Cracking binding solution, kit and extraction method for extracting free DNA (Deoxyribose Nucleic Acid) from plasma

Abstract

The invention relates to the technical field of plasma free DNA extraction, and particularly discloses a lysis binding solution, a kit and an extraction method for plasma free DNA extraction. The cracking binding liquid comprises the following components: chaotropic agent, chelating agent, buffering agent, defoaming agent and detergent; wherein the content of the defoaming agent in the cracking combination liquid is 0.0005 to 0.01 weight percent; the content of the detergent in the cracking combination liquid is 1 to 10 weight percent. The extraction kit comprises the lysis binding solution. In the extraction method, the cracking binding liquid is used. The synergistic effect of a defoaming agent and a detergent in the lysis binding liquid remarkably improves the extraction efficiency of free DNA in plasma, and later nucleic acid detection is facilitated.

Description

Technical Field

[0001] The present application relates to the technical field of plasma free DNA extraction, and in particular to a lysis and binding solution, a kit and an extraction method for plasma free DNA extraction. Background Art

[0002] Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biological macromolecules that carry genetic information. Cell free DNA (cfDNA) refers to DNA that is free from cells in circulating blood. Most of them are short chains, and the concentration is very low. They are easily contaminated and diluted by non-cfDNA with higher abundance, especially in patients with early cancer. In recent years, as liquid biopsy technology represented by cfDNA has become an important technology for molecular diagnosis or auxiliary diagnosis, the study of free DNA has become a hot field in genomic molecular diagnosis research. It has been gradually applied to various stages from the initial diagnosis of tumors to treatment and development, including: early screening, evaluation of tumor heterogeneity, risk of metastasis and recurrence, and prognosis evaluation, real-time monitoring of treatment response and drug resistance, tumor staging and grading, and guidance of treatment plan selection.

[0003] Due to the low content and short half-life of cfDNA, the extraction method of cfDNA has received much attention. The first step in using cfDNA for testing is to extract and purify cfDNA in human plasma. The quality of the extracted product directly affects the accuracy and reliability of subsequent test results. However, due to the low content of cfDNA and the short fragments, the yield of cfDNA is likely to be low due to insufficient cell lysis during cell lysis. Therefore, in order to increase the yield of cfDNA, a higher concentration of surfactant is added during cell lysis to fully destroy the cell membrane and fully release the nucleic acid.

[0004] For example, some prior art discloses a DNA extract solution, including the following components: 0.1-1M NaOH, 0.01%-0.1% sodium dodecyl sulfate, 10-500nM NaCl, 0.01mM disodium ethylenediaminetetraacetate, 0.05-0.5g / L gelatin and 0.001%-0.01% defoamer A. At the same time, a simple and efficient method for preparing plant genomic DNA is also disclosed: the method can extract plant DNA in only one step, and hundreds of DNA samples can be obtained in 5 minutes for subsequent PCR amplification reactions without organic reagent contamination. The method comprises: adding samples, steel beads and direct amplification DNA extract solution to a 96-well deep well plate; using a vibrating sample grinder for oscillation grinding to break up the tissue; aspirating the supernatant and diluting it 5-10 times for PCR amplification. However, the main research of the prior art is: adding defoaming agent A during the plant grinding process to reduce the large amount of foam generated during the grinding sample shaking process, reduce pollution, and facilitate subsequent operations; however, the prior art is aimed at the extraction of DNA from plant cells, and does not involve the extraction of free DNA from blood and the improvement of extraction efficiency.

[0005] Some prior art also discloses a low-foam inactivated viral nucleic acid preservation solution that is compatible with magnetic bead nucleic acid extraction. The inactivated viral nucleic acid preservation solution mainly contains the following ingredients: 0.1-2% (w / v) nuclease inhibitor, 0.1-0.5% (w / v) protein denaturant, 1-5% (w / v) buffer stabilizer, and 0.1-1% (w / v) defoaming agent. The preservation solution can directly inactivate viruses, preserve viral nucleic acid fragments, inhibit nuclease activity, protect medical staff, and reduce the risk of infection. Throat swabs, nasal swabs or tissue samples from specific parts can be collected, and the samples can be stored and transported for subsequent nucleic acid extraction. After strong shaking, the foam generated in the preservation solution will quickly disappear after standing for 20 seconds, and will not affect subsequent sampling operations. Although a defoaming agent is added to the preservation solution, the component achieves the technical effect by adding 5%-10% anhydrous ethanol and a polyether defoaming agent together; the presence of ethanol will make the subsequent DNA separation and purification steps more complicated; and the nucleic acid preservation solution is used for the preservation of virus samples and does not involve the extraction of free DNA in blood and the extraction efficiency.

[0006] In summary, at present, it is necessary to propose a cell lysis solution and extraction method for free DNA in plasma that is alcohol-free and has a high extraction rate. Summary of the invention

[0007] The present application provides a lysis binding solution, a kit and an extraction method for plasma free DNA extraction. In the lysis binding solution, since the addition of alcohol in the lysis binding solution will affect the accuracy of the later detection, no alcohol (such as ethanol, isopropanol, etc.) is added, and the step of removing alcohol during the later DNA purification is also avoided. When the plasma free DNA is extracted, during the cell lysis process, the combination of plasma components and the lysis binding solution will produce foams, which will not only affect the experimental operation, but also reduce the extraction efficiency and purity of nucleic acids; therefore, the addition of defoamers during the extraction process can effectively reduce or eliminate these foams, thereby improving the accuracy and repeatability of the experiment; in addition, the use of defoamers can also improve the efficiency of nucleic acid extraction, reduce sample loss caused by foam, and thus improve the sensitivity of detection. In addition, in the present application, a suitable type of defoamer (such as an organosilicon defoamer) is further screened, which synergizes with octyl-beta-D-thiopyranoglucoside to significantly improve the extraction rate of free DNA in plasma.

[0008] This application involves the following:

[0009] 1. A lysis and binding solution for extracting free DNA from plasma, comprising the following components: a chaotropic agent, a chelating agent, a buffer, a defoaming agent, and a detergent;

[0010] Wherein, the content of the defoaming agent in the lysis and binding solution is 0.0005-0.01wt%; the content of the detergent in the lysis and binding solution is 1-10wt%.

[0011] 2. According to the lysis and binding liquid of item 1, the defoaming agent is selected from any one or more of a polyether defoaming agent and a silicone defoaming agent;

[0012] and / or,

[0013] The detergent is selected from any one or more of octyl-beta-D-thiopyranoglucoside, sodium dodecyl sulfate, Triton and Tween NP40.

[0014] 3. The lysis and binding solution according to item 1 or 2, wherein the content of the chaotropic agent in the lysis and binding solution is 5-11M;

[0015] and / or, the amount of the chelating agent added to the lysis and binding solution is 5-11 M;

[0016] And / or, the amount of the buffer added to the lysis and binding solution is 10mM-1M.

[0017] 4. According to the lysis and binding solution of any one of items 1 to 3, the chaotropic agent is selected from any one or more combinations of guanidines and inorganic salts; preferably, the guanidines are selected from any one or more combinations of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate and guanidine hydrochloride; preferably, the inorganic salt is selected from any one or more combinations of sodium iodide, sodium perchlorate and sodium bromide; more preferably, the chaotropic agent is a combination of guanidine isothiocyanate and sodium iodide;

[0018] And / or, the chelating agent is selected from any one or more combinations of ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylenetriaminepentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, ethylenediaminetetraacetic acid, anhydrous citric acid, sodium citrate, calcium citrate, ammonium citrate, ammonium hydrogen citrate, citric acid, diammonium citrate, ammonium ferric citrate and lithium citrate;

[0019] And / or, the buffer is selected from any one or more combinations of Tris sulfuric acid and Tris hydrochloric acid.

[0020] 5. The lysis and binding solution according to any one of items 1 to 4, further comprising trehalose; preferably, the amount of trehalose added to the lysis and binding solution is 0.2-0.35 M;

[0021] And / or, the lysis and binding solution further comprises an osmotic pressure balancing agent; preferably, the osmotic pressure balancing agent is selected from any one or more combinations of glycerol, sodium citrate, mannitol and sorbitol; preferably, the amount of the osmotic pressure balancing agent added to the lysis and binding solution is 3-5wt%;

[0022] And / or, the lysis and binding solution further comprises an inorganic salt; preferably, the inorganic salt is selected from any one or more combinations of calcium chloride, sodium chloride, sodium sulfate, potassium sulfate and potassium chloride; preferably, the amount of the inorganic salt added to the lysis and binding solution is 0.5-2M.

[0023] 6. A kit for extracting free DNA from plasma, the kit comprising the lysis and binding solution of any one of items 1 to 5;

[0024] Preferably, the kit further comprises a solid phase adsorption matrix, a rinsing solution A, a rinsing solution B, and an eluting solution.

[0025] 7. The kit according to item 6, wherein the components of the rinse solution A include: a chaotropic agent, a buffer, a detergent and ethanol;

[0026] Preferably, the content of the chaotropic agent in the rinsing solution A is 2-6M;

[0027] and / or, the content of the buffer in the rinse solution A is 10 mM-1 M;

[0028] and / or, the content of the detergent in the rinsing liquid A is 3-8wt%;

[0029] and / or, the content of ethanol in the rinse solution A is 30-50 Vol.%;

[0030] Further preferably, the chaotropic agent is selected from any one or more combinations of guanidines and inorganic salts; preferably, the guanidines are selected from any one or more combinations of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate and guanidine hydrochloride; preferably, the inorganic salt is selected from any one or more combinations of sodium iodide, sodium perchlorate and sodium bromide; more preferably, the chaotropic agent is a combination of guanidine isothiocyanate and sodium iodide;

[0031] And / or, the buffer is selected from any one or more combinations of Tris sulfuric acid and Tris hydrochloric acid;

[0032] And / or, the detergent is selected from any one or more of octyl-beta-D-thiopyranoglucoside, sodium dodecyl sulfate, Triton and Tween NP40; preferably, the detergent is octyl-beta-D-thiopyranoglucoside.

[0033] 8. The kit according to item 6 or 7, wherein the components of the rinse solution B include: a buffer, polyethylene glycol and an inorganic salt;

[0034] Preferably, the content of the buffer in the rinse solution B is 10mM-1M;

[0035] and / or, the content of the polyethylene glycol in the rinsing liquid B is 12-15 wt %;

[0036] and / or, the content of the inorganic salt in the rinsing liquid B is 0.5-1.5M;

[0037] Further preferably, the buffer is selected from any one or more combinations of Tris sulfuric acid and Tris hydrochloric acid;

[0038] And / or, the inorganic salt is selected from any one or more combinations of calcium chloride, sodium chloride, sodium sulfate, potassium sulfate and potassium chloride.

[0039] 9. The kit according to any one of items 6 to 8, wherein the components of the eluent comprise a chelating agent and a buffer;

[0040] Preferably, the content of the chelating agent in the eluent is 0.01-10 mM;

[0041] and / or, the content of the buffer in the eluent is 1-100 mM;

[0042] Preferably, the chelating agent is selected from any one or more combinations of ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylenetriaminepentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, ethylenediaminetetraacetic acid, anhydrous citric acid, sodium citrate, calcium citrate, ammonium citrate, ammonium hydrogen citrate, citric acid, diammonium citrate, ammonium ferric citrate and lithium citrate;

[0043] And / or, any one or more combinations of the buffering agents Tris sulfuric acid and Tris hydrochloric acid.

[0044] 10. A method for extracting free DNA from plasma, comprising the steps of contacting and mixing the lysis and binding solution of any one of items 1 to 5 with a plasma sample and a solid phase adsorption matrix;

[0045] Preferably, the extraction method further comprises:

[0046] The solid phase adsorption matrix adsorbed with free DNA is washed with a washing solution A;

[0047] The solid phase adsorption matrix adsorbed with free DNA is washed with a washing solution B;

[0048] The solid phase adsorption matrix adsorbed with free DNA is washed with an elution solution.

[0049] Effects of the Invention

[0050] 1. The present application provides a lysis and binding solution for extracting free DNA in plasma, which does not contain alcohols (such as ethanol, isopropanol, etc.), thereby directly eliminating the step of dealcoholization during the subsequent DNA separation and purification; in addition, it also effectively avoids the problem of inaccurate test results caused by insufficient alcohol removal.

[0051] 2. The lysis and binding solution of the present application improves the extraction concentration of free DNA in plasma through the synergistic effect of defoaming agent and detergent, so as to further improve the accuracy of free DNA content detection in plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 .This application does not add defoaming agent to lyse and extract free DNA to extract fragment distribution.

[0053] Figure 2 .Add polyether defoaming agent to lyse and extract free DNA and extract fragment distribution.

[0054] Figure 3 .Add silicone defoaming agent to lyse and extract the distribution of free DNA fragments in plasma.

[0055] Figure 4 . Add anhydrous ethanol to lyse and extract the distribution of plasma free DNA fragments.

[0056] Figure 5 .Compare the recovery rate Ct value of cfDNA extracted by the lysis and binding solution containing defoaming agent in this application with that of cfDNA extracted by the reference kit.

[0057] Specific implementation methods

[0058] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in components' functions as the criterion for distinction.

[0059] As mentioned throughout the specification and claims, "including" or "comprising" is an open term and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0060] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments. References to "about" a value or parameter herein include (and describe) variations to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0061] As used herein, reference to a "not" value or parameter generally means and describes an "except" value or parameter. For example, the method is not used to treat cancer type X, meaning that the method is used to treat cancers other than type X.

[0062] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. This statement is therefore intended to serve as antecedent basis for the use of exclusive terminology such as "only", "only" and the like in connection with the recitation of claim elements, or the use of a limitation of "no".

[0063] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0064] The present application provides a lysis and binding solution for extracting plasma free DNA, which comprises the following components: a chaotropic agent, a chelating agent, a buffer, a defoaming agent, and a detergent. It should be noted that when the lysis and binding solution of the present application is used for extracting plasma free DNA, the extraction concentration of plasma free DNA can be significantly improved; however, this does not limit the application of the lysis and binding solution. For example, it can also be used for extracting DNA from other sources.

[0065] In the present application, the chaotropic agent can be a guanidine chaotropic agent, or any one or more of an inorganic salt chaotropic agent. For example, the guanidine chaotropic agent can be any one or more of a combination selected from guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate and guanidine hydrochloride. For example, the inorganic salt chaotropic agent can be any one or more of a combination of sodium iodide, sodium perchlorate and sodium bromide. In some embodiments, the chaotropic agent can be selected from any one or more of a combination of the following groups: guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate, guanidine hydrochloride, sodium iodide, sodium perchlorate and sodium bromide. In some preferred embodiments, the chaotropic agent is a combination of guanidine isothiocyanate and sodium iodide.

[0066] In some embodiments, the content of the chaotropic agent in the lysis binding solution is 5-11M, 6-10M, 7-9M or any content range in the range of 5-11M; for example, it can be 5M, 5.2M, 5.4M, 5.6M, 5.8M, 6M, 6.2M, 6.4M, 6.6M, 6.6M, 6.8M, 7M, 7.2M, 7.4M, 7.6M, 7.8M, 8M, 8.2M, 8.4M, 8.6M, 8.8M, 9M, 9.2M, 9.4M, 9.6M, 9.8M, 10M, 10.2M, 10.4M, 10.6M, 10.8M, 11M or any content in the range of 5-11M. Further, in some embodiments, when the chaotropic agent is a combination of a guanidine chaotropic agent and an inorganic salt chaotropic agent, the content of the guanidine chaotropic agent in the lysis and binding solution is 2-6M, 3-5M or any content in the range of 2-6M; for example, it can be 2M, 2.2M, 2.4M, 2.6M, 2.8M, 3M, 3.2M, 3.4M, 3.6M, 3.8M, 4M, 4.2M, 4.4M, 4.6M, 4.8M, 5M, 5.2M, 5.4M, 5.6M, 5.8M, 6M or any content in the range of 2-6M. In some embodiments, the content of the inorganic salt chaotropic agent in the lysis binding solution is 3-5M, 3.5-4.5M or any content in the range of 3-5M; for example, it can be 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, 5M or any content in the range of 3-5M.

[0067] In some embodiments, the chelating agent is selected from any one or more combinations of ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylenetriaminepentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, ethylenediaminetetraacetic acid, anhydrous citric acid, sodium citrate, calcium citrate, ammonium citrate, ammonium hydrogen citrate, citric acid, diammonium citrate, ammonium ferric citrate, and lithium citrate. In some embodiments, the chelating agent is ethylene glycol tetraacetic acid; in some embodiments, the chelating agent is sodium citrate; in some embodiments, the chelating agent is ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; in some embodiments, the chelating agent is ammonium hydrogen citrate; in some embodiments, the chelating agent is calcium citrate; in some embodiments, the chelating agent is ammonium ferric citrate; in some embodiments, the chelating agent is N,N-bis(carboxymethyl)glycine. In some embodiments, the amount of the chelating agent added to the lysis binding solution is 5-11M, for example, it can be 5M, 5.2M, 5.4M, 5.6M, 5.8M, 6M, 6.2M, 6.4M, 6.6M, 6.6M, 6.8M, 7M, 7.2M, 7.4M, 7.6M, 7.8M, 8M, 8.2M, 8.4M, 8.6M, 8.8M, 9M, 9.2M, 9.4M, 9.6M, 9.8M, 10M, 10.2M, 10.4M, 10.6M, 10.8M, 11M or any content in the range of 5-11M.

[0068] In the present application, there is no other restriction on the specific selection of the buffer, which can have the function of adjusting and controlling the pH value of the solution and keeping the pH of the solution relatively stable when acid or alkali is added. In some embodiments, the buffer is selected from any one or more combinations of Tris sulfuric acid and Tris hydrochloric acid. In some embodiments, the amount of the buffer added in the lysis binding solution is 10mM-1M, 50mMv1M, 100mM-900mM, 200mM-800mM, 300mM-700mM, 400mM-600mM, 450mM-550mM or any content range within the range of 10mM-1M; for example, it can be 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1M or any content within the range of 10mM-1M.

[0069] In the present application, the detergent and defoamer in the lysis and binding solution can significantly increase the extraction concentration of free DNA in plasma through synergistic action; for example, compared with the traditional lysis and extraction kit (the average Ct value of ACTB gene in plasma free DNA is 30.28), when the Ct value of ACTB gene in plasma free DNA is measured by the lysis and binding solution of the present application, the Ct value is about 29.33-29.93, which makes its Ct value advance by about 1.5-2, that is, qualitatively reflects that the extraction recovery rate is higher. In addition, in the lysis and binding solution, the extraction concentration of free DNA in plasma is maintained in the range of 1.18-1.36ng / μL or even as high as 1.38ng / μL, 1.4ng / μL, and 1.42ng / μL through the cooperation of the detergent and the defoamer, which is much higher than the extraction concentration of adding only detergent or defoamer alone (0.92ng / μL and 0.86ng / μL, respectively).

[0070] In some embodiments, the detergent is selected from any one or more of octyl-beta-D-thiopyranoglucoside, sodium dodecyl sulfate, Triton, and Tween NP40. In some preferred embodiments, the detergent is octyl-beta-D-thiopyranoglucoside.

[0071] In some embodiments, the defoaming agent is selected from any one or more of a polyether defoaming agent and a silicone defoaming agent; more preferably, the defoaming agent is a silicone defoaming agent.

[0072] In addition, the applicant further discovered that the lysis binding solution of the present application has good stability. For example, after the configured lysis binding solution is placed under a certain condition (e.g., room temperature) for a period of time (e.g., 1-6 months), the concentration of the target DNA in the same plasma sample is detected using the lysis binding solution after placement, and it is found that a high extraction concentration of the target DNA can still be obtained. For example, in some embodiments, the configured lysis binding solution can be placed at 45-55°C (e.g., 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C) for 5-15 days (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days) to monitor the stability of the reagent. In a specific embodiment, the configured lysis binding solution can be placed at 50°C for 7 days before the target DNA content in the plasma sample is determined. It should be understood by those skilled in the art that in this experiment, in order to avoid differences in data results caused by differences in the plasma samples of this application, it is necessary to ensure that the plasma samples before and after the accelerated experiment are consistent and that there are no significant differences in the components. For example, in some embodiments, multiple plasma samples can be mixed when preparing plasma samples to obtain a larger sample size; and the plasma samples are stored under the required conditions to ensure that there are no significant changes in the components therein; when testing, the same plasma sample is used for testing twice. The applicant found that in the lysis and binding solution, the type of defoamer has a greater impact on the experimental results: compared with polyether defoamers, silicone defoamers have more significant stability; further, among silicone defoamers, silicone defoamers with an active ingredient content of 15-25wt% have more excellent stability. Therefore, in some preferred embodiments, the content of the active ingredient in the silicone defoamer is 15-25wt%, 16-24wt%, 17-23wt%, 16-22wt% or any content range within the range of 15-25wt%; in a specific embodiment, the content of the active ingredient in the silicone defoamer is 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt% or any content within the range of 15-25wt%; in a specific embodiment, the silicone defoamer is purchased from Aladdin, item number S466558, containing 20wt% of active ingredients. Among them, the "active ingredient" can be understood as, for example, polysiloxane in the silicone defoamer, especially polydimethylsiloxane (PDMS); it can also be other types of polysiloxane, such as fluorosilicone and glycol siloxane; therefore, the content of the active ingredient is the sum of the mass proportion of the polysiloxane contained in the silicone defoamer relative to the silicone defoamer.

[0073] In some embodiments, the content of the defoaming agent in the lysis and binding solution is any content range within the range of 0.0005-0.01wt%, 0.001-0.005wt%, 0.002-0.004wt%, 0.0025-0.0035wt% or 0.0005-0.01wt%. In a specific embodiment, the content of the defoaming agent in the lysis and binding solution is 0.0005wt%, 0.0006wt%, 0.0007wt%, 0.0008wt%, 0.0009wt%, 0.001wt%, 0.0015wt%, 0.002wt%, 0.0025wt%, 0.003wt%, 0.0035wt%, 0.004wt%, 0.0045wt%, 0.005wt%, 0.006wt%, 0.007wt%, 0.008wt%, 0.009wt%, 0.01wt% or any content in the range of 0.0005-0.01wt%.

[0074] In some embodiments, the content of the detergent in the lysis and binding solution is 1-10wt%, 3-8wt%, 4-7wt%, 5-6wt% or any content range in the range of 1-10wt%. In a specific embodiment, the content of the detergent in the lysis and binding solution is 1wt%, 2wt%, 3wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, 5wt%, 5.2wt%, 5.4wt%, 5.6wt%, 5.8wt%, 6wt%, 6.2wt%, 6.4wt%, 6.6wt%, 6.8wt%, 7wt%, 7.2wt%, 7.4wt%, 7.6wt%, 7.8wt%, 8wt%, 9wt%, 10wt%, or any content in the range of 1-10wt%.

[0075] In some embodiments, the lysis and binding solution comprises the following components: 0.001-0.005 wt % of antifoaming agent, 3-8 wt % of detergent, 5-11 M of chelating agent, 5-11 M of chaotropic agent and 10 mM-1 M of buffer.

[0076] In some embodiments, the lysis and binding solution further comprises trehalose. In some embodiments, the amount of trehalose added to the lysis and binding solution is 0.2-0.35 M; for example, 0.2M, 0.21M, 0.22M, 0.23M, 0.24M, 0.25M, 0.26M, 0.27M, 0.28M, 0.29M, 0.3M, 0.31M, 0.32M, 0.33M, 0.34M, 0.35M or any content in the range of 0.2-0.35M.

[0077] In some embodiments, the lysis and binding solution further comprises an osmotic pressure balance agent. In some embodiments, the osmotic pressure balance agent is selected from any one or more combinations of glycerol, sodium citrate, mannitol and sorbitol. In some embodiments, the amount of the osmotic pressure balance agent added to the lysis and binding solution is 3-5wt%; for example, 3wt%, 3.1wt%, 3.2wt%, 3.3wt%, 3.4wt%, 3.5wt%, 3.6wt%, 3.7wt%, 3.8wt%, 3.9wt%, 4.0wt%, 4.1wt%, 4.2wt%, 4.3wt%, 4.4wt%, 4.5wt%, 4.6wt%, 4.7wt%, 4.8wt%, 4.9wt%, 5.0wt% or any content within the range of 3-5wt%.

[0078] In some embodiments, the lysis and binding solution further comprises an inorganic salt. In some embodiments, the inorganic salt is selected from any one or more combinations of calcium chloride, sodium chloride, sodium sulfate, potassium sulfate and potassium chloride. In some embodiments, the amount of the inorganic salt added to the lysis and binding solution is 0.5-2M; for example, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M or any content in the range of 0.5-2M.

[0079] The present application also provides a kit for extracting free DNA from plasma, wherein the kit comprises the lysis and binding solution.

[0080] In some embodiments, the kit further comprises a solid phase adsorption matrix, a rinse solution A, a rinse solution B, and an eluent.

[0081] In some embodiments, the components of the rinse solution A include: a chaotropic agent, a buffer, a detergent and ethanol.

[0082] In some embodiments, the content of the chaotropic agent in the rinse solution A is 2-6M; for example, it can be 2M, 2.2M, 2.4M, 2.6M, 2.8M, 3M, 3.2M, 3.4M, 3.6M, 3.8M, 4M, 4.2M, 4.4M, 4.6M, 4.8M, 5M, 5.2M, 5.4M, 5.6M, 5.8M, 6M or any content in the range of 2-6M.

[0083] In some embodiments, the content of the buffer in the rinse solution A is 10mM-1M; for example, it can be 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1M or any content in the range of 10mM-1M.

[0084] In some embodiments, the content of the detergent in the rinse liquid A is 3-8wt%; for example, it can be 3wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, 5wt%, 5.2wt%, 5.4wt%, 5.6wt%, 5.8wt%, 6wt%, 6.2wt%, 6.4wt%, 6.6wt%, 6.8wt%, 7wt%, 7.2wt%, 7.4wt%, 7.6wt%, 7.8wt%, 8wt% or any content in the range of 3-8wt%.

[0085] In some embodiments, the content of ethanol in the rinse liquid A is 30-50 Vol.%; for example, it can be 30 Vol.%, 31 Vol.%, 32 Vol.%, 33 Vol.%, 34 Vol.%, 35 Vol.%, 36 Vol.%, 37 Vol.%, 38 Vol.%, 39 Vol.%, 40 Vol.%, 41 Vol.%, 42 Vol.%, 43 Vol.%, 44 Vol.%, 45 Vol.%, 46 Vol.%, 47 Vol.%, 48 Vol.%, 49 Vol.%, 50 Vol.%, or any content within the range of 30-50 Vol.%.

[0086] In some embodiments, the components of the rinse solution B include: a buffer, polyethylene glycol, and an inorganic salt.

[0087] In some embodiments, the content of the buffer in the rinse solution B is 10mM-1M; for example, it can be 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1M or any content in the range of 10mM-1M.

[0088] In some embodiments, the content of polyethylene glycol in the rinse liquid B is 12-15wt%; for example, it can be 12.2wt%, 12.4wt%, 12.6wt%, 12.8wt%, 13wt%, 13.2wt%, 13.4wt%, 13.6wt%, 13.8wt%, 14wt%, 14.2wt%, 14.4wt%, 14.6wt%, 14.8wt%, 15wt% or any content in the range of 12-15wt%.

[0089] In some embodiments, the content of the inorganic salt in the rinse solution B is 0.5-1.5M; for example, it can be 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M or any content in the range of 0.5-1.5M.

[0090] In some embodiments, the components of the eluent include a chelating agent and a buffer.

[0091] In some embodiments, the content of the chelating agent in the eluent is 0.01-10 mM; for example, 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM or any content range within the range of 0.01-10 mM.

[0092] In some embodiments, the content of the buffer in the eluent is 1-100 mM; for example, 1 mM, 3 mM, 6 mM, 9 mM, 12 mM, 15 mM, 18 mM, 21 mM, 24 mM, 27 mM, 30 mM, 33 mM, 36 mM, 39 mM, 42 mM, 45 mM, 48 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM or any content range within the range of 1-100 mM.

[0093] In some embodiments, the chelating agent is selected from any one or more combinations of ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylenetriaminepentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, ethylenediaminetetraacetic acid, anhydrous citric acid, sodium citrate, calcium citrate, ammonium citrate, ammonium hydrogen citrate, citric acid, diammonium citrate, ammonium ferric citrate and lithium citrate; for example, in a preferred embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0094] In some embodiments, the buffer is any one or more of Tris sulfuric acid and Tris hydrochloric acid. In a preferred embodiment, the buffer is Tris sulfuric acid.

[0095] The present application also provides a method for extracting plasma free DNA, the extraction method comprising the step of contacting and mixing the lysis binding solution with the plasma sample and the solid phase adsorption matrix. In some embodiments, the contact and mixing time of the lysis binding solution with the plasma sample and the solid phase adsorption matrix is ​​10-15 minutes; for example, it can be 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes or any time within 10-15 minutes.

[0096] In some embodiments, the extraction method further comprises: washing the solid phase adsorption matrix adsorbed with free DNA with a washing solution A.

[0097] In some embodiments, the extraction method further comprises: washing the solid phase adsorption matrix adsorbed with free DNA with a washing solution B;

[0098] In some embodiments, the extraction method further comprises: washing the solid phase adsorption matrix adsorbed with the free DNA with an elution solution.

[0099] In some embodiments, the extraction method comprises:

[0100] The lysis binding solution is contacted and mixed with the plasma sample and the solid phase adsorption matrix;

[0101] The solid phase adsorption matrix adsorbed with free DNA is washed with a washing solution A;

[0102] washing the solid phase adsorption matrix adsorbed with free DNA with a washing solution B; and

[0103] The solid phase adsorption matrix adsorbed with free DNA is washed with an elution solution.

[0104] In some embodiments, when the solid phase adsorption matrix adsorbed with free DNA is washed with an eluent, the condition parameters include: the washing temperature is 20-60°C, 23-56°C, 26-53°C, 29-50°C or any temperature range within the range of 20-60°C, for example, 20°C, 23°C, 26°C, 29°C, 31°C, 33°C, 36°C, 39°C, 41°C, 44°C, 47°C, 50°C, 53°C, 56°C, 59°C, 60°C or any temperature within the range of 20-60°C; and

[0105] The washing time is 5-10 min, for example, 5 min, 5.2 min, 5.4 min, 5.6 min, 5.8 min, 6 min, 6.4 min, 6.8 min, 7.2 min, 7.6 min, 8 min, 8.5 min, 9 min, 9.5 min, 10 min or any time range within the range of 5-10 min. DETAILED DESCRIPTION

[0106] The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0107] The defoaming agent involved in the following embodiments comprises:

[0108] Polyether defoamer 1: Defoamer 204, purchased from Aladdin, product number A489541;

[0109] Polyether defoamer 2: Defoamer B emulsion, purchased from Aladdin, product number A489551, is a siloxane-based defoamer;

[0110] Silicone defoamer 1: Defoamer C emulsion, purchased from Aladdin, item number A485312, main component 30wt% polydimethylsiloxane concentrate aqueous emulsion;

[0111] Silicone defoamer 2: Defoamer SE1 5, main ingredients: 10wt% active silicon and aqueous emulsion of non-ionic emulsifier, product number A489557;

[0112] Silicone defoamer 3: Silicone defoamer, purchased from Aladdin, item number S466558, containing 20 wt% of active ingredients, and tolerant to pH.

[0113] Example 1.

[0114] The lysis and binding solution contains the following components and the content of each component: 4M guanidine thiocyanate, 4M sodium iodide, 500mM Tris-SO4, 4M sodium citrate, 4wt% glycerol, 1.5M calcium chloride, 6wt% octyl-beta-D-thiopyranoglucoside, 0.2M trehalose, and 0.002wt% polyether defoamer (defoamer 204, purchased from Aladdin, English name Antifoam 204, item number A489541, is a mixture of organic non-silicone polypropylene polyether dispersions).

[0115] The rinse solution A contains the following components and the content of each component: 4M guanidine isothiocyanate, 500mM Tris-SO4, 6wt% octyl-beta-D-thiopyranoglucoside and 40Vol.% anhydrous ethanol.

[0116] The rinse solution B contains the following components and the content of each component: 500 mM Tris-SO4, 18 wt% PEG6000 and 1 M sodium chloride.

[0117] The eluent contains the following components and the content of each component: 5 mM EGTA, 50 mM Tris-SO4.

[0118] The magnetic beads were purchased from Tianjin Huachen Bell Biotechnology Co., Ltd. The magnetic core is an iron-cobalt-nickel alloy Fe-Co-NiOX, and the particle size distribution is between 100-1000 nm. The surface of the magnetic core is coated with at least a carbon layer and an inorganic silicon dioxide layer from the inside to the outside, and polyethylene glycol is covalently grafted on the outermost layer.

[0119] The kit of the present application comprises the above-mentioned lysis and binding solution, rinsing solution A, rinsing solution B, elution solution and magnetic beads.

[0120] Method for extracting free DNA from plasma:

[0121] It uses ACTB as an internal reference to detect the content of cfDNA extracted from healthy human plasma samples, including the following steps:

[0122] 1) Lysis and binding: Place 2 mL of plasma sample in a 15 mL centrifuge tube, add 2 mL of lysis and binding solution, add 40 μL of magnetic beads, invert and mix several times, then shake and mix for 12 minutes (generally 10-15 minutes), place on a magnetic rack, and discard the supernatant.

[0123] 2) Washing and purification: Take out the centrifuge tube, add 1.5 mL of rinse solution A, shake and mix, transfer the rinse solution A and magnetic beads to a 2 mL centrifuge tube and place it on a magnetic rack, perform magnetic separation, and discard the waste liquid.

[0124] 3) Washing and purification: Take out the centrifuge tube, add 800 μL of rinsing solution B, shake and mix, transfer the rinsing solution and magnetic beads to a 2 mL centrifuge tube and place it on a magnetic rack for magnetic separation, and discard the waste liquid.

[0125] 4) Elution: Add 80 μL (usually 50-100 μL) of elution solution, place in a constant temperature oscillator, and elute at 37°C for 8 minutes (usually 23-56°C for 5-10 minutes);

[0126] 5) Place the centrifuge tube on a magnetic rack for magnetic separation, carefully pipette the supernatant into a new centrifuge tube, and store at -20°C for later use.

[0127] Example 2.

[0128] The difference between this embodiment and embodiment 1 is that the defoamer in the lysis and binding solution and its content are: 0.002wt% of organosilicon defoamer (purchased from Aladdin, item number S466558, main components 20% of active ingredients and 33% of solids, tolerant to different pH values). Others are the same as in embodiment 1.

[0129] Comparative Example 1.

[0130] The difference between this embodiment and embodiment 1 is that the defoaming agent and its content in the lysis and binding solution are: 10 Vol.% anhydrous ethanol. Others are the same as in embodiment 1.

[0131] Comparative Example 2.

[0132] The difference between this comparative example and Example 1 is that the lysis and binding solution does not contain a defoaming agent. Other steps are the same as those in Example 1.

[0133] Comparative Example 3.

[0134] The difference between this comparative example and Example 1 is that the lysis and binding solution does not contain a detergent. The other steps are the same as Example 1.

[0135] Effect verification:

[0136] To verify the extraction efficiency of cfDNA in plasma, it is necessary to perform detection by real-time fluorescence quantitative PCR (TaqMan probe). The human internal reference gene ACTB is used as the detection object, and primer probes are designed for the DNA sequence. The primer sequences are as follows:

[0137] Primers and probes for ACTB:

[0138] ACTB_F:5'-ACTATTCCACCTCCCACCC-3'

[0139] ACTB_R:5'-CACCCCCTCTCTCCACAACT-3'

[0140] ACTB_P:5'-TTCTCACCTCAACCCTCTCCCCC-3'

[0141] The product size is 65 bp.

[0142] The PCR amplification system used in this example is shown in Table 1.

[0143] Table 1

[0144]

[0145]

[0146] The PCR amplification program was: 95°C, 3 min; (93°C, 30 s; 59.7°C, 30 s; - read the fluorescence signal) 40 cycles; 40°C, 5 s. The threshold was set at 240.

[0147] (1) DNA fragment distribution and genomic DNA residue determination

[0148] The fragment distribution and genomic DNA residue of the extracted free DNA were determined using Agilent Technologies 2100 analyzer.

[0149] The ACTB gene was detected according to the above reaction system and reaction procedure. The distribution results of the extracted DNA fragments in Comparative Example 1 are as follows: Figure 1 The distribution results of the extracted DNA fragments in Examples 1 to 3 are as follows: Figures 2 to 4 .

[0150] from Figures 1 to 4 The results showed that when the defoaming agent system was added, there was a main peak at around 150bp, and no obvious main peak above 500bp, that is, the results of plasma DNA extraction were all cfDNA, and there was no genomic residue. This further shows that the increase in extraction yield is not due to genomic contamination, but real cfDNA.

[0151] (2) Extraction recovery rate and concentration test

[0152] Thermol×dsDNA HS Working Solution was used to detect the cfDNA concentration in plasma samples, and a comparative study was conducted with the free DNA magnetic bead extraction kit of Tiangen Biochemical Technology Co., Ltd. (i.e., the reference reagent in the table below). The results are shown in Table 2.

[0153] Table 2

[0154]

[0155] The concentration test of Qubit reagent showed that the extraction recovery rate increased by 1.77 times when the double-stranded cfDNA concentration was determined by adding defoaming agent compared with that without adding defoaming agent; and the present application added defoaming agent and then performed lysis and binding, which helped to improve the solution stability and magnetic bead binding capacity; in addition, the addition of anhydrous ethanol to reduce foam in the present application was not effective, and the solution was stratified, resulting in a low final extraction recovery rate.

[0156] (3) Fluorescence quantitative detection results

[0157] The average Ct value of ACTB gene in plasma free DNA was detected. The specific results are shown in Table 3.

[0158] Table 3

[0159]

[0160] Note: The smaller the average value of the detection Ct value, the higher the starting concentration, which reflects the higher recovery rate of the entire extraction.

[0161] As shown in Table 3 and Figure 5 As shown, the average Ct value of free DNA extraction of ACTB gene by the method without adding defoamer (i.e., comparative example 2) is 32.67, the average Ct value of free DNA extraction of ACTB gene by the method of adding anhydrous ethanol (i.e., comparative example 1) is 31.83, the average Ct value of free DNA extraction of ACTB gene by the method of adding polyether defoamer (i.e., example 1) is 29.93, and the average Ct value of free DNA extraction of ACTB gene by the method of adding silicone defoamer (i.e., example 2) is 29.33. Therefore, compared with the extraction method without adding defoamer, the Ct value of the extraction method of the present application (i.e., example 1 and example 2) is about 1.5-2 Ct values ​​ahead, and the extraction yield is significantly improved. The Ct value of the experimental group adding anhydrous ethanol for defoaming lags behind that of the experimental group adding defoamer. The reason may be that the reagent components of the anhydrous ethanol group are incompatible, resulting in a lag in the Ct value.

[0162] Examples 4-10 and Comparative Examples 2-11

[0163] The difference between the following examples and Example 2 is that the components and proportions of the lysis and binding solution are different, as shown in Table 4. Other reagents and compositions, as well as the extraction method are all referenced to Example 2.

[0164] Table 4

[0165]

[0166]

[0167] In Table 4, “-” means that the component was not added.

[0168] The above implementation schemes were used to detect the cfDNA concentration of plasma samples, and the results are shown in Table 5.

[0169] The lysis binding solution in the above implementation scheme was placed in a sealed electric constant temperature forced air drying oven at 50°C to accelerate its stability test. After 7 days, the cfDNA concentration of plasma samples from the same source was tested. The specific results are shown in Table 5.

[0170] Table 5

[0171]

[0172]

[0173] By comparing the data of Example 2, Comparative Example 3 and Comparative Example 2 in Table 5, it is found that when extracting free DNA in plasma, the silicone defoamer and octyl-beta-D-thiopyranoglucoside, both components in the lysis and binding solution, can work synergistically to significantly increase the extraction concentration, thereby further improving the detection accuracy of free DNA in plasma.

[0174] Examples 2-4 compare the effects of different organosilicon defoamers, and Examples 2 and 5-6 also verify the effects of different detergents on the results. It is found that by selecting different specific organosilicon defoamers and different specific detergents, a higher free DNA extraction concentration can be obtained. However, further combined with Example 1 and Example 11, it is found that organosilicon defoamers have better effects than polyether defoamers. And further through Comparative Examples 2-3 and Comparative Examples 8-11, it is found that different organosilicon defoamers and detergents can achieve good synergy, which has the effect of significantly improving the extraction concentration of free DNA in plasma.

[0175] Examples 2, 7 and 8 show that when the content of the defoamer in the lysis and binding solution is within the range of 0.001-0.005 wt%, a good extraction effect can be obtained. At the same time, combined with Comparative Examples 2-5, it is found that the defoamer used in the range of 0.001-0.005 wt% can have a significantly excellent synergistic effect with the detergent to significantly increase the extraction concentration of free DNA in plasma.

[0176] Further, the effect of the detergent content in the lysis and binding solution being in the range of 3-8 wt% was verified by Examples 2, 9 and 10, and it was found that within the range of the detergent dosage, a relatively ideal extraction concentration of free DNA in plasma could be obtained. Comparative Examples 2-3 and 6-7 also proved that within the range of the detergent addition amount, the detergent can synergistically improve the extraction concentration of free DNA with the defoaming agent.

[0177] Regarding the stability test of the lysis and binding solution, Examples 2-4 and 11 in the above data results show that the silicone defoaming agents therein have good stability; while other defoaming agents, such as defoamer C emulsion, defoamer SE15 and defoamer B emulsion, all showed poor stability, resulting in a significant decrease in the extraction concentration of the target DNA when they were used to lyse and bind the same plasma sample again after the stability acceleration experiment.

[0178] The description of the present disclosure is given for the purpose of illustration and description, and is not intended to be exhaustive or to limit the present disclosure to the disclosed form. 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 disclosure, and to enable those of ordinary skill in the art to understand the present disclosure and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A lysis and binding solution for extracting free DNA from plasma, comprising the following components: a chaotropic agent, a chelating agent, a buffer, a defoaming agent, and a detergent; in, The content of the defoaming agent in the lysis and binding solution is 0.0005-0.01wt%; the content of the detergent in the lysis and binding solution is 1-10wt%.

2. According to the lysis and binding solution of claim 1, the defoaming agent is selected from any one or more of a polyether defoaming agent and an organosilicon defoaming agent; and / or, The detergent is selected from any one or more of octyl-beta-D-thiopyranoglucoside, sodium dodecyl sulfate, Triton and Tween NP40.

3. The lysis and binding solution according to claim 1 or 2, wherein the content of the chaotropic agent in the lysis and binding solution is 5-11M; and / or, the amount of the chelating agent added to the lysis and binding solution is 5-11 M; And / or, the amount of the buffer added to the lysis and binding solution is 10mM-1M.

4. The lysis and binding solution according to any one of claims 1 to 3, wherein the chaotropic agent is selected from any one or more combinations of guanidines and inorganic salts; preferably, the guanidines are selected from any one or more combinations of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate and guanidine hydrochloride; preferably, the inorganic salts are selected from any one or more combinations of sodium iodide, sodium perchlorate and sodium bromide; more preferably, the chaotropic agent is a combination of guanidine isothiocyanate and sodium iodide; And / or, the chelating agent is selected from any one or more combinations of ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylenetriaminepentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, ethylenediaminetetraacetic acid, anhydrous citric acid, sodium citrate, calcium citrate, ammonium citrate, ammonium hydrogen citrate, citric acid, diammonium citrate, ammonium ferric citrate and lithium citrate; And / or, the buffer is selected from any one or more combinations of Tris sulfuric acid and Tris hydrochloric acid.

5. The lysis and binding solution according to any one of claims 1 to 4, further comprising trehalose; preferably, the amount of trehalose added to the lysis and binding solution is 0.2-0.35 M; And / or, the lysis and binding solution further comprises an osmotic pressure balancing agent; preferably, the osmotic pressure balancing agent is selected from any one or more combinations of glycerol, sodium citrate, mannitol and sorbitol; preferably, the amount of the osmotic pressure balancing agent added to the lysis and binding solution is 3-5wt%; And / or, the lysis and binding solution further comprises an inorganic salt; preferably, the inorganic salt is selected from any one or more combinations of calcium chloride, sodium chloride, sodium sulfate, potassium sulfate and potassium chloride; preferably, the amount of the inorganic salt added to the lysis and binding solution is 0.5-2M.

6. A kit for extracting free DNA from plasma, comprising the lysis and binding solution according to any one of claims 1 to 5; Preferably, the kit further comprises a solid phase adsorption matrix, a rinsing solution A, a rinsing solution B, and an eluting solution.

7. The kit according to claim 6, wherein the components of the rinse solution A include: a chaotropic agent, a buffer, a detergent and ethanol; Preferably, the content of the chaotropic agent in the rinsing solution A is 2-6M; and / or, the content of the buffer in the rinse solution A is 10 mM-1 M; and / or, the content of the detergent in the rinsing liquid A is 3-8wt%; and / or, the content of ethanol in the rinse solution A is 30-50 Vol.%; Further preferably, the chaotropic agent is selected from any one or more combinations of guanidines and inorganic salts; preferably, the guanidines are selected from any one or more combinations of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate and guanidine hydrochloride; preferably, the inorganic salt is selected from any one or more combinations of sodium iodide, sodium perchlorate and sodium bromide; more preferably, the chaotropic agent is a combination of guanidine isothiocyanate and sodium iodide; And / or, the buffer is selected from any one or more combinations of Tris sulfuric acid and Tris hydrochloric acid; And / or, the detergent is selected from any one or more of octyl-beta-D-thiopyranoglucoside, sodium dodecyl sulfate, Triton and Tween NP40; preferably, the detergent is octyl-beta-D-thiopyranoglucoside.

8. The kit according to claim 6 or 7, wherein the components of the rinse solution B include: a buffer, polyethylene glycol and an inorganic salt; Preferably, the content of the buffer in the rinse solution B is 10mM-1M; and / or, the content of the polyethylene glycol in the rinsing liquid B is 12-15 wt %; and / or, the content of the inorganic salt in the rinsing liquid B is 0.5-1.5M; Further preferably, the buffer is selected from any one or more combinations of Tris sulfuric acid and Tris hydrochloric acid; And / or, the inorganic salt is selected from any one or more combinations of calcium chloride, sodium chloride, sodium sulfate, potassium sulfate and potassium chloride.

9. The kit according to any one of claims 6 to 8, wherein the components of the eluent comprise a chelating agent and a buffer; Preferably, the content of the chelating agent in the eluent is 0.01-10 mM; and / or, the content of the buffer in the eluent is 1-100 mM; Preferably, the chelating agent is selected from any one or more combinations of ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylenetriaminepentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, ethylenediaminetetraacetic acid, anhydrous citric acid, sodium citrate, calcium citrate, ammonium citrate, ammonium hydrogen citrate, citric acid, diammonium citrate, ammonium ferric citrate and lithium citrate; And / or, any one or more combinations of the buffering agents Tris sulfuric acid and Tris hydrochloric acid.

10. A method for extracting free DNA from plasma, comprising the steps of contacting and mixing the lysis and binding solution according to any one of claims 1 to 5 with a plasma sample and a solid phase adsorption matrix; Preferably, the extraction method further comprises: The solid phase adsorption matrix adsorbed with free DNA is washed with a washing solution A; The solid phase adsorption matrix adsorbed with free DNA is washed with a washing solution B; The solid phase adsorption matrix adsorbed with free DNA is washed with an elution solution.

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