquor, 2.5 μL of Proclin 300 diluent, and the remainder is RNase-free water.

6. The method for preparing a nucleic acid releasing agent for isothermal amplification according to claim 1, characterized in that: In step four, the aseptic filtration process involves using a sterile syringe to push the mixture through a cellulose acetate membrane for filtration.

7. The method for preparing a nucleic acid releasing agent for isothermal amplification according to claim 1, characterized in that: In step four, the sterile container is a sterile centrifuge tube made of polypropylene.

8. A nucleic acid releasing agent for isothermal amplification, characterized in that: It is prepared by the preparation method according to any one of claims 1-7.

9. A method of using a nucleic acid releasing agent for isothermal amplification, characterized in that: The use of the nucleic acid releasing agent for isothermal amplification as described in claim 8 includes the following steps: Insert the swab sample into the container containing the nucleic acid release agent, rotate the swab to elute the sample into the nucleic acid release agent, remove the swab, and obtain a mixture; After incubating the container containing the mixture, if there is residue in the mixture, the mixture is centrifuged and the supernatant is taken as a template for isothermal amplification.

10. A method of using a nucleic acid releasing agent for isothermal amplification according to claim 9, characterized in that: The incubation temperature is 37℃ and the time is 15 minutes. The isothermal amplification is recombinase nuclease isothermal amplification, and the amplification process temperature is 39-42℃ and the amplification incubation time is 10-15 minutes.

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