Sample nucleic acid release, preservation and detection method

Abstract

The invention relates to a preparation for improving the release rate of nucleic acid in a sample and application thereof, specifically, the preparation comprises a preserving fluid and further comprises grinding filter beads. When the preparation is used for treating a sample, the nucleic acid release rate of the sample can be improved. Under the assistance of ultrasound and / or heating, the nucleic acid release rate of the sample is further improved, so that the sample collection and treatment procedures are simplified. The method for treating the sample by using the preparation is simple to operate, low in cost, rapid and convenient, and wide in application scene.

Description

Technical Field

[0001] The present invention relates to the technical field of sample detection, and in particular to a method for releasing, preserving and detecting sample nucleic acid. Background Art

[0002] Respiratory tract infection is the most common disease in people's daily lives. The pathogens that cause respiratory tract infection are usually contagious, and most hospitals have set up fever clinics for this purpose. Conventional molecular testing requires nucleic acid extraction and then PCR testing. This testing process usually takes about 2 hours, which is a long time overall. Fever patients are prone to pathogen transmission or cross-infection while waiting for the report.

[0003] In response to the above problems, technicians in this field have developed cartridge-type POCT detection instruments and reagents. Compared with the traditional magnetic bead method, cartridge-type reagents can shorten the entire detection time and reduce patient waiting time. However, the cartridge-type reagents have a high reagent cost due to their complex process, and their high testing costs have discouraged many patients. In addition, the quality of cartridge-type reagents cannot be fully controlled, and it is impossible to test every consumable. If there is a problem with the test, resampling may be required.

[0004] A release agent direct amplification method without nucleic acid extraction has also been developed. The swab sample is collected and immersed in a special release agent liquid to achieve the effect of releasing nucleic acid. However, this method is prone to false negative results. The main reason may be that this method lyses pathogens and releases nucleic acids, resulting in poor nucleic acid stability; in addition, RNA may be degraded before detection, resulting in false positives; at the same time, because this method has not been purified by nucleic acid, inhibitors collected from the patient's mouth, such as food residues, are easily introduced into the detection system, resulting in inhibition of the amplification reaction.

[0005] Therefore, there is an urgent need in the art for a method for sample preservation and nucleic acid release that is efficient, pollution-free, and does not damage nucleic acids. Summary of the invention

[0006] The purpose of the present invention is to provide a method for sample preservation and nucleic acid release which is efficient, pollution-free and does not damage nucleic acids.

[0007] Another object of the present invention is to provide a sample preservation and nucleic acid release solution that is highly efficient, pollution-free, and does not damage nucleic acids.

[0008] In a first aspect of the present invention, there is provided a preparation for sample preservation and nucleic acid release, the preparation comprising a preservation solution, the preservation solution comprising a release component, a nucleic acid stabilizing component and a buffer component;

[0009] Wherein, the release component is composed of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant;

[0010] The nucleic acid stabilizing component is composed of 0.01% to 10% (w / v) nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent;

[0011] The buffer component is 0.1-50 mM Tris;

[0012] Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof;

[0013] The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof;

[0014] The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof;

[0015] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.

[0016] In another preferred embodiment, the preservation solution consists of 0.01% to 5% (w / v) SDS, 0.01% to 5% (w / v) NP-40, 0.01% to 10% (w / v) trehalose, 0.1 to 20 mM EDTA and 0.1 to 50 mM Tris.

[0017] In another preferred embodiment, the preservation solution consists of 0.01% to 3% (w / v) SDS, 0.02% to 3% (w / v) NP-40, 0.05% to 5% (w / v) trehalose, 1 to 10 mM EDTA and 1 to 30 mM Tris.

[0018] In another preferred embodiment, the preservation solution consists of 0.01% to 1% (w / v) SDS, 0.02% to 2% (w / v) NP-40, 0.1% to 3% (w / v) trehalose, 3 to 8 mM EDTA and 10 to 20 mM Tris.

[0019] In another preferred embodiment, the storage solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0020] In another preferred embodiment, the storage solution contains no protein and no enzyme.

[0021] In another preferred embodiment, the preparation further comprises grinding filter beads.

[0022] In another preferred embodiment, the grinding filter beads are modified with hydroxyl groups or carboxyl groups.

[0023] In another preferred embodiment, the grinding filter beads are selected from the following group: quartz sand, zirconia beads, foam filter beads, activated carbon particles, bioceramic pellets, molecular sieves, fiber balls, or a combination thereof; preferably, bioceramic pellets and molecular sieves.

[0024] In another preferred embodiment, the concentration of the grinding filter beads in the preparation or the preservation solution is 0.01-5 g / mL, preferably 0.05-3 g / mL, preferably 0.1-2 g / mL, more preferably 0.2-1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL or 1 g / mL.

[0025] In another preferred embodiment, the pH of the preservation solution is 7-12, preferably 8-12, preferably 9-12, more preferably 10-12, for example about 11.

[0026] In a second aspect, the present invention provides a kit for sample preservation and nucleic acid release, the kit comprising:

[0027] A first container and a storage solution located in the first container, wherein the storage solution consists of a release component, a nucleic acid stabilizing component and a buffer component;

[0028] Wherein, the release component is composed of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant;

[0029] The nucleic acid stabilizing component is composed of 0.01% to 10% (w / v) nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent;

[0030] The buffer component is 0.1-50 mM Tris;

[0031] Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof;

[0032] The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof;

[0033] The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof;

[0034] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.

[0035] In another preferred embodiment, the kit further comprises grinding filter beads.

[0036] In another preferred embodiment, the preservation solution and the grinding filter beads are located in the same or different containers.

[0037] In another preferred embodiment, the grinding filter beads are modified with hydroxyl groups or carboxyl groups.

[0038] In another preferred embodiment, the grinding filter beads are selected from the following group: quartz sand, zirconia beads, foam filter beads, activated carbon particles, bioceramsite, molecular sieves, fiber balls, or a combination thereof.

[0039] In another preferred embodiment, the preservation solution consists of 0.01% to 5% (w / v) SDS, 0.01% to 5% (w / v) NP-40, 0.01% to 10% (w / v) trehalose, 0.1 to 20 mM EDTA and 0.1 to 50 mM Tris.

[0040] In another preferred embodiment, the preservation solution consists of 0.01% to 3% (w / v) SDS, 0.02% to 3% (w / v) NP-40, 0.05% to 5% (w / v) trehalose, 1 to 10 mM EDTA and 1 to 30 mM Tris;

[0041] Preferably, the preservation solution consists of 0.01% to 1% (w / v) SDS, 0.02% to 2% (w / v) NP-40, 0.1% to 3% (w / v) trehalose, 3 to 8 mM EDTA and 10 to 20 mM Tris;

[0042] More preferably, the preservation solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0043] In another preferred embodiment, when the preservation solution and the grinding filter beads are in the same container, the concentration of the grinding filter beads in the preservation solution is 0.01-5 g / mL, preferably 0.05-3 g / mL, preferably 0.1-2 g / mL, more preferably 0.2-1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL or 1 g / mL.

[0044] In another preferred embodiment, the pH of the preservation solution is 7-12, preferably 8-12, preferably 9-12, more preferably 10-12, for example about 11.

[0045] In another preferred embodiment, the storage solution contains no protein and no enzyme.

[0046] In another preferred embodiment, the kit further includes an instruction manual, which indicates that the kit is used for sample preservation and nucleic acid release, and also indicates a method of use for sample preservation and nucleic acid release.

[0047] In a third aspect, the present invention provides a method for increasing the release rate of nucleic acid in a sample, comprising the steps of:

[0048] (s1) providing a sample to be tested; and

[0049] (s2) adding the sample to a preservation solution and mixing the sample, thereby preserving the sample and increasing the release rate of nucleic acid in the sample;

[0050] The storage solution is composed of a release component, a nucleic acid stabilizing component and a buffer component;

[0051] Wherein, the release component is composed of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant;

[0052] The nucleic acid stabilizing component is composed of 0.01% to 10% (w / v) nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent;

[0053] The buffer component is 0.1-50 mM Tris;

[0054] Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof;

[0055] The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof;

[0056] The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof;

[0057] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.

[0058] In another preferred embodiment, the method further comprises step (s3) of adding grinding filter beads to the preservation solution containing the sample and mixing the mixture.

[0059] In another preferred embodiment, the grinding filter beads are modified with hydroxyl groups or carboxyl groups;

[0060] In another preferred embodiment, the grinding filter beads are selected from the following group: quartz sand, zirconia beads, foam filter beads, activated carbon particles, bioceramsite, molecular sieves, fiber balls, or a combination thereof.

[0061] In another preferred embodiment, the method further comprises step (s4) of subjecting the preservation solution containing the sample to ultrasonic and / or heating treatment.

[0062] In another preferred embodiment, the sample-containing preservation solution further contains grinding filter beads.

[0063] In another preferred embodiment, the preservation solution consists of 0.01% to 5% (w / v) SDS, 0.01% to 5% (w / v) NP-40, 0.01% to 10% (w / v) trehalose, 0.1 to 20 mM EDTA and 0.1 to 50 mM Tris.

[0064] In another preferred embodiment, the preservation solution consists of 0.01% to 3% (w / v) SDS, 0.02% to 3% (w / v) NP-40, 0.05% to 5% (w / v) trehalose, 1 to 10 mM EDTA and 1 to 30 mM Tris.

[0065] In another preferred embodiment, the preservation solution consists of 0.01% to 1% (w / v) SDS, 0.02% to 2% (w / v) NP-40, 0.1% to 3% (w / v) trehalose, 3 to 8 mM EDTA and 10 to 20 mM Tris.

[0066] In another preferred embodiment, the storage solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0067] In another preferred embodiment, the pH of the preservation solution is 7-12, preferably 8-12, preferably 9-12, more preferably 10-12, for example about 11.

[0068] In another preferred embodiment, in step (s3), 0.01 to 5 g of grinding filter beads are added to every 1 mL of the preservation solution.

[0069] In another preferred embodiment, in step (s3), 0.05-3 g of grinding filter beads are added to every 1 mL of preservation solution, preferably 0.1-2 g, more preferably 0.2-1 g, for example, about 0.2 g, 0.4 g, 0.6 g, 0.8 g or 1 g.

[0070] In another preferred embodiment, the sample is an oropharyngeal swab and / or a nasopharyngeal swab.

[0071] In another preferred embodiment, the sample contains pathogens, preferably viruses, bacteria and / or mycoplasmas, more preferably influenza A virus, adenovirus, Mycoplasma pneumoniae, human metapneumovirus, or a combination thereof.

[0072] In another preferred embodiment, the storage solution contains no protein and no enzyme.

[0073] In another preferred embodiment, in step (s4), the ultrasonic treatment and heating treatment are performed simultaneously or separately.

[0074] In another preferred embodiment, in step (s4), the frequency of the ultrasonic treatment is 30-50 kHZ, preferably 35-45 kHZ, and more preferably 35-40 kHZ.

[0075] In another preferred embodiment, in step (s4), the ultrasonic treatment is performed for 30 to 240 s, preferably 30 to 180 s, and more preferably 120 to 180 s.

[0076] In another preferred embodiment, in step (s4), the heating treatment is heating at 75-95°C for 1-5 min, preferably heating at 75-95°C for 1-3 min.

[0077] In another preferred embodiment, the nucleic acid release rate of the method is ≥85%, preferably ≥90%, and more preferably ≥95%.

[0078] In a fourth aspect, the present invention provides a method for detecting nucleic acid, comprising the steps of:

[0079] (z1) adding the sample to be tested into a preservation solution and mixing evenly, wherein the preservation solution comprises a release component, a nucleic acid stabilizing component and a buffer component;

[0080] Wherein, the release component is composed of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant;

[0081] The nucleic acid stabilizing component is composed of 0.01% to 10% (w / v) nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent;

[0082] The buffer component is 0.1-50 mM Tris;

[0083] Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof;

[0084] The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof;

[0085] The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof;

[0086] The metal ion chelator is selected from the group consisting of EDTA, EGTA, or a combination thereof; and

[0087] (z2) Performing nucleic acid testing on the storage solution containing the sample to be tested to obtain the nucleic acid test result.

[0088] In another preferred embodiment, between steps (z1) and (z2), there is also a step (z3) of adding grinding filter beads to the preservation solution containing the sample to be tested and mixing the mixture.

[0089] In another preferred embodiment, in step (z2), the preservation solution contains grinding filter beads and the sample to be tested.

[0090] In another preferred embodiment, between steps (z1) and (z2), there is further included a step (z4) of subjecting the storage solution containing the sample to be tested to ultrasonic and / or heat treatment.

[0091] In another preferred embodiment, between steps (z1) and (z2), both step (z3) and step (z4) are included.

[0092] In another preferred embodiment, step (z4) is performed after step (z3).

[0093] In another preferred embodiment, in the step (z4), the preservation solution contains grinding filter beads and the sample to be tested.

[0094] In another preferred embodiment, in step (z2), the preservation solution is diluted 1 to 20 times, preferably 5 to 15 times, more preferably 8 to 12 times, for example, about 10 times before nucleic acid detection.

[0095] In another preferred embodiment, the grinding filter beads are modified with hydroxyl groups or carboxyl groups.

[0096] In another preferred embodiment, the grinding filter beads are selected from the following group: quartz sand, zirconia beads, foam filter beads, activated carbon particles, bioceramsite, molecular sieves, fiber balls, or a combination thereof.

[0097] In another preferred embodiment, the anionic surfactant is sodium dodecyl sulfate; and the nonionic surfactant is NP-40.

[0098] In another preferred embodiment, the preservation solution consists of 0.01% to 5% (w / v) SDS, 0.01% to 5% (w / v) NP-40, 0.01% to 10% (w / v) trehalose, 0.1 to 20 mM EDTA and 0.1 to 50 mM Tris.

[0099] In another preferred embodiment, the preservation solution consists of 0.01% to 3% (w / v) SDS, 0.02% to 3% (w / v) NP-40, 0.05% to 5% (w / v) trehalose, 1 to 10 mM EDTA and 1 to 30 mM Tris.

[0100] In another preferred embodiment, the preservation solution consists of 0.01% to 1% (w / v) SDS, 0.02% to 2% (w / v) NP-40, 0.1% to 3% (w / v) trehalose, 3 to 8 mM EDTA and 10 to 20 mM Tris.

[0101] In another preferred embodiment, the storage solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0102] In another preferred embodiment, the pH of the preservation solution is 7-12, preferably 8-12, preferably 9-12, more preferably 10-12, for example about 11.

[0103] In another preferred embodiment, in step (z3), 0.01 to 5 g of grinding filter beads are added to every 1 mL of the preservation solution.

[0104] In another preferred embodiment, in step (z3), 0.05-3 g of grinding filter beads are added to every 1 mL of preservation solution, preferably 0.1-2 g, more preferably 0.2-1 g, for example, about 0.2 g, 0.4 g, 0.6 g, 0.8 g or 1 g.

[0105] In another preferred embodiment, the sample to be tested is an oropharyngeal swab and / or a nasopharyngeal swab.

[0106] In another preferred embodiment, the sample to be tested contains pathogens, preferably viruses, bacteria and / or mycoplasmas, more preferably influenza A virus, adenovirus, Mycoplasma pneumoniae, human metapneumovirus, or a combination thereof.

[0107] In another preferred embodiment, the storage solution contains no protein and no enzyme.

[0108] In another preferred embodiment, in step (z4), the ultrasonic treatment and heating treatment are carried out simultaneously or separately.

[0109] In another preferred embodiment, in step (z4), the frequency of the ultrasonic treatment is 30-50 kHZ, preferably 35-45 kHZ, and more preferably 35-40 kHZ.

[0110] In another preferred embodiment, in step (z4), the ultrasonic treatment is for 30 to 240 s, preferably 30 to 180 s, more preferably 120 to 180 s.

[0111] In another preferred embodiment, in step (z4), the heating treatment is heating at 75-95°C for 1-5 min, preferably heating at 75-95°C for 1-3 min.

[0112] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0113] After extensive and in-depth research, a large number of experiments and screening, the inventors unexpectedly discovered for the first time a preparation that combines sample preservation and processing, the preparation comprising a preservation solution and further comprising grinding filter beads. After a large number of experiments, the inventors optimized the components and concentrations of the preservation solution to increase the nucleic acid release rate of the sample. Experiments have shown that, with the assistance of ultrasound and / or heating, the use of the preparation of the present invention can further increase the nucleic acid release rate of the sample, thereby simplifying the sample collection and processing procedures. The method for processing samples using the preparation of the present invention is simple to operate, low in cost, fast and convenient, and has a wide range of application scenarios. The present invention was completed on this basis.

[0114] the term

[0115] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0116] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., are understood to include the stated elements or components but do not exclude other elements or components.

[0117] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0118] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and fractional values ​​thereof (e.g., one tenth and one hundredth of an integer) where appropriate.

[0119] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0120] As used herein, the term "SDS" is used interchangeably with "sodium dodecyl sulfate."

[0121] As used herein, the term "Tween-20" is used interchangeably with "Tween 20".

[0122] As used herein, the term "Triton-X-100" is used interchangeably with "Triton X-100".

[0123] As used herein, the term "Brij-35" is an ether compound generated by the reaction of lauryl alcohol and ethylene oxide, and is a nonionic surfactant having good emulsifying, wetting, spreading and solubilizing properties.

[0124] As used herein, the term "NP-40" is used interchangeably with "nonylphenol ethoxylate".

[0125] As used herein, the term "EDTA" is used interchangeably with "ethylenediaminetetraacetic acid."

[0126] As used herein, the term "EGTA" is used interchangeably with "ethylene glycol tetraacetic acid."

[0127] Magnetic bead method

[0128] The magnetic bead extraction reagent needs to release the pathogen nucleic acid from the pathogen and then adsorb it onto the magnetic beads; transfer the magnetic beads with the adsorbed nucleic acid to a washing solution containing salt to further remove protein impurities and lysate; wash the magnetic beads after one wash for a second time with a salt-free alcohol solution; dry the washed magnetic beads to remove ethanol residues; transfer the dried magnetic beads to a low-salt elution solution for elution. Each step takes at least 2-5 minutes and requires the use of magnets and other devices. If run on an instrument, it needs to be matched with a specific extraction instrument, and the entire process takes between 15 minutes and 30 minutes.

[0129] Preparations of the present invention

[0130] The preparation of the present invention is used for sample preservation and nucleic acid release, and comprises a preservation solution, wherein the preservation solution is composed of a release component, a nucleic acid stabilizing component and a buffer component;

[0131] Wherein, the release component is composed of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant;

[0132] The nucleic acid stabilizing component is composed of 0.01% to 10% (w / v) nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent;

[0133] The buffer component is 0.1-50 mM Tris;

[0134] Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof;

[0135] The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof;

[0136] The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof;

[0137] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.

[0138] In the preservation solution of the present invention, the combination of ionic surfactant and nonionic surfactant can reduce the pressure of the system and make the reaction system tolerate higher concentration of ionic surfactant. The preservation solution of the present invention includes nucleic acid protectant and metal chelator, and the combination of the two can further improve the tolerance of the amplification system to anionic surfactant.

[0139] In a preferred embodiment, the preservation solution consists of 0.01% to 5% (w / v) SDS, 0.01% to 5% (w / v) NP-40, 0.01% to 10% (w / v) trehalose, 0.1 to 20 mM EDTA and 0.1 to 50 mM Tris; preferably, the preservation solution consists of 0.01% to 3% (w / v) SDS, 0.02% to 3% (w / v) NP-40, 0.05% to 5% (w / v) trehalose, 1 to 10 mM EDTA and 1 to 30 mM Tris; preferably, the preservation solution consists of 0.01% to 1% (w / v) SDS, 0.02% to 2% (w / v) NP-40, 0.1% to 3% (w / v) trehalose, 3 to 8 mM EDTA and 10 to 20 mM More preferably, the preservation solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5mM EDTA, and 5mM Tris.

[0140] In a preferred embodiment, the pH of the preservation solution is 7-12, preferably 8-12, preferably 9-12, more preferably 10-12, for example about 11. When the pH is 11, the amplification effect of the sample treated with the preservation solution of the present invention is the best.

[0141] When the sample is preserved using the preservation solution of the present invention, the RNA nucleic acid remains stable when preserved at room temperature for 6 hours and remains stable when preserved at 2-8°C for 6 days.

[0142] In a preferred embodiment, the preparation further includes grinding filter beads. In a preferred embodiment, the grinding filter beads are modified with hydroxyl groups or carboxyl groups. In a preferred embodiment, the grinding filter beads are selected from the following group: quartz sand, zirconia beads, foam filter beads, activated carbon particles, biological ceramsite, molecular sieves, fiber balls, or a combination thereof; preferably, biological ceramsite, molecular sieves. Different grinding filter beads are selected according to different application scenarios. The preparation of the present invention contains grinding filter beads, which can effectively remove impurities in the sample and improve the efficiency of nucleic acid release through physical collision. The grinding filter beads can not only assist in the release of nucleic acids, but also adsorb impurities in the sample and reduce inhibitors in the system.

[0143] In a preferred embodiment, the concentration of the grinding filter beads is 0.01-5 g / mL, preferably 0.05-3 g / mL, preferably 0.1-2 g / mL, more preferably 0.2-1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL or 1 g / mL.

[0144] Method of the present invention

[0145] The method of the present invention is used to increase the release rate of nucleic acid in a sample, comprising the steps of:

[0146] (s1) provide a sample; and

[0147] (s2) adding the sample to a preservation solution and mixing the sample, wherein the preservation solution comprises a release component, a nucleic acid stabilizing component and a buffer component;

[0148] Wherein, the release component is composed of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant;

[0149] The nucleic acid stabilizing component is composed of 0.01% to 10% (w / v) nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent;

[0150] The buffer component is 0.1-50 mM Tris;

[0151] Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof;

[0152] The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof;

[0153] The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof;

[0154] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.

[0155] In a preferred embodiment, the method further comprises step (s3) of adding grinding filter beads to the preservation solution containing the sample and mixing the mixture.

[0156] In a preferred embodiment, the preservation solution consists of 0.01% to 5% (w / v) SDS, 0.01% to 5% (w / v) NP-40, 0.01% to 10% (w / v) trehalose, 0.1 to 20 mM EDTA and 0.1 to 50 mM Tris; preferably, the preservation solution consists of 0.01% to 3% (w / v) SDS, 0.02% to 3% (w / v) NP-40, 0.05% to 5% (w / v) trehalose, 1 to 10 mM EDTA and 1 to 30 mM Tris; preferably, the preservation solution consists of 0.01% to 1% (w / v) SDS, 0.02% to 2% (w / v) NP-40, 0.1% to 3% (w / v) trehalose, 3 to 8 mM EDTA and 10 to 20 mM More preferably, the preservation solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5mM EDTA, and 5mM Tris.

[0157] In a preferred embodiment, the preparation further comprises grinding filter beads. In a preferred embodiment, the grinding filter beads are modified with hydroxyl groups or carboxyl groups. In a preferred embodiment, the grinding filter beads are selected from the following group: quartz sand, zirconium oxide beads, foam filter beads, activated carbon particles, biological ceramsite, molecular sieve, fiber balls, or a combination thereof; preferably, biological ceramsite, molecular sieve.

[0158] In a preferred embodiment, the concentration of the grinding filter beads is 0.01-5 g / mL, preferably 0.05-3 g / mL, preferably 0.1-2 g / mL, more preferably 0.2-1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL or 1 g / mL.

[0159] In a preferred embodiment, the method further comprises step (s4) of ultrasonically and / or heat-treating the preservation solution containing the sample. In another preferred example, in step (s4), the ultrasonic treatment and the heat treatment are performed simultaneously or separately. In a preferred embodiment, in step (s4), the frequency of the ultrasonic treatment is 30 to 50 kHZ, preferably 35 to 45 kHZ, and more preferably 35 to 40 kHZ. In a preferred embodiment, in step (s4), the ultrasonic treatment is 30 to 240 s, preferably 30 to 180 s, and more preferably 120 to 180 s. In a preferred embodiment, in step (s4), the heat treatment is heating at 75 to 95 ° C for 1 to 5 min, preferably heating at 75 to 95 ° C for 1 to 3 min. The release rate of nucleic acids can be further improved by ultrasonic and / or heat treatment.

[0160] In a preferred embodiment, the sample is an oropharyngeal swab and / or a nasopharyngeal swab. In a preferred embodiment, the sample contains pathogens, preferably viruses, bacteria and / or mycoplasmas, more preferably influenza A virus, adenovirus, Mycoplasma pneumoniae, human metapneumovirus, or a combination thereof.

[0161] The main advantages of the present invention include:

[0162] 1. The method of the present invention combines grinding filter beads to assist physical fragmentation while adsorbing impurities in the system, thereby achieving purification, reducing inhibitors introduced into the amplification system, and effectively reducing the occurrence of false negative samples.

[0163] 2. The method of the present invention combines the steps of sample collection and nucleic acid processing, effectively shortening the detection time of respiratory pathogens in fever clinics and shortening the entire detection process by about 20 to 30 minutes.

[0164] 3. The method of the present invention can be tested on demand, which increases the test turnover efficiency and reduces the waiting time of patients.

[0165] 4. The method of the present invention can be combined with physical methods such as ultrasound or heating to further improve the release efficiency of sample nucleic acid and denature inhibitors in the collected sample. The reliability of the test results of samples processed by this method can be continuously improved by grinding filter beads and assisting with physical methods.

[0166] 5. The method of the present invention does not require additional sophisticated instruments and consumables with complex structures, thus reducing detection costs.

[0167] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0168] Example 1

[0169] The pseudovirus particles of influenza A virus were treated with nuclease before use. In order to study the sample release conditions, different surfactants were tested. Therefore, in this application, after the pseudovirus was diluted with sample preservation solutions of different components, a portion of the pseudovirus was diluted with water by the same multiple, and extracted with nucleic acid extraction and purification reagents (Shanghai Fengxiebei No. 20180202) as a control.

[0170] Select different types of surfactants and prepare different preservation solutions according to Table 1.

[0171] Table 1

[0172] Serial number Main Ingredients concentration Serial number Main Ingredients concentration 1 Sodium Lauryl Sulfate 0.01％ 17 Hexadecyl ammonium bromide 0.10％ 2 Sodium Lauryl Sulfate 0.10％ 18 Hexadecyl ammonium bromide 1％ 3 Sodium Lauryl Sulfate 1％ 19 tween-20 0.02％ 4 Lithium dodecyl sulfate 0.01％ 20 tween-20 0.20％ 5 Lithium dodecyl sulfate 0.10％ 21 tween-20 2％ 6 Lithium dodecyl sulfate 1％ 22 NP-40 0.02％ 7 Sodium laurylamine 0.01％ 23 NP-40 0.20％ 8 Sodium laurylamine 0.10％ 24 NP-40 2％ 9 Sodium laurylamine 1％ 25 triton-X-100 0.02％ 10 Sodium Tetradecyl Sulfate 0.01％ 26 triton-X-100 0.20％ 11 Sodium Tetradecyl Sulfate 0.10％ 27 triton-X-100 2％ 12 Sodium Tetradecyl Sulfate 1％ 28 Brij-35 0.02％ 13 Sodium cetyl sulfate 0.01％ 29 Brij-35 0.20％ 14 Sodium cetyl sulfate 0.10％ 30 Brij-35 2％ 15 Sodium cetyl sulfate 1％ 31 DEPC treated water / 16 Hexadecyl ammonium bromide 0.01％

[0173] The specific operation process is as follows:

[0174] (1) Sample processing

[0175] Take influenza A virus pseudovirus and treat the pseudovirus with nuclease to ensure that the free nucleic acid in it is completely digested and only complete virus particles remain.

[0176] (2) Sample release and nucleic acid extraction

[0177] The pseudovirus digested with nuclease was diluted 20 times using the above-prepared preservation solution formula, and the pseudovirus was diluted with DEPC-treated water in two portions, one with a dilution factor of 20 times and the other with a dilution factor of 40 times. The preservation solution after adding the pseudovirus was fully mixed and placed at room temperature for 5 minutes. The pseudovirus treated with DEPC water was extracted using magnetic bead nucleic acid extraction and purification reagents.

[0178] (3) Sample testing

[0179] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively. To prevent the surfactant concentration from being too high, the samples in the above preservation solution were diluted 10 times with water and tested simultaneously.

[0180] The results are shown in Table 2.

[0181] Table 2

[0182] Serial number Main Ingredients concentration Repeat 1 Repeat 2 Mean Release rate 1 Sodium Lauryl Sulfate 0.01％ 27.65 27.90 27.77 27.36％ 2 Sodium Lauryl Sulfate 0.10％ / / / / 3 Sodium Lauryl Sulfate 1％ / / / / 4 Lithium dodecyl sulfate 0.01％ 27.73 27.94 27.84 26.23％ 5 Lithium dodecyl sulfate 0.10％ / / / / 6 Lithium dodecyl sulfate 1％ / / / / 7 Sodium laurylamine 0.01％ 27.83 27.99 27.91 24.88％ 8 Sodium laurylamine 0.10％ / / / / 9 Sodium laurylamine 1％ / / / / 10 Sodium Tetradecyl Sulfate 0.01％ 27.89 27.65 27.77 27.45％ 11 Sodium Tetradecyl Sulfate 0.10％ / / / / 12 Sodium Tetradecyl Sulfate 1％ / / / / 13 Sodium cetyl sulfate 0.01％ 27.89 27.84 27.86 25.71％ 14 Sodium cetyl sulfate 0.10％ / / / / 15 Sodium cetyl sulfate 1％ / / / / 16 Hexadecyl ammonium bromide 0.01％ 27.60 27.94 27.77 27.44％ 17 Hexadecyl ammonium bromide 0.10％ / / / / 18 Hexadecyl ammonium bromide 1％ / / / / 19 tween-20 0.02％ 28.11 28.46 28.28 19.21％ 20 tween-20 0.20％ 28.17 28.24 28.20 20.35％ 21 tween-20 2％ 28.74 28.60 28.67 14.74％ 22 NP-40 0.02％ 28.11 28.06 28.08 22.07％ 23 NP-40 0.20％ 28.36 28.15 28.25 19.65％ 24 NP-40 2％ 28.74 28.60 28.67 14.74％ 25 triton-X-100 0.02％ 28.45 28.21 28.33 18.59％ 26 triton-X-100 0.20％ 28.24 28.10 28.17 20.81％ 27 triton-X-100 2％ 28.74 28.60 28.67 14.74％ 28 Brij-35 0.02％ 28.39 28.40 28.40 17.77％ 29 Brij-35 0.20％ 28.42 28.18 28.30 18.98％ 30 Brij-35 2％ 28.74 28.60 28.67 14.74％ 31 DEPC treated water / 36.21 / / / 32 Nucleic acid extraction / 24.95 24.86 24.91 Set to 100%

[0183] The treated samples were diluted 10 times and the results are shown in Table 3.

[0184] Table 3

[0185]

[0186] According to the above results, different surfactants have certain nucleic acid release capabilities relative to DEPC-treated water. Among them, ionic surfactants have better release effects than non-ionic surfactants, and through dilution and amplification, it was found that within a certain range, the higher the concentration, the better the release effect. However, if the concentration of ionic surfactants is too high, it may affect the amplification efficiency of the PCR reaction.

[0187] Based on the above experiments, the release effects of different anionic surfactants were basically consistent. Based on availability, sodium dodecyl sulfate was selected as the main surfactant for subsequent studies.

[0188] Example 2

[0189] Sodium dodecyl sulfate was used in combination with different nonionic surfactants to test its release ability. The specific ratios are shown in Table 4.

[0190] Table 4

[0191] Serial number Sodium Lauryl Sulfate tween-20 NP-40 Triton-X-100 Brij-35 1 0.01％ 0.02％ 2 0.01％ 0.20％ 3 0.10％ 0.02％ 4 0.10％ 0.20％ 5 0.01％ 0.02％ 6 0.01％ 0.20％ 7 0.10％ 0.02％ 8 0.10％ 0.20％ 9 0.01％ 0.02％ 10 0.01％ 0.20％ 11 0.10％ 0.02％ 12 0.10％ 0.20％ 13 0.01％ 0.02％ 14 0.01％ 0.20％ 15 0.10％ 0.02％ 16 0.10％ 0.20％ 17 DEPC treated water 18 Nucleic acid extraction

[0192] The specific operation process is as follows:

[0193] (1) Sample processing

[0194] Take influenza A virus pseudovirus and treat the pseudovirus with nuclease to ensure that the free nucleic acid in it is completely digested and only complete virus particles remain.

[0195] (2) Sample release and nucleic acid extraction

[0196] The pseudovirus digested with nuclease was diluted 20 times using the above-prepared preservation solution formula, and the pseudovirus was diluted with DEPC-treated water in two portions, one with a dilution factor of 20 times and the other with a dilution factor of 40 times. The preservation solution after adding the pseudovirus was fully mixed and placed at room temperature for 5 minutes. The pseudovirus treated with DEPC water was extracted using magnetic bead nucleic acid extraction and purification reagents.

[0197] (3) Sample testing

[0198] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively. To prevent the surfactant concentration from being too high, the samples in the above preservation solution were diluted 10 times with water and tested simultaneously.

[0199] The results are shown in Tables 5 and 6.

[0200] Table 5

[0201]

[0202]

[0203] The test results after the sample was diluted 10 times are shown in Table 6.

[0204] Table 6

[0205] Serial number Repeat 1 Repeat 2 Mean Release efficiency 1 31.20 31.05 31.13 36.25％ 2 30.83 31.21 31.02 39.09％ 3 30.56 30.69 30.63 51.35％ 4 30.61 30.58 30.60 52.45％ 5 31.12 30.83 30.98 40.29％ 6 31.22 31.10 31.16 35.53％ 7 30.72 30.59 30.65 50.36％ 8 30.55 30.79 30.67 49.75％ 9 30.77 30.75 30.76 46.77％ 10 31.40 31.34 31.37 30.70％ 11 30.81 30.68 30.75 47.23％ 12 30.80 30.78 30.79 45.94％ 13 30.86 31.19 31.03 38.93％ 14 30.82 30.87 30.85 44.10％ 15 30.81 30.64 30.72 47.98％ 16 30.87 30.54 30.70 48.66％ 17 / / / 18 28.77 28.56 28.67 100％

[0206] According to the above results, when nonionic surfactants and anionic surfactants are used together, the effects are basically the same. After adding nonionic surfactants, the anionic surfactant concentration that could not detect nucleic acid release (such as 0.10% sodium dodecyl sulfate in Table 2) can detect nucleic acid release. NP-40 was used for exploration in subsequent experiments.

[0207] Example 3

[0208] Surfactants can denature proteins, so surfactants are also a type of nucleic acid protectant. Since most of the application scenarios are oropharyngeal swabs, the samples will contain various impurities in the oral cavity, which may degrade nucleic acids, especially RNA nucleic acids. Add RNA protectants to the above components with lysis function, and the concentration is configured to 2×. Collect 20 oropharyngeal swabs and mix them thoroughly in 20mL DEPC-treated water.

[0209] The prepared 2× solution was mixed with DEPC-treated water from the collected swab in a 1:1 ratio and used to dilute the extracted pseudovirus nucleic acid.

[0210] In this example, the basic formula used is 0.1% SDS, 0.2% NP-40, and specific experimental settings as shown in Table 7 are further set based on the basic formula.

[0211] Table 7

[0212] Serial number Formamide Trehalose DTT TCEP β-Mercaptoethanol Other treatments 1 1％ 2 2％ 3 3％ 4 0.50％ 5 1％ 6 1.50％ 7 2％ 8 0.5mM 9 1mM 10 1.5mM 11 5mM 12 10mM 13 15mM 14 0.50％ 15 1％ 16 1.50％ 17 DEPC treated water + swab

[0213] The specific operation process is as follows:

[0214] (1) Sample processing

[0215] Take influenza A pseudovirus, extract it with a nucleic acid extraction reagent, and obtain influenza A pseudovirus nucleic acid.

[0216] Take 20 negative swabs collected from different people, add 20 mL of DEPC-treated water, mix thoroughly and set aside.

[0217] (2) Sample release and nucleic acid extraction

[0218] Dilute the prepared influenza A pseudovirus nucleic acid 5 times with the above-prepared formula with a concentration of 2×, and then dilute it 2 times with the mixed negative swab treated with DEPC water. At the same time, dilute the nucleic acid 10 times with DEPC-treated water. After the preservation solution with pseudovirus nucleic acid added is fully mixed, place it at room temperature for 30 minutes. Use pseudovirus nucleic acid treated with DEPC water, and follow the above operation to ensure that the nucleic acid dilution multiple is consistent.

[0219] (3) Sample testing

[0220] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0221] The results are shown in Table 8.

[0222] Table 8

[0223]

[0224]

[0225] According to the above results, the addition of formamide and trehalose has little inhibitory effect on the detection system and can ensure effective amplification.

[0226] Example 4

[0227] Further studies were conducted on formamide and trehalose combined with metal ion chelators. RNA protectants were added to the above components with lysis function, and the concentration was configured to 2×. 20 oropharyngeal swabs were collected and thoroughly mixed in 20 mL of DEPC-treated water.

[0228] The prepared 2× solution was mixed with DEPC-treated water from the collected swab in a 1:1 ratio and used to dilute the extracted pseudovirus nucleic acid.

[0229] In this example, the basic formula used is 0.1% SDS, 0.2% NP-40, and specific experimental settings as shown in Table 9 are further set based on the basic formula.

[0230] Table 9

[0231] Serial number Formamide Trehalose 0.5M EDTA 0.5M EGTA Other treatment methods 1 1％ 2 2％ 3 3％ 4 0.50％ 5 1％ 6 1.50％ 7 2％ 8 0.50％ 9 1％ 10 1.50％ 11 0.50％ 12 1％ 13 1.50％ 14 2％ 1％ 15 1％ 1％ 16 2％ 1％ 1％ 17 2％ 1％ 18 1％ 1％ 19 2％ 1％ 1％ 20 DEPC treated water + swab

[0232] The specific operation process is as follows:

[0233] (4) Sample processing

[0234] Take influenza A pseudovirus, extract it with a nucleic acid extraction reagent, and obtain influenza A pseudovirus nucleic acid.

[0235] Take 24 negative swabs collected from different people, add 24 mL of DEPC-treated water, mix thoroughly and set aside.

[0236] (5) Sample release and nucleic acid extraction

[0237] Dilute the prepared influenza A pseudovirus nucleic acid 5 times using the above-prepared formula with a concentration of 2×, and then dilute it 2 times with the mixed negative swab treated with DEPC water. At the same time, dilute the pseudovirus 10 times with DEPC-treated water. After fully mixing the preservation solution with the pseudovirus nucleic acid added, place it at room temperature for 30 minutes. Use the pseudovirus nucleic acid treated with DEPC water in the same way as above to ensure that the nucleic acid dilution multiple is consistent. Divide each of the above samples into 8 portions, 4 portions are placed at room temperature for 0, 3, 6, and 9 hours for testing; 4 portions are placed at 2-8°C for testing at 0, 3, 6, and 9 days.

[0238] (6) Sample testing

[0239] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0240] The Ct values ​​are shown in Table 10.

[0241] Table 10

[0242] Serial number Normal temperature 0h Normal temperature 3h Normal temperature 6h Normal temperature 9h Serial number Day 0 3 days 6 days 9 days 1 30.43 30.76 31.33 32.30 1 30.74 30.52 31.76 32.60 2 30.35 30.65 31.34 31.99 2 30.03 30.77 31.17 32.21 3 30.21 30.81 30.89 31.33 3 30.25 31.12 30.63 31.64 4 30.23 30.44 30.56 32.24 4 30.21 30.12 30.51 32.56 5 30.34 30.57 30.52 31.98 5 30.77 30.86 30.87 31.78 6 30.43 30.66 30.89 31.24 6 30.84 30.62 31.02 31.53 7 30.63 30.87 30.99 32.67 7 30.99 30.45 31.02 32.83 8 30.56 30.68 30.91 31.21 8 30.67 31.06 31.33 31.33 9 30.53 30.55 30.98 32.33 9 30.82 30.72 30.80 32.18 10 30.53 30.78 30.99 31.56 10 30.63 30.95 31.43 31.27 11 30.59 30.77 30.72 31.76 11 30.12 30.86 31.06 32.26 12 30.65 30.67 30.89 31.24 12 30.60 30.61 30.45 30.80 13 30.28 30.67 31.02 31.44 13 30.73 31.10 31.23 31.86 14 26.79 26.74 26.77 26.93 14 26.73 27.04 26.86 26.94 15 26.34 26.35 26.60 26.77 15 25.86 26.13 26.37 27.16 16 26.45 26.61 26.33 26.93 16 26.24 26.65 26.39 26.89 17 26.39 26.39 26.73 26.67 17 25.97 26.76 26.71 26.66 18 26.48 26.41 26.74 26.87 18 26.46 25.98 26.52 27.13 19 26.53 26.67 26.42 26.43 19 26.20 26.40 26.34 26.09 20 26.31 30.21 36.87 / 20 26.17 32.86 / /

[0243] According to the above results, according to the comparison of Ct values, it can be seen that the Ct values ​​of sequence numbers 14-19 in Table 10 are significantly advanced (i.e., the Ct values ​​are low), so adding at least one of formamide or trehalose as a nucleic acid stabilizer, and then adding one of EDTA or EGTA, can not only improve the nucleic acid preservation performance of the entire system, but also reduce the inhibitory effect of high-concentration surfactants on the entire reaction system. Considering the toxicity and availability of the components, trehalose and EDTA were selected for use in subsequent experiments.

[0244] Example 5

[0245] The pH value has a certain effect on the release of pathogen nucleic acid, and the pH value also affects the amplification of the reaction system. A stable pH can ensure that the preservation solution works under various conditions. The basic formula in this embodiment is 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, and 1% (v / v) 0.5M EDTA (i.e., 5mM EDTA). The specific experimental settings are shown in Table 11.

[0246] Table 11

[0247] Serial number pH 1M Tris concentration (v / v) 1 9 0.50％ 2 9 1.50％ 3 10 0.50％ 4 10 1.50％ 5 11 0.50％ 6 11 1.50％ 7 12 0.50％ 8 12 1.50％ 9 No pH adjustment 10 Nucleic acid extraction

[0248] The specific operation process is as follows:

[0249] (1) Sample processing

[0250] Take influenza A virus pseudovirus and treat the pseudovirus with nuclease to ensure that the free nucleic acid in it is completely digested and only complete virus particles remain.

[0251] (2) Sample release and nucleic acid extraction

[0252] The pseudovirus digested with nuclease was diluted 20 times using the above-prepared preservation solution formula, and the pseudovirus was diluted 20 times with DEPC-treated water. The preservation solution after adding the pseudovirus was fully mixed and placed at room temperature for 5 minutes. The pseudovirus treated with DEPC water was extracted using magnetic bead nucleic acid extraction and purification reagents.

[0253] (3) Sample testing

[0254] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0255] The results are shown in Table 12.

[0256] Table 12

[0257]

[0258]

[0259] According to the results, the pH value between 9-12 can be effectively amplified, but when the pH is higher than 11, adding 1M Tris at 1.5% (v / v) will affect the amplification results. According to the results, it was found that the detection effect was the best when the pH was 11 and 1M Tris was added at 1.5% (v / v), so the subsequent test conditions selected the addition of 0.5% (v / v) 1M Tris (i.e. 5mM Tris) and the pH was adjusted to 11.0.

[0260] Example 6

[0261] The basic formula in this embodiment is 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 1% (v / v) 0.5M EDTA (i.e., 5mM EDTA), and 0.5% (v / v) 1M Tris, and the pH of the solution is 11.0.

[0262] Based on the above results, the release capacity of samples of common respiratory pathogens was verified, including influenza A virus, adenovirus, bronchiolitis, and human metapneumovirus.

[0263] The specific operation process is as follows:

[0264] (1) Sample processing

[0265] The sample is an oropharyngeal swab. Samples of influenza A virus, adenovirus, mycoplasma pneumoniae, and human metapneumovirus are mixed and prepared into a mixed sample.

[0266] (2) Sample release and nucleic acid extraction

[0267] The above samples were diluted 20 times with preservation solution and DEPC-treated water respectively. After being fully mixed, they were allowed to stand at room temperature for 5 minutes. The samples diluted with DEPC-treated water were extracted using magnetic bead-based nucleic acid extraction and purification reagents.

[0268] (3) Sample testing

[0269] The above samples were tested using the influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method), the Mycoplasma pneumoniae nucleic acid detection kit (fluorescence PCR method), and the six respiratory virus nucleic acid detection kit (fluorescence PCR method).

[0270] The results are shown in Table 13.

[0271] Table 13

[0272]

[0273] According to the above results, the release effect of nucleic acid of DNA pathogens is better than that of RNA, which may be due to the high pre-denaturation and denaturation temperature in the amplification process, which assists the release of DNA nucleic acid. According to the above results, the existing preservation solution has a certain release effect on both DNA and RNA.

[0274] Example 7

[0275] Compared with the purer simulated samples, real clinical samples may contain more inhibitors. Therefore, the present application adds grinding filter beads made by a special process, whose surface is modified with negatively charged hydroxyl or carboxyl groups. At the same time, the physical collision of the grinding filter beads improves the release efficiency of the sample nucleic acid.

[0276] The components of the preservation solution used in this example are 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, 0.5% (v / v) 1M Tris, and the pH of the solution is 11.0. Take 22mL of the preservation solution, add 11 sampling swabs and mix thoroughly. Divide the treated preservation solution into 2mL per tube, and add an equal amount of influenza A sample to each tube. Add grinding filter beads of different masses to 10 tubes and vortex mix thoroughly. The other tube does not add grinding filter beads as a control, and 200μL is taken as a sample for nucleic acid extraction. The amount of grinding filter beads added is shown in Table 14.

[0277] Table 14

[0278]

[0279] The specific operation process is as follows:

[0280] (1) Sample processing

[0281] Take influenza virus sample

[0282] (2) Sample release and nucleic acid extraction

[0283] Take 22 mL of preservation solution, add 11 sampling swabs and mix thoroughly. Divide the treated preservation solution into 2 mL per tube, and add an equal amount of influenza A sample to each tube. Add grinding beads of different masses to 10 tubes and vortex thoroughly to mix. The other tube does not add grinding beads as a control, and 200 μL is taken from it as a sample for nucleic acid extraction.

[0284] (3) Sample testing

[0285] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0286] The results are shown in Table 15.

[0287] Table 15

[0288]

[0289] According to the above results, adding grinding filter beads of different masses can improve the release efficiency of the sample.

[0290] Example 8

[0291] According to the principle of grinding filter beads, other materials of grinding filter bead analogs were selected for testing, including quartz sand, zirconium oxide beads, foam filter beads, activated carbon particles, ceramsite, molecular sieves, fiber balls and other adsorbents, and their impurity adsorption and sample release effects were evaluated after adding the preservation solution. The preservation solution used in this example is composed of 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, 0.5% (v / v) 1M Tris, and the pH of the solution is 11.0. The amount of grinding filter beads added is shown in Table 16.

[0292] Table 16

[0293] Serial number Group Mass(g) 1 Hydroxyl 0.8 2 Quartz sand 0.8 3 Zirconia beads 0.8 4 Foam filter beads 0.8 5 Activated carbon granules 0.8 6 Bio-ceramsite 0.8 7 Molecular sieve 0.8 8 Fiber Ball 0.8 9 No grinding beads 10 Nucleic acid extraction

[0294] The specific operation process is as follows:

[0295] (1) Sample processing

[0296] Take influenza virus sample

[0297] (2) Sample release and nucleic acid extraction

[0298] Take 18 mL of preservation solution, add 9 sampling swabs and mix thoroughly. Divide the processed preservation solution into 2 mL per tube, and add an equal amount of influenza A sample to each tube. 8 of the tubes are processed according to the above table, and the other tube does not add grinding filter beads as a control, and 200 μL is taken from it as a sample for nucleic acid extraction.

[0299] (3) Sample testing

[0300] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0301] The results are shown in Table 17.

[0302] Table 17

[0303]

[0304] According to the above results, grinding filter beads of different materials can effectively improve the amplification effect after the sample nucleic acid is released. Therefore, mixing and matching these materials can play a role in different scenarios in practical applications.

[0305] Example 9

[0306] After adding grinding filter beads, the amplification effect of the sample released by the preservation solution is still worse than that of the nucleic acid extracted by the magnetic bead method nucleic acid extraction reagent. Considering that chemical reagents have a greater impact on the reaction system, physical methods are used to improve the pathogen detection effect. The preservation solution components used in this embodiment are 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, 0.5% (v / v) 1M Tris, and the pH of the solution is 11.0. In the treatment group containing grinding filter beads, hydroxyl grinding filter beads are used for grinding filter beads with a mass of 0.8g. The parameters of the ultrasonic instrument used in this study are power 100W and frequency 35-40kHZ. The experimental scheme is shown in Table 18.

[0307] Table 18

[0308] Serial number Experimental treatment Grinding filter beads 1 No sonication none 2 No sonication Hydroxyl 3 Ultrasound 150s none 4 Ultrasound 150s Hydroxyl 5 Nucleic acid extraction none

[0309] The specific operation process is as follows:

[0310] (1) Sample processing

[0311] Take influenza virus sample

[0312] (2) Sample release and nucleic acid extraction

[0313] Take 10mL of preservation solution, add 5 sampling swabs and mix thoroughly. Divide the treated preservation solution into 2mL per tube, and add an equal amount of influenza A sample to each tube. Treat 4 tubes according to the above table, and use the other tube as a control, and take 200μL from it as a sample for nucleic acid extraction.

[0314] (3) Sample testing

[0315] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0316] The results are shown in Table 19.

[0317] Table 19

[0318]

[0319] According to the above results, the amplification detection effect of ultrasonic treatment combined with preservation solution and grinding filter beads is basically consistent with that of magnetic bead method. However, after ultrasonic treatment, if the preservation solution does not contain grinding filter beads, the amplification effect will become worse. This may be due to the high efficiency of nucleic acid release after ultrasound, but impurities are not removed, resulting in RNA nucleic acid degradation.

[0320] Example 10

[0321] After adding grinding filter beads, the amplification effect of the sample released by the preservation solution is still worse than that of the nucleic acid extracted by the magnetic bead method nucleic acid extraction reagent. Considering that chemical reagents have a greater impact on the reaction system, physical methods are used to assist in improving the pathogen detection effect. The preservation solution components used in this embodiment are 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, 0.5% (v / v) 1M Tris, and the pH of the solution is 11.0. In the treatment group containing grinding filter beads, hydroxyl grinding filter beads are used for grinding filter beads with a mass of 0.8g. The parameters of the ultrasonic instrument used in this study are power 100W and frequency 35-40kHZ. The experimental scheme is shown in Table 20.

[0322] Table 20

[0323] Serial number Experimental treatment Grinding filter beads 1 Ultrasound 30s Hydroxyl 2 Ultrasound 60s Hydroxyl 3 Ultrasound 90s Hydroxyl 4 Ultrasound 120s Hydroxyl 5 Ultrasound 150s Hydroxyl 6 Ultrasound 180s Hydroxyl 7 Ultrasound 210s Hydroxyl 8 Ultrasound 240s Hydroxyl 9 No ultrasound Hydroxyl 10 Nucleic acid extraction Hydroxyl

[0324] The specific operation process is as follows:

[0325] (1) Sample processing

[0326] Take influenza virus sample

[0327] (2) Sample release and nucleic acid extraction

[0328] Take 18 mL of preservation solution, add 9 sampling swabs and mix thoroughly. Divide the treated preservation solution into 2 mL per tube, and add an equal amount of influenza A sample to each tube. 8 of the tubes are treated according to the above table, and the other tube is used as a control, and 200 μL is taken from it as a sample for nucleic acid extraction.

[0329] (3) Sample testing

[0330] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0331] The results are shown in Table 21.

[0332] Table 21

[0333] Serial number Experimental treatment Grinding filter beads Repeat 1 Repeat 2 average value Release rate 1 Ultrasound 30s Hydroxyl 28.73 28.74 28.73 84.52％ 2 Ultrasound 60s Hydroxyl 28.65 28.83 28.74 84.18％ 3 Ultrasound 90s Hydroxyl 28.52 28.98 28.75 83.38％ 4 Ultrasound 120s Hydroxyl 28.66 28.42 28.54 96.58％ 5 Ultrasound 150s Hydroxyl 28.44 28.82 28.63 90.86％ 6 Ultrasound 180s Hydroxyl 28.52 28.56 28.54 96.50％ 7 Ultrasound 210s Hydroxyl 28.58 29.11 28.84 78.33％ 8 Ultrasound 240s Hydroxyl 29.11 29.03 29.07 66.96％ 9 No ultrasound Hydroxyl 29.43 29.36 29.40 53.40％ 10 Nucleic acid extraction Hydroxyl 27.34 27.64 27.49 Set to 100%

[0334] According to the above results, different ultrasonic times can improve the detection effect. Within 30-240s of ultrasound, the release rate of the samples after ultrasound reached more than 65%, but too long ultrasound time may lead to poor detection effect.

[0335] Embodiment 11

[0336] After adding grinding filter beads, the amplification effect after the sample is released by the preservation solution is still worse than the nucleic acid extracted by the magnetic bead method nucleic acid extraction reagent. Considering that chemical reagents have a greater impact on the reaction system, physical methods are used to improve the pathogen detection effect. The preservation solution components used in this embodiment are 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, 0.5% (v / v) 1M Tris, and the pH of the solution is 11.0. In the treatment group containing grinding filter beads, hydroxyl grinding filter beads were used for grinding filter beads with a mass of 0.8g (2ml). A metal bath was used for heating in this study. The experimental scheme is shown in Table 22.

[0337] Table 22

[0338]

[0339]

[0340] The specific operation process is as follows:

[0341] (1) Sample processing

[0342] Take influenza virus sample

[0343] (2) Sample release and nucleic acid extraction

[0344] Take 10mL of preservation solution, add 5 sampling swabs and mix thoroughly. Divide the treated preservation solution into 2mL per tube, and add an equal amount of influenza A sample to each tube. Treat 4 tubes according to the above table, and use the other tube as a control, and take 200μL from it as a sample for nucleic acid extraction.

[0345] (3) Sample testing

[0346] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0347] The results are shown in Table 23.

[0348] Table 23

[0349] Serial number Experimental treatment Grinding filter beads Repeat 1 Repeat 2 average value Release rate 1 No treatment none 29.34 29.63 29.49 45.06％ 2 No treatment Hydroxyl 28.85 28.87 28.86 69.37％ 3 Heat at 85℃ for 3min none 30.26 30.48 30.37 24.40％ 4 Heat at 85℃ for 3min Hydroxyl 28.16 28.55 28.36 98.62％ 5 Nucleic acid extraction none 27.25 27.42 27.34 Set to 100%

[0350] According to the above results, the amplification detection effect of heating treatment combined with preservation solution and grinding filter beads is basically consistent with the magnetic bead method. However, after heating, if the preservation solution does not contain grinding filter beads, the amplification effect will become worse. This may be due to the high efficiency of nucleic acid release after heating, but impurities are not removed, resulting in RNA nucleic acid degradation.

[0351] Example 12

[0352] After adding grinding filter beads, the amplification effect after the sample is released by the preservation solution is still worse than the nucleic acid extracted by the magnetic bead method nucleic acid extraction reagent. Considering that chemical reagents have a greater impact on the reaction system, physical methods are used to improve the pathogen detection effect. The preservation solution components used in this embodiment are 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, 0.5% (v / v) 1M Tris, and the pH of the solution is 11.0. In the treatment group containing grinding filter beads, hydroxyl grinding filter beads were used for grinding filter beads with a mass of 0.8g. A metal bath was used for heating in this study. The experimental scheme is shown in Table 24.

[0353] Table 24

[0354]

[0355]

[0356] The specific operation process is as follows:

[0357] (1) Sample processing

[0358] Take influenza virus sample

[0359] (2) Sample release and nucleic acid extraction

[0360] Take 20mL of preservation solution, add 10 sampling swabs and mix thoroughly. Divide the treated preservation solution into 2mL tubes and add an equal amount of influenza A sample to each tube. Add grinding beads of different masses to 9 tubes and vortex thoroughly to mix. The other tube without grinding beads is used as a control, and 200μL is taken from it as a sample for nucleic acid extraction.

[0361] (3) Sample testing

[0362] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0363] The results are shown in Table 25.

[0364] Table 25

[0365]

[0366] According to the above results, different heating times and temperatures can improve the detection effect. When heated at 75-95°C for 1-3 minutes, the sample release rate reaches more than 80%.

[0367] Therefore, in actual use, combining ultrasound or heating can further improve the nucleic acid release efficiency of this method and improve the detection sensitivity of the entire detection system.

[0368] Comparative Example 1

[0369] To evaluate the reliability of this test program in hospital outpatient testing, the differences in test results between the preservation solution alone, the preservation solution + grinding filter beads, and the preservation solution + grinding filter beads plus ultrasonic treatment and magnetic bead method nucleic acid extraction were compared. The specific arrangements are as follows:

[0370] 1. Collect samples using a sampling tube containing preservation solution and test directly. Take 200 μL for nucleic acid extraction using the magnetic bead method.

[0371] 2. Add 0.8 g of hydroxyl grinding filter beads to the sample in 1 and let it stand for 5 minutes before testing.

[0372] 3. After the samples in 2 are sonicated, they are directly amplified.

[0373] The above samples were tested using the Influenza A Virus / Influenza B Virus Nucleic Acid Detection Kit (fluorescence PCR method), and a total of 80 samples were compared.

[0374] The specific operation process is as follows:

[0375] (1) Sample processing and release

[0376] After collecting real oropharyngeal swab samples at the outpatient clinic and letting them stand for 5 minutes, 5 μL was taken out for testing, and then 200 μL was taken out for magnetic bead nucleic acid extraction reagent; after adding grinding filter beads to the remaining sample and mixing thoroughly, 5 μL was taken out for testing; after the remaining sample was processed with ultrasonic equipment for 150S, 5 μL was taken out for testing.

[0377] (2) Sample release and nucleic acid extraction

[0378] After taking out the above 200 μL sample, use nucleic acid extraction and purification reagent (Shanghai Fengxiebei No. 20180202) for extraction.

[0379] (3) Sample testing

[0380] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0381] The results are shown in Table 26.

[0382] Table 26

[0383]

[0384]

[0385] According to the above results, the detection results using preservation solution alone are the worst, and the total consistency rate with the magnetic bead method is only 87.5%; using preservation solution with grinding filter beads, the total consistency rate with the magnetic bead method can reach 93.75%; using preservation solution with grinding filter beads and additional ultrasonic treatment can increase the total consistency rate with the magnetic bead method to 98.75%.

[0386] Comparative Example 2

[0387] To evaluate the reliability of this test program in hospital outpatient testing, and to compare the differences in test results between preservation solution superimposed ultrasonic treatment and magnetic bead method nucleic acid extraction, the specific arrangements are as follows:

[0388] 1. Collect the sample using a sampling tube containing preservation solution and take 200 μL for magnetic bead-based nucleic acid extraction.

[0389] 2. After ultrasonicating the sample in the preservation solution, take the sample and amplify it directly.

[0390] The above samples were tested using the influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method), and a total of 30 samples were compared.

[0391] The specific operation process is as follows:

[0392] (1) Sample processing and release

[0393] After collecting real oropharyngeal swab samples at the outpatient clinic and letting them stand for 5 minutes, 200 μL was taken out for magnetic bead nucleic acid extraction reagent. The remaining samples were processed with an ultrasonic device for 150 seconds, and 5 μL was taken out for testing.

[0394] (2) Sample release and nucleic acid extraction

[0395] After taking out the above 200 μL sample, use nucleic acid extraction and purification reagent (Shanghai Fengxiebei No. 20180202) for extraction.

[0396] (3) Sample testing

[0397] The above samples were tested using influenza A virus / influenza B virus nucleic acid detection kit (fluorescence PCR method) respectively.

[0398] The results are shown in Table 27.

[0399] Table 27

[0400] Positive sample number Preservative solution + ultrasound Magnetic bead method 2 24.15 21.04 3 27.32 24.14 4 29.38 29.16 7 28.28 27.25 10 25.47 25.51 13 \ 35.82 14 26.47 24.30 16 \ 26.56 24 24.97 25.61 26 \ 35.70 28 34.65 32.58 30 32.69 31.70 Positive coincidence rate 75.00％ \ Negative compliance rate 100.00％ \ Total compliance rate 90.00％ \

[0401] According to the above results, the total consistency rate of the magnetic bead method using the preservation solution combined with ultrasonic treatment can reach 90.00%. The test results are better than those using only the preservation solution, but worse than the preservation solution combined with grinding filter beads and the preservation solution combined with grinding filter beads and additional ultrasonic treatment.

[0402] discuss

[0403] In addition to having an excellent nucleic acid release rate, the method of the present invention has a short sample processing time. Compared with the magnetic bead method, the method of the present invention can be detected by a nucleic acid detection kit after the sample is added to the preservation solution and placed for 5 minutes, while the magnetic bead method requires steps such as magnetic bead adsorption, magnetic bead washing, and elution, which takes about 15 to 30 minutes.

[0404] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A preparation for sample preservation and nucleic acid release, characterized in that: The preparation comprises a preservation solution, which is composed of a release component, a nucleic acid stabilizing component and a buffer component; Wherein, the release component is composed of 0.01% to 5% anionic surfactant and 0.01% to 5% nonionic surfactant; The nucleic acid stabilizing component is composed of 0.01% to 10% nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent; The buffer component is 0.1-50 mM Tris; Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof; The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof; The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof; The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.

2. The preparation according to claim 1, characterized in that The preservation solution consists of 0.01% to 5% SDS, 0.01% to 5% NP-40, 0.01% to 10% trehalose, 0.1 to 10 mM EDTA and 0.1 to 50 mM Tris.

3. The preparation according to claim 1, characterized in that The preservation solution consists of 0.01%-3% SDS, 0.02%-3% NP-40, 0.05%-5% trehalose, 3-8mM EDTA and 1-30mM Tris.

4. The preparation according to claim 1, characterized in that The formulation also includes grinding filter beads.

5. The preparation according to claim 4, characterized in that The grinding filter beads are modified with hydroxyl groups or carboxyl groups.

6. The preparation according to claim 4, characterized in that The concentration of the grinding filter beads in the preparation or the preservation solution is 0.01-5 g / mL, preferably 0.05-3 g / mL, preferably 0.1-2 g / mL, more preferably 0.2-1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL or 1 g / mL.

7. A kit for sample preservation and nucleic acid release, characterized in that: The kit comprises: A first container and a storage solution located in the first container, wherein the storage solution consists of a releasing component, a nucleic acid stabilizing component and a buffer component; Wherein, the release component is composed of 0.01% to 5% anionic surfactant and 0.01% to 5% nonionic surfactant; The nucleic acid stabilizing component is composed of 0.01% to 10% nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent; The buffer component is 0.1-50 mM Tris; Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof; The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof; The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof; The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.

8. A method for increasing the release rate of nucleic acid in a sample, characterized in that: Includes steps: (s1) provide a sample; and (s2) adding the sample to a preservation solution and mixing the sample, thereby preserving the sample and increasing the release rate of nucleic acid in the sample; The storage solution is composed of a release component, a nucleic acid stabilizing component and a buffer component; Wherein, the release component is composed of 0.01% to 5% anionic surfactant and 0.01% to 5% nonionic surfactant; The nucleic acid stabilizing component is composed of 0.01% to 10% nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent; The buffer component is 0.1-50 mM Tris; Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof; The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof; The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof; The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or a combination thereof.

9. The method according to claim 8, characterized in that The method further comprises step (s3) of adding grinding filter beads to the preservation solution containing the sample and mixing the mixture.

10. A method for nucleic acid detection, characterized in that: Includes steps: (z1) adding the sample to be tested into a preservation solution and mixing evenly, wherein the preservation solution comprises a release component, a nucleic acid stabilizing component and a buffer component; Wherein, the release component is composed of 0.01% to 5% anionic surfactant and 0.01% to 5% nonionic surfactant; The nucleic acid stabilizing component is composed of 0.01% to 10% nucleic acid protective agent and 0.1 to 20 mM metal ion chelating agent; The buffer component is 0.1-50 mM Tris; Wherein, the anionic surfactant is selected from the following group: sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or a combination thereof; The nonionic surfactant is selected from the group consisting of NP-40, Tween-20, Triton-X-100, Brij-35, or a combination thereof; The nucleic acid protecting agent is selected from the group consisting of formamide, trehalose, or a combination thereof; The metal ion chelator is selected from the group consisting of EDTA, EGTA, or a combination thereof; and (z2) Performing nucleic acid testing on the storage solution containing the sample to be tested to obtain the nucleic acid test result.

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