Extraction-free virus swab sample nucleic acid releasing agent with preserving fluid function and application thereof

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to an extraction-free virus swab sample nucleic acid releasing agent with a preserving fluid function and application thereof. The invention provides an extraction-free virus swab sample nucleic acid releasing agent with a preserving fluid function and application thereof. The invention provides an extraction-free virus swab sample nucleic acid releasing agent with a preserving fluid function. The swab rinsing solution is characterized in that the swab rinsing solution can rapidly release viruses at room temperature (20 + / -5 DEG C), does not need complicated extraction steps, can be directly used for subsequent amplification detection of samples such as new coronavirus, can well preserve nucleic acid samples at room temperature (20 + / -5 DEG C), and has the function of a stable preserving solution.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to an extraction-free virus swab sample nucleic acid releaser with the function of a preservation fluid and an application thereof. Background Art

[0003] The nucleic acid test of the new coronavirus mainly includes the steps of collection and transportation, nucleic acid purification, and amplification detection. As an upper respiratory tract infection virus, the current collection of the new coronavirus mainly uses swab samples, mainly including nasal swabs, pharyngeal swabs, oral swabs, and anal swabs. Next, the collected swab samples are placed in the sample preservation solution. The current sample preservation solution mainly includes two categories, namely inactivated and non-inactivated preservation solutions. Before the amplification test, the collected samples must be extracted for nucleic acid to remove a large amount of harmful substances such as guanidine salts and metal chelators in the preservation solution that have an inhibitory effect on the amplification reaction, so as to avoid the occurrence of false negative results.

[0004] The experimental operation steps of nucleic acid extraction are cumbersome and the process is complicated, which greatly limits its universality in achieving bedside testing and increases the time from sample to result. In addition, conventional nucleic acid extraction also places extremely high demands on the test operators. The operators need to master professional experimental skills, and the positive or negative test results largely depend on the purity of the nucleic acid extracted by the experimenter. How to achieve the amplification test step directly on the collected swab samples without complicated extraction steps for nucleic acid samples is a key problem that needs to be solved urgently. Summary of the invention

[0005] In view of this, the present invention provides an extraction-free virus swab sample nucleic acid releaser having the function of a preservation fluid and an application thereof.

[0006] The present invention provides a nucleic acid releaser for virus swab samples that does not require extraction and has the function of a preservation solution, and its application. The present invention provides a nucleic acid releaser for virus swab samples that does not require extraction and has the function of a preservation solution. It is characterized by a swab washing solution that can quickly release viruses at room temperature (20°C ± 5°C), without the need for complicated extraction steps, and can be directly used for subsequent amplification detection of samples such as the new coronavirus. At the same time, it can also well preserve nucleic acid samples at room temperature (20°C ± 5°C), and has the function of a stable preservation solution.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a reagent combination, comprising 0.25-5% (v / v) of a membrane permeabilizing agent, 0-5% (v / v) of a nucleic acid stabilizer, 0-5% (v / v) of a reaction enhancer, and 0-50 mM of a reducing agent;

[0009] The above components are not 0 at the same time.

[0010] In some specific embodiments of the present invention, the reagent combination includes 1-5% (v / v) of a membrane permeabilizing agent, 1-5% (v / v) of a nucleic acid stabilizer, 0.5-2% (v / v) of a reaction enhancer, and 10-50 mM of a reducing agent;

[0011] Preferably, the reagent combination comprises 2-2.5% (v / v) of a membrane permeabilizing agent, 2% (v / v) of a nucleic acid stabilizer, 1% (v / v) of a reaction enhancer and 25 mM of a reducing agent.

[0012] In some specific embodiments of the present invention, the membrane-breaking agent includes one or more of TritonX-100, NP 40, Tween-20 or CA630;

[0013] The nucleic acid stabilizer includes one or more of DMSO, betaine, BSA, formamide or spermidine;

[0014] The reaction enhancer includes one or more of glycerol, L-proline, glycine, taurine or PEG8000;

[0015] The reducing agent includes TCEP and / or DTT.

[0016] In some specific embodiments of the present invention, the membrane permeabilizing agent includes TritonX-100 and Tween-20.

[0017] In some specific embodiments of the present invention, the TritonX-100 is 2-5% (v / v); the Tween-20 is 1-4% (v / v).

[0018] In some specific embodiments of the present invention, the TritonX-100 is 2% (v / v); the Tween-20 is 2.5% (v / v).

[0019] In some specific embodiments of the present invention, the nucleic acid stabilizer is betaine.

[0020] In some specific embodiments of the present invention, the reaction enhancer is glycerol.

[0021] In some specific embodiments of the present invention, the reducing agent is DTT.

[0022] The present invention also provides the use of the reagent combination in preparing a nucleic acid releasing agent and / or a preservation solution.

[0023] Based on the above research, the present invention also provides a nucleic acid release agent and / or a preservation solution, including the reagent combination.

[0024] The present invention also provides the use of any of the following items in preparing a kit for detecting nucleic acid, amplifying nucleic acid and / or preserving nucleic acid:

[0025] (1), the reagent combination; and / or

[0026] (2) The nucleic acid releasing agent and / or storage solution.

[0027] The present invention also provides a kit, comprising any of the following items:

[0028] (1), the reagent combination; and / or

[0029] (2) The nucleic acid releasing agent and / or storage solution.

[0030] The present invention also provides a nucleic acid amplification method, which comprises releasing nucleic acid in a nucleic acid sample to be tested by any of the following methods, and amplifying the obtained nucleic acid:

[0031] (1), the reagent combination; and / or

[0032] (2) the nucleic acid releasing agent and / or storage solution; and / or

[0033] (3) The kit.

[0034] In some specific embodiments of the present invention, the CRISPR / Cas12a detection system includes:

[0035]

[0036] In some specific embodiments of the present invention, the Reporter comprises / ROX / TTATTATT / BHQ2 / .

[0037] In some specific embodiments of the present invention, the action time of any of the following items is 1 min and the action temperature is 15-25° C.:

[0038] (1), the reagent combination; and / or

[0039] (2) the nucleic acid releasing agent and / or storage solution; and / or

[0040] (3) The reagent combination or nucleic acid releasing agent in the kit.

[0041] The present invention also provides a method for preserving nucleic acid, comprising mixing nucleic acid with any of the following:

[0042] (1), the reagent combination; and / or

[0043] (2) the nucleic acid releasing agent and / or storage solution; and / or

[0044] (3) The reagent combination or storage solution in the kit.

[0045] The present invention provides a virus swab sample nucleic acid release agent with the function of a preservation solution and its application. The beneficial effect of the present invention is that the swab washing solution of the present invention can quickly release the virus at room temperature (20℃±5℃), and the virus sample can be inactivated in 1 minute, which effectively reduces the infection risk of the experimenter; the present invention does not require complicated extraction steps, and can be directly used for subsequent amplification detection of samples such as the new coronavirus, which greatly simplifies the operation of nucleic acid extraction; the present invention can also well preserve nucleic acid samples at room temperature (20℃±5℃), and has the function of a stable preservation solution, which is convenient for the storage and transportation of sample sampling tubes. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0047] Figure 1-a1 The test results of different concentrations and types of membrane permeabilizing agents (TritonX-100, NP 40) in the nucleic acid sample release agent of the embodiment are shown;

[0048] Figure 1-a2 The test results of different concentrations and types of membrane permeabilizing agents (Tween-20, CA630) in the nucleic acid sample release agent of the embodiment are shown;

[0049] Figure 1-b1 The test results of nucleic acid stabilizers (DMSO, betaine, BSA) of different concentrations and types in the nucleic acid sample releaser of the embodiment are shown;

[0050] Figure 1-b2 The test results of nucleic acid stabilizers (formamide, spermidine) of different concentrations and types in the nucleic acid sample releaser of the embodiment are shown;

[0051] Figure 1-c1 The test results of the nucleic acid sample release agent of the embodiment for different concentrations and types of reaction enhancers (glycerol, L-proline, glycine);

[0052] Figure 1-c2 The test results of the nucleic acid sample release agent of the embodiment for different concentrations and types of reaction enhancers (taurine, PEG8000);

[0053] Figure 1-d The test results of the nucleic acid sample release agent of the embodiment for different concentrations and types of reducing agents;

[0054] Figure 2 The test results of the nucleic acid sample release agent of the embodiment at different release times at room temperature;

[0055] Figure 3-a The results of the nucleic acid sample releaser test for the sensitivity of the novel coronavirus RT-LAMP (0 copies per milliliter to 500 copies per milliliter) are shown in the examples;

[0056] Figure 3-b The results of the nucleic acid sample releaser test for the sensitivity of the novel coronavirus RT-LAMP (1000 copies per milliliter to 5000 copies per milliliter);

[0057] Figure 4-a1 The test results of the storage effect of the nucleic acid sample of the nucleic acid sample release agent at room temperature (20°C ± 5°C) are shown in the embodiment;

[0058] Figure 4-a2 The test results of the storage effect of the nucleic acid sample of the nucleic acid sample release agent at room temperature (20°C ± 5°C) are shown in the embodiment;

[0059] Figure 4-b1 The test results of the storage effect of the nucleic acid sample of the nucleic acid sample release agent at 4°C are shown in the embodiment;

[0060] Figure 4-b2 The test results of the storage effect of the nucleic acid sample of the nucleic acid sample release agent at 4°C are shown in the embodiment;

[0061] Figure 4-c1 Shows the test results of the preservation effect of nucleic acid samples in commercial preservation solution at room temperature (20℃±5℃);

[0062] Figure 4-c2 Shows the test results of the preservation effect of nucleic acid samples in commercial preservation solution at room temperature (20℃±5℃);

[0063] Figure 4-d1 Shows the test results of the preservation effect of nucleic acid samples in commercial preservation solution at 4°C;

[0064] Figure 4-d2 Shows the test results of the storage effect of commercial preservation solution nucleic acid samples at 4°C. DETAILED DESCRIPTION

[0065] The present invention discloses an extraction-free virus swab sample nucleic acid releaser with the function of a preservation fluid and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be pointed out in particular that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0066] The purpose of the present invention is to provide a virus swab sample nucleic acid release agent that does not require extraction and has the function of a preservation solution. It is characterized by being a swab rinse solution that can quickly release the virus at room temperature (20°C ± 5°C), without the need for complicated extraction steps, and can be directly used for subsequent amplification detection of samples such as the new coronavirus. At the same time, it can also well preserve nucleic acid samples at room temperature (20°C ± 5°C), and has the function of a stable preservation solution.

[0067] The purpose of the present invention is to provide a swab rinse solution that can quickly release the virus at room temperature (20°C ± 5°C), without complicated extraction steps, and can be directly used for subsequent amplification detection of samples such as the new coronavirus. Therefore, the main components of the present invention need to meet two basic conditions: (1) they can effectively achieve virus lysis; (2) they will not cause a strong inhibitory effect on subsequent detection reactions. Based on the above two basic conditions, the present invention screens the concentration and type of the main components.

[0068] The nucleic acid release agent for the virus swab sample without extraction comprises a membrane disrupting agent, a nucleic acid stabilizer, a reaction enhancer, and a reducing agent.

[0069] Preferably, the membrane-breaking agent includes but is not limited to one or a combination of TritonX-100, NP 40, Tween-20 or CA630.

[0070] Preferably, the nucleic acid stabilizer includes but is not limited to one or a combination of DMSO, betaine, BSA, formamide or spermidine.

[0071] Preferably, the reaction enhancer includes, but is not limited to, one or a combination of glycerol, L-proline, glycine, taurine or PEG8000.

[0072] Preferably, the reducing agent includes but is not limited to TCEP or DTT, or a combination of the two.

[0073] Preferably, the concentration range of each component in the extraction-free virus swab sample nucleic acid releaser is: one membrane-breaking agent 2%-5% (v / v), another membrane-breaking agent 1%-4% (v / v), nucleic acid stabilizer 1%-5% (v / v), reaction enhancer 0.5%-2% (v / v), and reducing agent 10-50mM.

[0074] More preferably, the concentrations of the components in the extraction-free virus swab sample nucleic acid releaser are: 2% (v / v) of the one membrane-breaking agent, 2.5% (v / v) of the other membrane-breaking agent, 2% (v / v) of the nucleic acid stabilizer, 1% (v / v) of the reaction enhancer, and 25 mM of the reducing agent.

[0075] As a preferred embodiment, the extraction-free virus swab sample nucleic acid releaser of the present invention is composed of TritonX-100, Tween-20, betaine, glycerol, DTT and DEPC water.

[0076] Preferably, the concentration range of each component in the extraction-free virus swab sample nucleic acid releaser is: TritonX-100 is 2%-5% (v / v), Tween-20 is 1%-4% (v / v), betaine is 1%-5% (v / v), glycerol is 0.5%-2% (v / v), and DTT is 10-50mM.

[0077] Most preferably, the concentrations of the components in the extraction-free virus swab sample nucleic acid releaser are: TritonX-100 is 2% (v / v), Tween-20 is 2.5% (v / v), betaine is 2% (v / v), glycerol is 1% (v / v), and DTT is 25mM.

[0078] The method of using the nucleic acid releaser for virus swab samples without extraction of the present invention is as follows: after the collected nasal / pharyngeal swab is completely immersed in 1 mL of nucleic acid sample releaser, the long handle of the swab is broken, and the sample with the swab head is immersed in the sampling tube, and the sample is left to stand at room temperature (20°C ± 5°C) for more than 1 minute, and the nucleic acid of the nasal / pharyngeal swab is released in the sample releaser, and the liquid in the tube can be directly used for subsequent nucleic acid detection by absorbing. The released nucleic acid solution can be directly added to the amplification reaction solution as a virus template, and the nucleic acid solution of the same volume as the amplification system can also be absorbed to directly dissolve the amplification reaction freeze-dried pellet for amplification reaction.

[0079] Table 1

[0080]

[0081]

[0082] Due to the requirements of the software used in the sequence table, U is represented by T. The actual sequence is subject to SEQ ID No. 1 in the specification.

[0083] The extraction-free virus swab sample nucleic acid release agent with the function of a preservation solution provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0084] The present invention will be further described below in conjunction with embodiments:

[0085] Example 1 Screening and use of the main components of nucleic acid sample release agents

[0086] The test method is as follows: prepare 40 μL of liquid with a single component of the corresponding final concentration, dilute the new coronavirus pseudovirus containing the Orf1ab gene fragment (Cat. No.: FNV-2019-ncov-abEN, Fubaiao Biopharmaceutical Technology Co., Ltd.) to 1000 copies / μL, then take 1 μL of the diluted new coronavirus pseudovirus and add it to 40 μL of RT-LAMP freeze-dried balls (Cat. No.: A3828-02, Xinhai Gene Detection Co., Ltd.), the system contains primers of the Orf1ab gene (SEQ ID No. 2-SEQ ID No. 7), the primer sequences are shown in Table 1, and the final concentrations of each primer in the system are as follows: 0.2 μM F3, 0.2 μM B3, 1.6 μM FIP, 1.6 μM BIP, 0.8 μM LF, 0.8 μM LB. At this time, the liquid completely dissolves the corresponding freeze-dried system, amplifies at 65°C in a fluorescent quantitative PCR instrument for 30 minutes, and observes the peak time of the amplification curve. After the reaction, 3 μL of the RT-LAMP reaction product was aspirated and added to 10 μL of the CRISPR / Cas12a detection system (Table 2), and the reaction was carried out at 37°C for 30 min in a fluorescence quantitative PCR instrument, and the fluorescence increment was monitored in real time.

[0087] Test results such as Figure 1-a1 , Figure 1-a2 , Figure 1-b1 , Figure 1-b2 , Figure 1-c1 , Figure 1-c2 As shown in Figure 1d, for different single components, the main observations are whether there is a large delay in the peak time of the first step RT-LAMP reaction, and whether the final fluorescence value of the second step CRISPR / Cas12a after the 20-min reaction is low. Figure 1-a1 , Figure 1-a2 The results are for the detection of different types and concentrations of membrane permeabilization agents. It can be considered that TritonX-100, NP 40, Tween-20 or CA630 will not have a significant impact on the detection reaction. Figure 1-b1 , Figure 1-b2 The test results of nucleic acid stabilizers of different types and concentrations show that DMSO, betaine and BSA will not have a significant effect on the detection reaction, and formamide will not have a significant effect on the detection reaction at a concentration below 1% (v / v). The addition of spermidine directly leads to reaction inhibition. Figure 1-c1 , Figure 1-c2 The results of the detection of reaction enhancers of different types and concentrations show that the four types of reaction enhancers, glycerol, L-proline, glycine, taurine or PEG8000, have the effect of enhancing the detection reaction. Figure 1-dThe results of the detection of reducing agents of different types and concentrations show that TCEP will not have a significant effect on the detection reaction at a concentration below 10 mM, while TCEP will inhibit the reaction at a concentration of 25 mM or above, and DTT will have a significant effect at a concentration of 100 mM or above, but will not inhibit the reaction at 50 mM or below. According to the screening of the above components, the most preferred formula of the novel coronavirus nucleic acid sample releaser of the present invention includes the components shown in Table 3:

[0088] The CRISPR / Cas12a detection system is shown in Table 2:

[0089] Table 2 CRISPR / Cas12a detection system

[0090]

[0091] Buffer 2.1 and Cas12a enzyme were purchased from New England Biotechnology (Beijing) Co., Ltd., with the catalog number M0653T. The reporter sequence was / ROX / TTATTATT / BHQ2 / , synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the gRNA was SEQ ID No. 1 in Table 1.

[0092] Table 3 Formal formula of nucleic acid sample release agent

[0093]

[0094] According to the formula in Table 3, 1 mL of nucleic acid sample release agent is prepared, and the mixed solution is shaken and mixed to obtain the finished nucleic acid release agent. After the collected nasal / pharyngeal swab is completely immersed in 1 mL of nucleic acid sample release agent, the long handle of the swab is broken, and the sample with the swab head is immersed in the sampling tube. Let it stand at room temperature (20℃±5℃) for 10 minutes, and the nasal / pharyngeal swab nucleic acid will be released into the sample release agent. The liquid in the tube can be directly used for subsequent nucleic acid testing. The released nucleic acid solution can be directly added to the amplification reaction solution as a virus template, and the same volume of nucleic acid solution as the amplification system can be directly dissolved in the amplification reaction freeze-dried beads for amplification reaction.

[0095] Example 2 Different release time test

[0096] The release time and release temperature of the nucleic acid sample release agent in Example 1 were tested.

[0097] The nucleic acid sample release agent in Example 1 was configured in a sample storage tube, and the throat swabs of volunteers were collected and rinsed in the release agent. After the new coronavirus pseudovirus containing the Orf1ab gene fragment (Cat. No.: FNV-2019-ncov-abEN, Fubaiao Biopharmaceutical Technology Co., Ltd.) was diluted to 1000 copies / μL, 1 μL of the diluted new coronavirus pseudovirus was drawn and added to the nucleic acid sample release agent rinsed with the throat swab, so that the final concentration of the new coronavirus nucleic acid in each tube of the sample storage tube was 1000 copies / mL. After lysing for 1min, 5min, 10min, and 15min at room temperature (20°C ± 5°C), a 40μL volume of the nucleic acid releaser solution containing the pharyngeal swab was drawn to dissolve the RT-LAMP freeze-dried reagent (Article No.: A3828-02, Xinhai Gene Detection Co., Ltd.) of the 40μL reaction system, the system contains primers for the Orf1ab gene (SEQ ID No. 2-SEQ ID No. 7), the primer sequences are shown in Table 1, and the final concentrations of each primer in the system are as follows: 0.2μM F3, 0.2μM B3, 1.6μM FIP, 1.6μM BIP, 0.8μM LF, 0.8μM LB. Amplify at 65°C for 30min. After the reaction, 3μL of the RT-LAMP reaction product was drawn and added to 10μL of the CRISPR / Cas12a detection system (same as Example 1), and the fluorescence quantitative PCR instrument was reacted at 37°C for 20min and the fluorescence increment was monitored in real time.

[0098] Test results such as Figure 2 As shown in the detection results of the new coronavirus Orf1ab gene, the nucleic acid sample releaser of the present invention can detect the nucleic acid of the new coronavirus by standing and lysing at room temperature (20℃±5℃) for 1 minute, and the detection limit can reach 1000 copies / mL, that is, 40 copies / reaction.

[0099] Example 3: Single RT-LAMP sensitivity detection of novel coronavirus

[0100] The nucleic acid sample releaser in Example 1 was tested for its sensitivity to the single RT-LAMP of the new coronavirus.

[0101] Using the same method as in Example 2, the new crown pseudovirus was diluted to 250 copies / μL, 500 copies / μL, 1000 copies / μL, 2000 copies / μL and 5000 copies / μL. 1μL of the diluted new crown pseudovirus was sequentially drawn and added to the release agent rinsed with the throat swab, so that the final concentration of the new crown nucleic acid in each tube of sample storage tube was 250 copies / mL, 500 copies / mL, 1000 copies / mL, 2000 copies / mL and 5000 copies / mL. Lyse at room temperature (20°C ± 5°C) for 1min, and perform the same method as in Example 2 to perform a single RT-LAMP amplification reaction on each concentration of the new crown nucleic acid lysate and then perform CRISPR / Cas12a detection.

[0102] Test results such as Figure 3-a , Figure 3-b As shown, the detection limit of the single-plex RT-LAMP for the Orf1ab gene of the new coronavirus of the present invention can reach 250 copies / mL, that is, 10 copies / reaction.

[0103] Example 4 Preservation performance of nucleic acid sample release agent

[0104] The preservation effect of the nucleic acid sample release agent in Example 1 on the nucleic acid sample was tested and compared with a commercial preservation solution.

[0105] Using the same method as in Example 3, the new crown pseudovirus was diluted to 500 copies / μL, 1000 copies / μL, 2000 copies / μL and 5000 copies / μL. 1 μL of the new crown pseudovirus was drawn in sequence and added to the release agent of the pharyngeal swab, and 1 μL of the new crown pseudovirus was also drawn in sequence and added to the commercial preservation solution (Article No.: CWY072H, Kangwei Century Biotechnology Co., Ltd.). The final concentration of the new crown nucleic acid in each tube of sample preservation tube was 500 copies / mL, 1000 copies / mL, 2000 copies / mL and 5000 copies / mL, respectively. After the nucleic acid sample release agent of the present invention and the commercial preservation solution were stored at room temperature (20°C ± 5°C) and 4°C refrigerator for 1 day, 2 days, 3 days, and 4 days, the single RT-LAMP amplification reaction of the new crown nucleic acid lysate of each concentration was performed and then CRISPR / Cas12a detection was performed.

[0106] Figure 4-a1 , Figure 4-a2 and Figure 4-b1 , Figure 4-b2The storage effects of the nucleic acid sample releaser of the present invention at room temperature (20°C ± 5°C) and in a 4°C refrigerator were demonstrated. The peak time of the first step RT-LAMP reaction of the present invention did not show a significant delay after 4 days of storage compared with 0 days. It can be considered that the nucleic acid sample releaser of the present invention has a good storage effect on nucleic acid samples, and the viral nucleic acid does not show significant degradation. Whether stored at room temperature (20°C ± 5°C) or in a 4°C refrigerator, the nucleic acid sample can be stably stored for 4 days.

[0107] Figure 4-c1 , Figure 4-c2 and Figure 4-d1 , Figure 4-d2 The storage effect of the commercial storage solution at room temperature (20℃±5℃) and in a 4℃ refrigerator was demonstrated. On the 4th day at room temperature (20℃±5℃), the nucleic acid sample with a low copy number (500 copies / mL) showed a significant delay in the peak time, and the viral nucleic acid was degraded. Compared with the commercial storage solution of the control group, the nucleic acid sample releaser of the present invention showed a better storage effect under the storage conditions of room temperature (20℃±5℃) and for nucleic acid samples with low viral load.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A reagent combination, characterized in that: The method comprises 0.25-5% (v / v) of a membrane permeabilizing agent, 0-5% (v / v) of a nucleic acid stabilizer, 0-5% (v / v) of a reaction enhancer, and 0-50 mM of a reducing agent; The above components are not 0 at the same time.

2. The reagent combination according to claim 1, characterized in that The method comprises 1-5% (v / v) of a membrane-breaking agent, 1-5% (v / v) of a nucleic acid stabilizer, 0.5-2% (v / v) of a reaction enhancer and 10-50 mM of a reducing agent.

3. The reagent combination according to claim 1 or 2, characterized in that The membrane-breaking agent includes one or more of TritonX-100, NP40, Tween-20 or CA630; The nucleic acid stabilizer includes one or more of DMSO, betaine, BSA, formamide or spermidine; The reaction enhancer includes one or more of glycerol, L-proline, glycine, taurine or PEG8000; The reducing agent includes TCEP and / or DTT.

4. Use of the reagent combination according to any one of claims 1 to 3 in the preparation of a nucleic acid releasing agent and / or a preservation solution.

5. A nucleic acid releasing agent and / or storage solution, characterized in that: The method comprises the reagent combination according to any one of claims 1 to 3.

6. Use of any of the following in the preparation of a kit for detecting nucleic acid, amplifying nucleic acid and / or preserving nucleic acid: (1) The reagent combination according to any one of claims 1 to 3; and / or (2) The nucleic acid releasing agent and / or storage solution according to claim 5.

7. A kit, characterized in that Include any of the following: (1) The reagent combination according to any one of claims 1 to 3; and / or (2) The nucleic acid releasing agent and / or storage solution according to claim 5.

8. A nucleic acid amplification method, characterized in that: The nucleic acid in the nucleic acid sample to be tested is released by any of the following methods, and the obtained nucleic acid is amplified: (1) The reagent combination according to any one of claims 1 to 3; and / or (2) The nucleic acid releasing agent and / or storage solution according to claim 5; and / or (3) The kit according to claim 7.

9. A nucleic acid detection method, characterized in that: The nucleic acid in the nucleic acid sample to be tested is released by any of the following methods, the obtained nucleic acid is amplified, and CRISPR / Cas12a is used for detection: (1) The reagent combination according to any one of claims 1 to 3; and / or (2) The nucleic acid releasing agent and / or storage solution according to claim 5; and / or (3) The kit according to claim 7.

10. A method for preserving nucleic acid, characterized in that: Mix the nucleic acid with any of the following: (1) The reagent combination according to any one of claims 1 to 3; and / or (2) The nucleic acid releasing agent and / or storage solution according to claim 5; and / or (3) The reagent combination or storage solution in the kit according to claim 7.

Images