Nucleic acid releasing agent and use method thereof

Abstract

The invention provides a nucleic acid releasing agent and a use method, and relates to the technical field of biological research, by screening main influence factors of the nucleic acid releasing agent and optimizing the concentration, the nucleic acid releasing agent can fully release nucleic acid of pathogens in a clinical sample while ensuring that the nucleic acid releasing agent does not generate an inhibition effect on a subsequent detection system, and the detection accuracy is improved. The nucleic acid releasing agent provided by the invention can be directly used for sampling and storage, and can be stably stored for 3 days at room temperature and 7 days at 2-8 DEG C according to stability research, and the nucleic acid releasing agent can be used for subsequent detection, so that the detection sensitivity is improved. The nucleic acid releasing agent can efficiently release nucleic acid and ensure the quality and quantity of nucleic acid extraction by accurately adjusting the concentration and proportion of each component, a reliable basis is provided for subsequent detection and analysis, accurate diagnosis and research are facilitated, Tris-Cl in the nucleic acid releasing agent controls the pH value within a proper range, the natural structure and function of nucleic acid are kept, and the nucleic acid releasing agent has good application prospects. And degradation and denaturation are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biological research technology, and more specifically, particularly relates to a nucleic acid releasing agent and a use method thereof. Background Art

[0002] Nucleic acid extraction is one of the necessary processes in the current molecular diagnostic product testing. By lysing and breaking human cells and pathogens (bacteria, viruses, etc.) to release the nucleic acid (DNA / RNA) substances inside, and then undergoing certain purification steps, nucleic acids with higher concentrations and purity can be obtained for subsequent molecular detection processes such as fluorescent PCR, constant temperature amplification (Lamp, etc.), and gene sequencing. The quality (concentration and purity) of the nucleic acid obtained has a great impact on the success or failure of the subsequent detection process. Therefore, research on nucleic acid extraction is also a hot topic in the current molecular diagnostic industry.

[0003] At present, there are three main nucleic acid extraction methods and kits commonly used on the market: magnetic bead method, column method and sample release agent. The extraction principles and steps of the magnetic bead method and the column method are similar. The difference lies in the medium used for nucleic acid adsorption. The magnetic bead method uses magnetic beads to adsorb and purify nucleic acids, and the column method uses adsorption columns as nucleic acid adsorption media. The main processes of the two are generally three steps, namely lysis / adsorption, washing and elution. The extraction and purification of nucleic acids are completed through a series of transfer, mixing, separation and other steps, and the obtained nucleic acid concentration and purity are high. Sample release agent is a nucleic acid extraction reagent that has been increasingly widely used in recent years. It uses a one-step method to lyse and break cells, pathogens, etc. in clinical samples. The released nucleic acids can be used in subsequent molecular detection processes without purification. Its advantages are simple and fast operation, suitable for rapid detection application scenarios such as POCT. The disadvantage is that the concentration and purity of the nucleic acid obtained are lower than those of the magnetic bead method and column method, which has a certain impact on the sensitivity of subsequent molecular detection. At present, conventional sample release agents on the market are mainly used in fluorescent PCR detection projects. Its main operating steps include:

[0004] 1. The sample release agent is mixed with the clinical sample in a certain proportion;

[0005] 2. Leave at room temperature for a few minutes to complete the release of nucleic acid;

[0006] 3. Directly take a smaller volume of the mixture for subsequent molecular testing.

[0007] The existing magnetic bead method and column method nucleic acid extraction reagents have complicated operation processes, require professional equipment and personnel to operate, have high reagent and labor costs, and have low detection efficiency. The existing nucleic acid releasers need to be added with clinical samples, and the sample addition process has a certain dilution effect on the clinical samples. In order to avoid the inhibitory effect of the components in the nucleic acid releaser on the subsequent detection, the amount of nucleic acid template added is small, resulting in low sensitivity of subsequent detection. The first purpose of the present invention is to provide a nucleic acid releaser and a method of use that can be directly used for sampling, storage, nucleic acid release and subsequent detection, which solves the problems of complicated operation, high cost and low detection efficiency of magnetic bead method and column method nucleic acid extraction reagents. The second object of the present invention is to provide a nucleic acid releaser and a method of use that can be directly used for sampling, preservation, nucleic acid release and subsequent detection, which solves the disadvantage that conventional sample releasers on the market require an additional sample addition process and cannot be used for sample and nucleic acid preservation. The third object of the present invention is to provide a nucleic acid releaser and a method of use that can be directly used for sampling, preservation, nucleic acid release and subsequent detection. Since the nucleic acid releaser has a small inhibitory effect on subsequent detection, a nucleic acid template of up to 100% can be added to the subsequent detection system, which solves the disadvantage that the conventional sample releasers on the market have a small sample addition amount resulting in insufficient sensitivity. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a nucleic acid releasing agent and a method of use to solve the above problems.

[0009] A nucleic acid releaser comprises Tris-Cl, KOH, KCl, EDTA, BSA, TritonX-100, Tween-20, SDS, NaCl and Proclin-300. Tris-Cl is a buffer used to maintain the pH value of a solution within the range of 7.2-7.8. KOH can adjust the pH value of the solution. KCl helps to maintain the ionic strength and osmotic pressure. EDTA can chelate metal ions and play a stabilizing role. BSA can stabilize and protect nucleic acids and prevent the degradation of nucleic acids. TritonX-100 is a non-ionic surfactant that helps to lyse cells and promote the release of nucleic acids. Tween-20 is a surfactant that assists in cell lysis and nucleic acid release. SDS is a strong anionic surfactant that can destroy cell membranes and release nucleic acids. NaCl maintains a certain ionic environment. Proclin-300 is a preservative that prevents reagents from deteriorating.

[0010] Preferably, the pH of Tris-Cl is 7.2-7.8. Controlling the pH of Tris-Cl at 7.2-7.8, which is close to the physiological pH range, is conducive to maintaining the natural structure and function of nucleic acids, reducing the degradation and denaturation of nucleic acids, and providing a relatively stable and suitable chemical environment for various biochemical reactions in the process of nucleic acid release, so that related reactions can be carried out efficiently and accurately, and are compatible with the activity conditions of many biological molecules and enzymes, which is convenient for subsequent analysis and detection operations. The relatively mild pH environment can reduce the adverse effects on cells and other sample components, and help to release nucleic acids completely.

[0011] Preferably, the content of Tris-Cl is 25-100mM, the content of KOH is 1-10mM, the content of KCl is 5-20mM, the content of EDTA is 0.1-2mM, and the content of BSA is 0.1-10mg / mL. As a buffer, Tris-Cl can not only maintain pH stability, but also buffer pH fluctuations caused by external factors such as temperature changes and reaction progress. In the complex biochemical process of nucleic acid release, slight pH changes may affect the activity of the enzyme and the direction of the reaction. Tris-Cl ensures that these changes are minimized, thereby ensuring the consistency and reliability of nucleic acid release. The pH adjustment effect of KOH is not just a simple acid-base neutralization. It can fine-tune the pH value of the solution so that it falls precisely within the range most suitable for nucleic acid release and stability. Moreover, in some specific reaction mechanisms, KOH may also participate in specific chemical reactions, such as activating certain enzymes or inducing the breakage of specific chemical bonds, thereby promoting the release of nucleic acids from cell structures.

[0012] Preferably, the volume percentage of TritonX-100 is 0.01% to 0.1%. As a nonionic surfactant, the mechanism of TritonX-100 in lysing cells is not only to destroy the cell membrane. It can also interact with various lipids and proteins in the cell, change their conformation and function, thereby promoting cell lysis and nucleic acid release. Moreover, the mildness of TritonX-100 makes it relatively less damaging to nucleic acids while lysing cells, which helps to maintain the integrity of nucleic acids.

[0013] Preferably, the volume percentage of Tween-20 is 0.01% to 0.2%. In addition to assisting cell lysis and nucleic acid release, the Tween-20 surfactant can also improve the surface tension and wettability of the solution. This helps the nucleic acid release agent to better contact and penetrate the sample, thereby improving the reaction efficiency. At the same time, Tween-20 can also reduce the adsorption between the sample and the container wall, further improving the recovery rate of the nucleic acid.

[0014] Preferably, the mass percentage of SDS is 0.0001% to 0.01%. The strong anionic surfactant properties of SDS give it a unique advantage in destroying cell membranes. It can form complexes with lipids and proteins on the cell membrane, causing the rupture and dissolution of the cell membrane. In addition, SDS can also bind to nucleic acids, changing the charge distribution and conformation of nucleic acids, thereby facilitating the release of nucleic acids from cell structures. However, the strong effect of SDS also needs to be carefully controlled, and excessive use may lead to excessive denaturation or damage of nucleic acids.

[0015] Preferably, the mass percentage of NaCl is 0.01% to 0.5%. The ionic environment maintained by NaCl is not just a simple electrolyte background. It can affect the electrostatic interaction and the formation of ionic bonds in the solution, thereby affecting the folding and function of proteins and nucleic acids. During the release of nucleic acids, a suitable ionic environment can promote the activity of related enzymes and proteins and improve the reaction efficiency. At the same time, NaCl can also adjust the osmotic pressure of the solution, synergistically with KCl to ensure the normal physiological state of the cells.

[0016] Preferably, the volume percentage of Proclin-300 is 0.005% to 0.1%. As a preservative, Proclin-300 not only prevents the reagent from deteriorating, but also inhibits the growth and reproduction of microorganisms, reduces nucleic acid degradation and erroneous results caused by microbial contamination. In the case of long-term storage and multiple uses, Proclin-300 can ensure the quality and stability of the nucleic acid release agent, and provide a guarantee for the reliability of the experimental results.

[0017] Another technical problem to be solved by the present invention is to provide a method for using a nucleic acid releasing agent, comprising the following steps:

[0018] S1: sampling;

[0019] S2: Save;

[0020] S3: Detection.

[0021] Preferably, the swab after sampling is broken and placed in a storage tube containing a nucleic acid release agent for storage at room temperature for no more than 3 days, and at 2-8°C for no more than 7 days.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention screens the main influencing factors of the nucleic acid releaser and optimizes the concentration, so that it can fully release the nucleic acid of the pathogen in the clinical sample while ensuring that it does not have an inhibitory effect on the subsequent detection system. The maximum proportion of sampling can be used for subsequent detection, which increases the amount of nucleic acid template in the detection system and improves the detection sensitivity.

[0024] 2. The nucleic acid releaser of the present invention can be directly used for sampling and storage. Through stability research, it is determined that it can be stably stored for 3 days at room temperature and for 7 days at 2-8°C.

[0025] 3. The nucleic acid releaser of the present invention can efficiently release nucleic acid by accurately adjusting the concentration and ratio of each component, ensuring the quality and quantity of nucleic acid extraction, providing a reliable basis for subsequent detection and analysis, and facilitating accurate diagnosis and research.

[0026] 4. Tris-Cl in the nucleic acid releaser controls the pH in an appropriate range, maintains the natural structure and function of nucleic acids, reduces degradation and denaturation, enables various biochemical reactions to proceed efficiently and accurately, adapts to the activity conditions of many biological molecules and enzymes, and is very beneficial for subsequent analysis and detection operations.

[0027] 5. Tris-Cl as a buffer not only maintains pH stability, but also buffers pH fluctuations caused by external factors, ensuring the consistency and reliability of nucleic acid release and the repeatability of experimental results.

[0028] 6. In addition to acid-base neutralization, KOH can also fine-tune the pH of the solution so that it falls precisely within the appropriate range, and may participate in specific reactions to promote nucleic acid release, thereby improving the effect and controllability of nucleic acid release.

[0029] 7. KCl is essential for maintaining ionic strength and osmotic pressure, which can affect protein folding and function, maintain cell integrity and normal morphology, and avoid affecting the efficiency and quality of nucleic acid release due to osmotic pressure problems.

[0030] 8. EDTA creates a pure chemical environment for nucleic acid release by chelating metal ions, improving the controllability and accuracy of the release process and avoiding the adverse effects of metal ions on nucleic acids.

[0031] 9. In addition to stabilizing and protecting nucleic acids, BSA can also form a protective network, bind and isolate harmful substances, increase solution viscosity, reduce physical losses, and improve nucleic acid recovery rates.

[0032] 10. As a non-ionic surfactant, TritonX-100 has a comprehensive cell lysis mechanism, causes little damage to nucleic acids, maintains the integrity of nucleic acids, and its mildness is also conducive to its wide application.

[0033] 11. Tween-20 improves the surface tension and wettability of the solution, increases reaction efficiency, reduces adsorption of samples to the container wall, and further increases nucleic acid recovery rate.

[0034] 12. SDS has unique advantages in destroying cell membranes. It can change the charge distribution and conformation of nucleic acids, which is beneficial to the release of nucleic acids. At the same time, appropriate dosage control ensures the quality of nucleic acids. The ionic environment maintained by NaCl affects the folding and function of proteins and nucleic acids, and works synergistically with KCl to ensure the normal physiological state of cells and improve reaction efficiency.

[0035] 13. As a preservative, Proclin-300 not only prevents the reagents from deteriorating, but also inhibits the growth and reproduction of microorganisms, ensuring the quality and stability of nucleic acid releasers and the reliability of experimental results.

[0036] 14. Performance verification tests have proven that the optimized nucleic acid releaser can release pathogen nucleic acids more efficiently, has high detection sensitivity, and is consistent for different pathogen samples, broadening the scope of application. Stability verification tests have determined the stable storage time of the nucleic acid releaser under different conditions, providing clear guidance for practical applications and ensuring the effectiveness of the reagent within a reasonable time. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the components of the nucleic acid releasing agent of the present invention. DETAILED DESCRIPTION

[0038] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0039] See also Figure 1 The present invention provides a nucleic acid releasing agent, comprising 25-100 mM Tris-Cl (PH7.2-7.8), 1-10 mM KOH, 5-20 mM KCl, 0.1-2 mM EDTA, 0.1-10 mg / mL BSA, 0.01%-0.1% by volume of TritonX-100, 0.01%-0.2% by volume of Tween-20, 0.0001%-0.01% by mass of SDS, 0.01%-0.5% by mass of NaCl, and 0.005%-0.1% by volume of Proclin-300.

[0040] Tris-Cl is a buffer used to maintain the pH of the solution within the range of 7.2-7.8. KOH can adjust the pH of the solution. KCl helps maintain ionic strength and osmotic pressure. EDTA can chelate metal ions and play a stabilizing role. BSA can stabilize and protect nucleic acids and prevent their degradation. TritonX-100 is a non-ionic surfactant that helps lyse cells and promote nucleic acid release. Tween-20 is a surfactant that assists in cell lysis and nucleic acid release. SDS is a strong anionic surfactant that can destroy cell membranes and release nucleic acids. NaCl maintains a certain ionic environment. Proclin-300 is a preservative that prevents reagents from deteriorating.

[0041] Controlling the pH of Tris-Cl at 7.2-7.8, close to the physiological pH range, is conducive to maintaining the natural structure and function of nucleic acids, reducing the degradation and denaturation of nucleic acids, and providing a relatively stable and suitable chemical environment for various biochemical reactions in the process of nucleic acid release, so that related reactions can be carried out efficiently and accurately, and adapting to the activity conditions of many biological molecules and enzymes, which is convenient for subsequent analysis and detection operations. The milder pH environment can reduce the adverse effects on cells and other sample components and help to release nucleic acids completely.

[0042] As a buffer, Tris-Cl can not only maintain pH stability, but also buffer pH fluctuations caused by external factors such as temperature changes, reaction progress, etc. In the complex biochemical process of nucleic acid release, small pH changes may affect enzyme activity and reaction direction. Tris-Cl ensures that these changes are minimized, thereby ensuring the consistency and reliability of nucleic acid release.

[0043] The pH adjustment of KOH is more than a simple acid-base neutralization. It can fine-tune the pH of the solution so that it falls precisely within the range that is most suitable for nucleic acid release and stabilization. Moreover, in some specific reaction mechanisms, KOH may also participate in specific chemical reactions, such as activating certain enzymes or initiating the breaking of specific chemical bonds, thereby promoting the release of nucleic acids from cell structures.

[0044] KCl is essential for maintaining ionic strength and osmotic pressure. Appropriate ionic strength can affect the folding and function of proteins. Some enzymes and proteins related to nucleic acid release can only exert their best activity under specific ionic strength. At the same time, stable osmotic pressure helps maintain the integrity and normal morphology of cells, avoiding cell rupture or shrinkage caused by osmotic pressure imbalance, which affects the efficiency and quality of nucleic acid release.

[0045] The metal ion chelation of EDTA does more than just prevent metal ion-catalyzed nucleic acid degradation. Many metal ions are involved in various signal transduction and metabolic pathways within cells, and their presence may interfere with the process of nucleic acid release or cause unwanted side reactions. By chelating these metal ions, EDTA creates a pure chemical environment for nucleic acid release, improving the controllability and accuracy of the release process.

[0046] In addition to stabilizing and protecting nucleic acids, BSA can also interact with other components to form a protective network. It can bind and isolate substances that may damage nucleic acids, such as free radicals, heavy metal ions, etc. In addition, BSA can increase the viscosity of the solution, reduce the physical loss of nucleic acids during operation, and improve the recovery rate of nucleic acids.

[0047] As a non-ionic surfactant, TritonX-100's mechanism of cell lysis is not just to destroy the cell membrane. It can also interact with various lipids and proteins in the cell, changing their conformation and function, thereby promoting cell lysis and nucleic acid release. Moreover, the mildness of TritonX-100 makes it relatively less damaging to nucleic acids while lysing cells, which helps to maintain the integrity of nucleic acids.

[0048] In addition to assisting cell lysis and nucleic acid release, Tween-20 surfactant can also improve the surface tension and wettability of the solution. This helps the nucleic acid release agent to better contact and penetrate the sample, improving the reaction efficiency. At the same time, Tween-20 can also reduce the adsorption between the sample and the container wall, further improving the recovery rate of nucleic acids.

[0049] The strong anionic surfactant properties of SDS give it a unique advantage in destroying cell membranes. It can form complexes with lipids and proteins on the cell membrane, leading to the rupture and dissolution of the cell membrane. In addition, SDS can also bind to nucleic acids, changing the charge distribution and conformation of nucleic acids, thereby facilitating the release of nucleic acids from cell structures. However, the strong effect of SDS also needs to be carefully controlled, and excessive use may lead to excessive denaturation or damage of nucleic acids.

[0050] The ionic environment maintained by NaCl does more than just provide a simple electrolyte background. It can affect the electrostatic interactions and ionic bond formation in the solution, thereby affecting the folding and function of proteins and nucleic acids. During the release of nucleic acids, a suitable ionic environment can promote the activity of related enzymes and proteins and improve the reaction efficiency. At the same time, NaCl can also regulate the osmotic pressure of the solution, synergizing with KCl to ensure the normal physiological state of the cell.

[0051] As a preservative, Proclin-300 does more than just prevent reagents from spoiling. It can also inhibit the growth and reproduction of microorganisms, reducing nucleic acid degradation and erroneous results caused by microbial contamination. In the case of long-term storage and multiple uses, Proclin-300 can ensure the quality and stability of nucleic acid release agents, providing a guarantee for the reliability of experimental results.

[0052] The performance verification test of nucleic acid releaser was carried out to verify the release effect of nucleic acid releaser on nucleic acid of different pathogens, the influence on subsequent detection system and detection sensitivity. The test materials were: samples of various known pathogens (such as bacteria, viruses, etc.), nucleic acid releaser, nucleic acid extraction kit, real-time fluorescence quantitative PCR detection reagents and instruments, blank control samples. The test process is as follows:

[0053] Step 1: Prepare pathogen samples with different concentration gradients, such as high, medium, and low concentrations;

[0054] Step 2: Take equal amounts of pathogen samples with different concentrations and divide them into several groups, with the same sample size in each group;

[0055] Step 3: Add different formulations or concentrations of nucleic acid releasers (including formulations before and after optimization) to each group of samples, and set up a blank control group (only adding samples without adding nucleic acid releasers);

[0056] Step 4: Incubate for a certain period of time under appropriate conditions to allow the nucleic acid releaser to fully function;

[0057] Step 5: Use a nucleic acid extraction kit to extract nucleic acid from each group of samples;

[0058] Step 6: Use real-time fluorescence quantitative PCR detection reagent to detect the extracted nucleic acid and record the Ct value (cycle threshold).

[0059] The test results show that the optimized nucleic acid releaser treatment group can release pathogen nucleic acids more efficiently than the unoptimized one, which is manifested by a smaller Ct value corresponding to the same concentration of pathogen samples. In high, medium and low concentration pathogen samples, as the sample concentration decreases, the Ct value gradually increases, but the optimized nucleic acid releaser can still effectively release nucleic acids in low concentration samples, and the Ct value is relatively small, indicating that the detection sensitivity is high. The blank control group should have no obvious amplification signal, and the Ct value should be none or extremely large, indicating that the nucleic acid releaser itself has no inhibitory effect on the subsequent detection system. The test results of different pathogen samples should be consistent, that is, the nucleic acid releaser has improved the release and detection sensitivity of nucleic acids of multiple pathogens.

[0060] Conduct a nucleic acid releaser stability verification test to determine the stable storage time of the nucleic acid releaser under different conditions. The test materials include nucleic acid releaser, known nucleic acid samples, nucleic acid detection kits, incubators or environmental settings under different temperature conditions (room temperature, 2-8°C). The test process is as follows:

[0061] Step 1: Prepare multiple identical known nucleic acid samples;

[0062] Step 2: Immediately mix a portion of the nucleic acid sample with the nucleic acid releaser as the initial control group, immediately conduct nucleic acid testing, and record the test results;

[0063] Step 3: Mix other nucleic acid samples with the nucleic acid releaser and place them in room temperature and 2-8°C environment respectively;

[0064] Step 4: Take out samples for nucleic acid testing on the first, second and third days at room temperature;

[0065] Step 5: Under the condition of 2-8℃, samples were taken out for nucleic acid testing on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th days respectively;

[0066] Step 6: Use the nucleic acid detection kit to conduct nucleic acid detection on the samples taken out and compare the results with those of the initial control group.

[0067] The experimental results show that the nucleic acid test results of the initial control group should be normal and stable. Under room temperature conditions, there is no significant difference in the test results on the 1st and 2nd days compared with the initial control group, and the test results on the 3rd day may be slightly lower but still within an acceptable range, indicating that it can be stably stored for 3 days. At 2-8°C, there is no significant difference in the test results of the first 6 days compared with the initial control group, and the test results on the 7th day may begin to show a certain degree of change, indicating that it can be stably stored for 7 days.

[0068] The present invention screens the main influencing factors of the nucleic acid releaser and optimizes the concentration, so that the nucleic acid of the pathogen in the clinical sample can be fully released while ensuring that no inhibition is exerted on the subsequent detection system, and the maximum proportion of sampling can be used for subsequent detection, thereby increasing the amount of nucleic acid template in the detection system and improving the detection sensitivity. The nucleic acid releaser of the present invention can be directly used for sampling and storage, and it is determined through stability research that the nucleic acid releaser can be stably stored for 3 days at room temperature and 7 days at 2-8°C. The nucleic acid releaser of the present invention can efficiently release nucleic acid by accurately adjusting the concentration and proportion of each component, thereby ensuring the quality and quantity of nucleic acid extraction, providing a reliable basis for subsequent detection and analysis, and facilitating accurate diagnosis and research.

[0069] Tris-Cl in the nucleic acid releaser controls the pH within a suitable range, maintains the natural structure and function of nucleic acids, reduces degradation and denaturation, enables various biochemical reactions to be carried out efficiently and accurately, adapts to the activity conditions of many biological molecules and enzymes, and is very beneficial for subsequent analysis and detection operations. Tris-Cl as a buffer not only maintains pH stability, but also buffers pH fluctuations caused by external factors, ensures the consistency and reliability of nucleic acid release, and ensures the repeatability of experimental results. In addition to acid-base neutralization, KOH can also fine-tune the solution pH to make it fall precisely within the appropriate range, and may participate in specific reactions to promote nucleic acid release, thereby improving the effect and controllability of nucleic acid release. KCl is essential for maintaining ionic strength and osmotic pressure, can affect protein folding and function, and maintain cell The integrity and normal morphology of cells can be maintained, and the efficiency and quality of nucleic acid release can be avoided by osmotic pressure problems. EDTA creates a pure chemical environment for nucleic acid release by chelating metal ions, which improves the controllability and accuracy of the release process and avoids the adverse effects of metal ions on nucleic acids. In addition to stabilizing and protecting nucleic acids, BSA can also form a protective network, bind and isolate harmful substances, increase solution viscosity, reduce physical losses, and improve nucleic acid recovery. TritonX-100, as a non-ionic surfactant, has a comprehensive cell lysis mechanism, causes little damage to nucleic acids, maintains nucleic acid integrity, and its mildness is also conducive to wide application. Tween-20 improves the surface tension and wettability of the solution, improves reaction efficiency, reduces the adsorption of samples and container walls, and further improves the nucleic acid recovery rate.

[0070] SDS has unique advantages in destroying cell membranes. It can change the charge distribution and conformation of nucleic acids, which is beneficial to the release of nucleic acids. At the same time, appropriate dosage control ensures the quality of nucleic acids. The ionic environment maintained by NaCl affects the folding and function of proteins and nucleic acids, and works synergistically with KCl to ensure the normal physiological state of cells and improve reaction efficiency. Proclin-300, as a preservative, not only prevents reagent deterioration, but also inhibits the growth and reproduction of microorganisms, ensures the quality and stability of nucleic acid releasers, and guarantees the reliability of experimental results. Performance verification tests have proved that the optimized nucleic acid releaser can release pathogen nucleic acids more efficiently, has high detection sensitivity, and is consistent for different pathogen samples, broadening the scope of application. The stability verification test determines the stable storage time of nucleic acid releasers under different conditions, provides clear guidance for practical applications, and ensures the effectiveness of the reagents within a reasonable time.

[0071] A method for using a nucleic acid releasing agent comprises the following steps:

[0072] S1: Sampling: collect nasopharyngeal and oropharyngeal swabs using disposable sampling swabs according to the standard sampling process;

[0073] S2: Storage: break the swab after sampling and place it in a storage tube containing a nucleic acid release agent. Store it at room temperature for no more than 3 days, and at 2-8°C for no more than 7 days;

[0074] S3: Detection, direct sampling for subsequent fluorescence PCR, Lamp constant temperature and other tests. The sampling ratio refers to the instructions for subsequent detection reagents, and the maximum loading ratio is 100%.

[0075] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A nucleic acid releasing agent, characterized in that Including Tris-Cl, KOH, KCl, EDTA, BSA, TritonX-100, Tween-20, SDS, NaCl, Proclin-300.

2. A nucleic acid releasing agent as claimed in claim 1, characterized in that: The pH of Tris-Cl is 7.2~7.

8.

3. A nucleic acid releasing agent as claimed in claim 1, characterized in that: The Tris-Cl content is 25-100 mM, the KOH content is 1-10 mM, the KCl content is 5-20 mM, the EDTA content is 0.1-2 mM, and the BSA content is 0.1-10 mg / mL.

4. A nucleic acid releasing agent as claimed in claim 1, characterized in that: The volume percentage of TritonX-100 is 0.01% to 0.1%.

5. A nucleic acid releasing agent as claimed in claim 1, characterized in that: The volume percentage of Tween-20 is 0.01% to 0.2%.

6. A nucleic acid releasing agent as claimed in claim 1, characterized in that: The mass percentage of SDS is 0.0001% to 0.01%.

7. A nucleic acid releasing agent as claimed in claim 1, characterized in that: The mass percentage of NaCl is 0.01% to 0.5%.

8. A nucleic acid releasing agent as claimed in claim 1, characterized in that: The volume percentage of Proclin-300 is 0.005% to 0.1%.

9. A method for using a nucleic acid releasing agent, characterized in that: The following steps are involved: S1: sampling; S2: Save; S3: Detection.

10. A method for using a nucleic acid releasing agent as claimed in claim 9, characterized in that: After sampling, the swab should be broken and placed in a tube containing a nucleic acid releaser. It should be stored at room temperature for no more than 3 days and at 2-8°C for no more than 7 days.

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