Sulfite conversion solution, kit and sulfite conversion method of DNA (Deoxyribose Nucleic Acid)

Abstract

The invention relates to the technical field of sulfite conversion of DNA, and particularly discloses a sulfite conversion solution, a kit and a sulfite conversion method of DNA. The sulfite conversion solution comprises sulfite, DMSO and urea; wherein in the sulfite conversion solution, the content of the DMSO is 2 to 13 weight percent, and the content of the urea is 4 to 7 M; the extraction kit comprises the sulfite conversion solution; the sulfite conversion solution is used in the conversion method. The extraction concentration of the free DNA in the plasma after sulfite conversion is remarkably improved through the synergistic effect of DMSO and urea in the sulfite conversion solution, so that the detection accuracy is higher in the methylation detection of the free DNA.

Description

Technical Field

[0001] This application relates to the technical field of bisulfite conversion of DNA, and particularly relates to a bisulfite conversion solution, a kit, and a method for bisulfite conversion of DNA. Background Art

[0002] Nucleic acids include two categories: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and are a type of biological macromolecule carrying genetic information. Cell-free DNA (cfDNA) refers to DNA that is free outside cells in circulating blood. Most of it is short-chain, with a very low concentration, and is extremely vulnerable to contamination and dilution by non-cfDNA with a higher abundance, especially in early cancer patients. In recent years, with liquid biopsy technology represented by cfDNA becoming an important technology for molecular diagnosis or auxiliary diagnosis, the research on cell-free DNA has become a hot field in genomic molecular diagnosis research. It has currently been gradually applied to various stages from the initial diagnosis to treatment and development of tumors, including: early screening, evaluating tumor heterogeneity, metastasis and recurrence risks, and prognosis assessment, real-time monitoring of treatment responses and drug resistance, tumor staging and grading, and guiding the selection of treatment plans, etc.

[0003] Due to the characteristics of low content and short half-life of cfDNA, the extraction method of cfDNA has attracted much attention. The first step in using cfDNA for detection is to extract and purify cfDNA in human plasma, and the quality of the extraction product directly affects the accuracy and reliability of subsequent detection results. The characteristics of small fragment size and low content of cell-free DNA in serum and plasma have put forward higher requirements for cfDNA extraction. Existing products on the market cannot directly obtain BisDNA. It is necessary to obtain cell-free DNA through "lysis → adsorption → three purifications → elution", and then obtain BisDNA through "conversion → adsorption → purification → desulfonation → two purifications → elution". The two processes before and after are not only cumbersome, time-consuming, and reagent-consuming, but also cannot be fully connected, increasing the loss of cell-free DNA in vain. Moreover, ethanol is generally used for purification, which will cause a series of problems, such as ethanol residue: after ethanol washing, if the drying time is insufficient, the residual ethanol will inhibit the downstream PCR reaction; if the drying time is too long, the recovery rate of cell-free DNA will be greatly reduced, resulting in a situation where it cannot be detected. And the stability of the reagent bottle opening, ethanol is volatile, and the extraction reagent added with ethanol needs to be used immediately after opening. The evaporation of ethanol in the non-closed state will reduce the extraction efficiency. Transportation problem: Ethanol is a flammable and volatile liquid, and the cleaning solution contains a high concentration of ethanol, making the transportation of nucleic acid extraction reagents a major problem. These disadvantages undoubtedly bring difficulties to the popularization of methylation detection.

[0004] Therefore, at present, it is necessary to propose a bisulfite conversion solution and a conversion method that do not contain alcohol and have a high extraction rate of cell-free DNA in plasma. Summary of the Invention

[0005] The present application provides a sulfite conversion solution, a kit, and a method for sulfite conversion of DNA. In this sulfite conversion solution, urea and DMSO act synergistically to significantly increase the extraction concentration of the sulfite conversion product of free DNA in plasma, thereby significantly improving the detection accuracy.

[0006] The present application relates to the following:

[0007] 1. A sulfite conversion solution, comprising: sulfite, DMSO, and urea;

[0008] Wherein, in the sulfite conversion solution, the content of DMSO is 2 - 13 wt%, and the content of urea is 4 - 7 M.

[0009] 2. According to the sulfite conversion solution described above, in the sulfite conversion solution, the content of sulfite is 7 - 10 M.

[0010] 3. According to the sulfite conversion solution described above, it further comprises trehalose;

[0011] Preferably, in the sulfite conversion solution, the content of trehalose is 0.2 - 1 M.

[0012] 4. A kit for DNA methylation conversion and extraction, comprising the sulfite conversion solution described above;

[0013] Preferably, the kit further comprises: a lysis-binding solution, a wash solution, and an elution solution.

[0014] 5. According to the kit described above, the lysis-binding solution comprises a chaotropic agent, a buffer, a detergent, phenylmethylsulfonyl fluoride, an osmotic pressure balancer, and a surfactant;

[0015] Preferably, in the lysis-binding solution, the content of the chaotropic agent is 5 - 11 M;

[0016] And / or, in the lysis-binding solution, the content of the buffer is 10 - 50 mM;

[0017] And / or, in the lysis-binding solution, the content of the detergent is 1 - 5 wt%;

[0018] And / or, in the lysis-binding solution, the content of phenylmethylsulfonyl fluoride is 3 - 5 mM;

[0019] And / or, in the lysis-binding solution, the content of the osmotic pressure balancer is 3 - 5 wt%;

[0020] And / or, in the lysis binding solution, the content of the surfactant is 15-35 wt%;

[0021] More preferably, the chaotropic agent is selected from any one or a combination of guanidine chaotropic agents and inorganic salt chaotropic agents; preferably, the guanidine chaotropic agent is selected from any one or a combination of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate, and guanidine hydrochloride; preferably, the inorganic salt chaotropic agent is selected from any one or a combination of sodium iodide, sodium perchlorate, and sodium bromide;

[0022] And / or, the buffer is selected from any one or a combination of Tris sulfate, Tris hydrochloride, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);

[0023] And / or, the detergent is selected from any one or a combination of sodium deoxycholate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, and sodium cholate;

[0024] And / or, the osmotic pressure balancer is selected from any one or a combination of glycerol, sodium citrate, mannitol, and sorbitol;

[0025] And / or, the surfactant is selected from any one or a combination of lauryl polyoxyethylene ether, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate, sodium dodecyl sulfate, polyethylene glycol octylphenyl ether, sodium alkylbenzene sulfonate, and N-lauroylsarcosine.

[0026] 6. According to the kit described above, the wash solution contains a chaotropic agent, a buffer, a sodium salt, and polyethylene glycol;

[0027] Preferably, in the wash solution, the content of the chaotropic agent is 2-6 M;

[0028] And / or, in the wash solution, the content of the buffer is 10-50 mM;

[0029] And / or, in the wash solution, the content of the sodium salt is 0.5-1 M;

[0030] And / or, in the wash solution, the content of the polyethylene glycol is 2-20 wt%;

[0031] More preferably, the chaotropic agent is selected from any one or a combination of guanidine chaotropic agents and inorganic salt chaotropic agents; preferably, the guanidine chaotropic agent is selected from any one or a combination of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate, and guanidine hydrochloride; preferably, the inorganic salt chaotropic agent is selected from any one or a combination of sodium iodide, sodium perchlorate, and sodium bromide;

[0032] And / or, the buffer is selected from any one or a combination of more than one of Tris sulfate, Tris hydrochloride, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);

[0033] And / or, the sodium salt is selected from any one or a combination of more than one of sodium chloride, potassium chloride, calcium chloride, sodium sulfate, and potassium sulfate.

[0034] 7. According to the kit described above, the eluent contains a chelating agent and a buffer;

[0035] Preferably, in the eluent, the content of the chelating agent is 0.01 - 10 mM;

[0036] And / or, in the eluent, the content of the buffer is 1 - 100 mM;

[0037] More preferably, the chelating agent is selected from any one or a combination of more than one of ethylene glycol tetraacetic acid, hydroxyethyl ethylenediamine triacetic acid, diethylenetriamine pentaacetic 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;

[0038] And / or, the buffer is selected from any one or a combination of more than one of Tris sulfate, Tris hydrochloride, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

[0039] 8. A method for bisulfite conversion of DNA, the conversion method comprising:

[0040] A step of contacting and mixing the bisulfite conversion solution with the purified DNA to obtain a conversion product;

[0041] Preferably, the conditions for the contacting and mixing include: contacting and mixing at a temperature of 80 - 95 °C;

[0042] And / or, the time for the contacting and mixing is 5 - 10 min.

[0043] 9. According to the bisulfite conversion method described above, it further comprises:

[0044] Lyzing and binding the conversion product with the lysis binding solution to obtain a second lysis binding product;

[0045] Rinsing the second lysis binding product with the rinsing solution to obtain a second rinsing product;

[0046] Eluting the second rinsing product with the eluent to obtain the bisulfite-converted DNA.

[0047] 10. According to the sulfite conversion method described above, the method for obtaining the purified DNA comprises the following steps:

[0048] Mix the lysis binding solution with the plasma sample and perform lysis binding to obtain a first lysis binding product;

[0049] Rinse the first lysis binding product with the rinsing solution to obtain a first rinsing product;

[0050] And elute the first rinsing product with the elution solution to obtain purified DNA.

[0051] Advantages of the Invention

[0052] The present application provides a sulfite conversion solution, which mainly contains a high concentration of urea and DMSO. The two act together to improve the conversion rate. When this sulfite conversion is used for DNA methylation conversion and extraction, a relatively high concentration of the target DNA can be obtained. Brief Description of the Drawings

[0053] Figure 1 . Distribution of free DNA extraction fragments obtained by the method of Example 1.

[0054] Figure 2 . Distribution of free DNA extraction fragments obtained by extraction and conversion with a reference reagent.

[0055] Figure 3 . Comparison of the Ct values of the recovery rates of cfDNA extracted and converted by the method of Example 1 and by extraction and conversion with a reference kit.

[0056] Detailed Implementation Methods

[0057] It should be noted that in the description of the specification and claims, certain terms are used to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. The specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of components as the criterion for distinction.

[0058] As mentioned throughout the specification and claims, the term "comprising" or "including" is an open-ended term and should be interpreted as "including but not limited to". The subsequent description in the specification is for the purpose of describing the preferred implementation mode of the present application, but the description is for the general principle of the specification and is not used to limit the scope of the present application. The protection scope of the present application shall be determined by the scope defined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs.

[0059] It should be understood that the embodiments of the present application described herein include embodiments of "consisting of" and / or "consisting essentially of". References herein to "about" values or parameters include (and describe) variations to that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0060] 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 element. Thus, this statement is intended to serve as antecedent basis for use in conjunction with the recitation of claim elements, such exclusive terms as "only", "solely", etc., or use of a "negative" limitation.

[0061] 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.

[0062] Sulfite can deaminate unmethylated cytosine in DNA to uracil, while methylated cytosine remains unchanged. Subsequently, after amplification with special primers, the products are sequenced to determine whether the CpG site is methylated. Therefore, bisulfite conversion is an indispensable step for determining the presence of methylation sites in DNA.

[0063] In the present application, the term "cell-free DNA", also known as "cfDNA", refers to extracellular DNA present in body fluids such as plasma or serum, cerebrospinal fluid, and synovial fluid.

[0064] As used herein, DNA methylation is a form of chemical modification of DNA that can change genetic expression without changing the DNA sequence; under the action of DNA methyltransferase, a methyl group is covalently bonded to the 5th carbon position of cytosine in genomic CpG dinucleotides. A large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.

[0065] The present application provides a bisulfite conversion solution, which comprises: sulfite, DMSO, and urea, wherein, in the bisulfite conversion solution, the content of DMSO is 2-13 wt%, and the content of urea is 4-7 M.

[0066] In some embodiments, in the sulfite conversion solution, the content of DMSO is in the range of 2-13 wt%, 3-10 wt%, 3-9 wt%, 4-8 wt%, 5-7 wt% or any content range within the range of 2-13 wt%; for example, it can be 2 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5 wt%, 5.2 wt%, 5.4 wt%, 5.6 wt%, 5.8 wt%, 6.0 wt%, 6.2 wt%, 6.4 wt%, 6.6 wt%, 6.8 wt%, 7.0 wt%, 7.2 wt%, 7.4 wt%, 7.6 wt%, 7.8 wt%, 8.0 wt%, 8.2 wt%, 8.4 wt%, 8.6 wt%, 8.8 wt%, 9.0 wt%, 9.2 wt%, 9.4 wt%, 9.6 wt%, 9.8 wt%, 10 wt%, 10.5 wt%, 11 wt%, 11.5 wt%, 12 wt%, 12.5 wt%, 13 wt% or any content within the range of 2-13 wt%.

[0067] In some embodiments, in the sulfite conversion solution, the content of urea is in the range of 4-7 M, 4-6 M, 4-5 M or any content range within the range of 4-7 M; for example, it can be 4 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M, 4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, 7 M or any content within the range of 4-7 M.

[0068] In the sulfite conversion solution of the present application, the inventors found that after adding DMSO and urea thereto, these two substances can significantly increase the detection concentration during the methylation detection of free DNA (especially free DNA in plasma) through synergistic effects, that is, it has a significantly improved conversion efficiency. For example: when only DMSO or only urea is added to the sulfite conversion solution, the detection concentration of methylated DNA of free DNA in plasma obtained is only 0.72 ng / μL, or even as low as 0.56 ng / μL. However, for the solution of the present application with both DMSO and urea added, the detection concentration of methylated DNA of free DNA in plasma obtained is generally 1.03 - 1.12 ng / μL, or even as high as 1.15, 1.2 ng / μL, or higher. The applicant found that if other substances similar to urea, such as sucrose, gelatin, polyvinylpyrrolidone, etc., are used, it is difficult to exert a synergistic effect with DMSO; similarly, if other substances similar to DMSO, such as hydroquinone, glucomannan, water-soluble vitamin E, etc., are used, it is also difficult to exert a synergistic effect with urea; only when urea and DMSO are used together can a higher concentration of the target DNA be obtained. The technical solution of the present application significantly increases the extraction concentration of the target DNA, providing a basis for more accurate content detection thereof.

[0069] In some embodiments, in the sulfite conversion solution, the content of the sulfite is any content range within 7 - 10 M, 8 - 9 M, or 7 - 10 M; for example, it can be 7 M, 7.1 M, 7.2 M, 7.3 M, 7.4 M, 7.5 M, 7.6 M, 7.7 M, 7.8 M, 8 M, 8.1 M, 8.2 M, 8.3 M, 8.4 M, 8.5 M, 8.6 M, 8.7 M, 8.8 M, 8.9 M, 9 M, 9.1 M, 9.2 M, 9.3 M, 9.4 M, 9.5 M, 9.6 M, 9.7 M, 9.8 M, 9.9 M, 10 M, or any content within the range of 7 - 10 M.

[0070] In some embodiments, the sulfite conversion solution further contains trehalose, and its content is 0.2 - 1 M; for example, it can be 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, or any content range within 0.2 - 1 M.

[0071] The sulfite solution of the present application can be used for the sulfite conversion of DNA. It can be used as a separate sulfite conversion reagent for the sulfite conversion of DNA, and then subsequent related purification, detection, etc. can be carried out; it can also be used as one of the reagents in products such as kits related to DNA methylation detection.

[0072] Therefore, the present application also provides a DNA methylation conversion and extraction kit, which contains the above-mentioned bisulfite conversion solution.

[0073] Those skilled in the art can understand that to achieve DNA methylation, it is necessary to first obtain isolated DNA (such as cfDNA, etc.), and the isolated DNA can also be purified and enriched as required. Suitable reagents for isolating DNA include but are not limited to: one or more lysis solutions and binding solutions (or lysis-binding solutions), one or more washing solutions, one or more elution solutions, etc. Other reagents adapted to the bisulfite conversion solution of the present application are not restrictive. Other reagents can be, for example, lysis solutions, binding solutions, washing solutions, elution solutions, lysis-binding solutions, etc.

[0074] In some embodiments, the kit further contains: a lysis-binding solution, a washing solution, and an elution solution.

[0075] In some embodiments, the lysis-binding solution contains a chaotropic agent, a buffer, a detergent, phenylmethylsulfonyl fluoride, an osmotic pressure balancer, and a surfactant.

[0076] In some embodiments, in the lysis-binding solution, the content of the chaotropic agent is in the range of 5 - 11M, 6 - 10M, 7 - 9M, or any content range within 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 within the range of 5 - 11M.

[0077] In some embodiments, in the lysis-binding solution, the content of the buffer is in the range of 10 - 50mM, 15 - 45mM, 20 - 40mM, 25 - 35mM, or any content range within 10 - 50mM: for example, it can be 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 21mM, 22mM, 24mM, 25mM, 27mM, 28mM, 30mM, 33mM, 36mM, 39mM, 42mM, 45mM, 48mM, 50mM, or any content range within 10 - 50mM.

[0078] In some embodiments, in the lysis binding solution, the content of the detergent is 1-5 wt%. In a specific embodiment, the content of the detergent in the lysis binding solution is 1 wt%, 1.2 wt%, 1.4 wt%, 1.6 wt%, 1.8 wt%, 2 wt%, 2.2 wt%, 2.4 wt%, 2.6 wt%, 2.8 wt%, 3 wt%, 3.2 wt%, 3.4 wt%, 3.6 wt%, 3.8 wt%, 4 wt%, 4.2 wt%, 4.4 wt%, 4.6 wt%, 4.8 wt%, 5 wt% or any content within the range of 1-5 wt%.

[0079] In some embodiments, in the lysis binding solution, the content of the desulfobenzoyl fluoride is 3-5 mM or any content range within this range; in a specific embodiment, the content of the desulfobenzoyl fluoride in the lysis binding solution can be, for example, 3 mM, 3.2 mM, 3.4 mM, 3.6 mM, 3.8 mM, 4 mM, 4.2 mM, 4.4 mM, 4.6 mM, 4.8 mM, 5 mM or any content within the range of 3-5 mM.

[0080] In some embodiments, in the lysis binding solution, the content of the osmotic pressure balancer is 3-5 wt%; for example, it is 3 wt%, 3.2 wt%, 3.4 wt%, 3.6 wt%, 3.8 wt%, 4 wt%, 4.2 wt%, 4.4 wt%, 4.6 wt%, 4.8 wt%, 5 wt% or any content within the range of 3-5 wt%.

[0081] In some embodiments, in the lysis binding solution, the content of the surfactant is 15-35 wt%, 17-32 wt%, 20-30 wt%, 23-27 wt% or any content range within the range of 15-35 wt%. In a specific embodiment, the content of the surfactant in the lysis binding solution is 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt% or any content within the range of 15-35 wt%.

[0082] In some embodiments, the chaotropic agent is selected from any one or a combination of more than one of guanidine chaotropic agents and inorganic salt chaotropic agents; in some embodiments, the guanidine chaotropic agent is selected from any one or a combination of more than one of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate, and guanidine hydrochloride; in some embodiments, the inorganic salt chaotropic agent is selected from any one or a combination of more than one of sodium iodide, sodium perchlorate, and sodium bromide. In some preferred embodiments, the chaotropic agent is a combination of guanidine hydrochloride and sodium iodide.

[0083] In some embodiments, the buffer is selected from any one or a combination of more than one of Tris sulfate, Tris hydrochloride, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); in some preferred embodiments, the buffer is HEPES.

[0084] In some embodiments, the detergent is selected from any one or a combination of more than one of sodium deoxycholate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, and sodium cholate; in some preferred embodiments, the detergent is sodium deoxycholate.

[0085] In some embodiments, the osmotic pressure balancer is selected from any one or a combination of more than one of glycerol, sodium citrate, mannitol, and sorbitol.

[0086] In some embodiments, the surfactant is selected from any one or a combination of more than one of lauryl polyoxyethylene ether, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate, sodium dodecyl sulfate, octylphenoxypolyethoxyethanol, sodium alkylbenzenesulfonate, and N-lauroylsarcosine.

[0087] In some embodiments, the washing solution contains a chaotropic agent, a buffer, a sodium salt, and polyethylene glycol.

[0088] In some embodiments, in the washing solution, the content of the chaotropic agent is any content within the range of 2-6 M, 3-5 M, or 2-6 M; for example, it can be 2 M, 2.2 M, 2.4 M, 2.6 M, 2.8 M, 3 M, 3.2 M, 3.4 M, 3.6 M, 3.8 M, 4 M, 4.2 M, 4.4 M, 4.6 M, 4.8 M, 5 M, 5.2 M, 5.4 M, 5.6 M, 5.8 M, 6 M, or any content within the range of 2-6 M.

[0089] In some embodiments, in the washing solution, the content of the buffer is in any content range within 10 - 50 mM, 15 - 45 mM, 20 - 40 mM, 25 - 35 mM, or 10 - 50 mM; for example, it can be 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 21 mM, 22 mM, 24 mM, 25 mM, 27 mM, 28 mM, 30 mM, 33 mM, 36 mM, 39 mM, 42 mM, 45 mM, 48 mM, 50 mM, or any content range within 10 - 50 mM.

[0090] In some embodiments, in the washing solution, the content of the sodium salt is 0.5 - 1 M; for example, it can be 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, or any range within 0.5 - 1 M.

[0091] In some embodiments, in the washing solution, the content of the polyethylene glycol is in any content range within 2 - 20 wt%, 4 - 18 wt%, 6 - 16 wt%, 8 - 14 wt%, 10 - 12 wt%, or 2 - 20 wt%; for example, it can be 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, or any content within 2 - 20 wt%. In the present application, the polyethylene glycol can be obtained commercially; it can also be prepared by methods known to those skilled in the art. In the present application, the polyethylene glycol can specifically be polyethylene glycol 6000.

[0092] In some embodiments, the chaotropic agent is selected from any one or a combination of more than one of guanidine chaotropic agents and inorganic salt chaotropic agents; in some embodiments, the guanidine chaotropic agent is selected from any one or a combination of more than one of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate, and guanidine hydrochloride; in some embodiments, the inorganic salt chaotropic agent is selected from any one or a combination of more than one of sodium iodide, sodium perchlorate, and sodium bromide. In some preferred embodiments, the chaotropic agent is guanidine hydrochloride.

[0093] In some embodiments, the buffer is selected from any one or a combination of more than one of Tris sulfate, Tris hydrochloride, and 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid (HEPES); in some preferred embodiments, the buffer is HEPES.

[0094] In some embodiments, the sodium salt is selected from any one or a combination of more than one of sodium chloride, potassium chloride, calcium chloride, sodium sulfate, and potassium sulfate.

[0095] In some embodiments, in the eluent, the content of the chelating agent 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.

[0096] In some embodiments, in the eluent, the content of the buffer 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.

[0097] In some embodiments, the chelating agent is selected from any one or a combination of more than one of ethylene glycol tetraacetic acid, hydroxyethyl ethylenediamine triacetic acid, diethylenetriamine pentaacetic 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 preferred embodiments, the chelating agent is ethylenediaminetetraacetic acid.

[0098] In some embodiments, the buffer is selected from any one or a combination of more than one of Tris sulfate, Tris hydrochloride, and 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid (HEPES). In some preferred embodiments, the buffer is Tris hydrochloride.

[0099] This application also provides a method for bisulfite conversion of DNA, and the conversion method includes:

[0100] A step of contacting and mixing the bisulfite conversion solution described above with purified DNA to obtain a conversion product.

[0101] In some embodiments, the conditions for contacting and mixing include: contacting and mixing at any temperature range within the range of 80 - 95 °C, 82 - 93 °C, 85 - 90 °C, or 80 - 95 °C; for example, contacting and mixing can be carried out at 80 °C, 81 °C, 82 °C, 83 °C, 84 °C, 85 °C, 86 °C, 87 °C, 88 °C, 89 °C, 90 °C, 92 °C, 93 °C, 94 °C, 95 °C, or any temperature within the range of 80 - 95 °C.

[0102] In some embodiments, the time for contacting and mixing is any time range within the range of 5 - 10 min, 6 - 9 min, 7 - 8 min, or 5 - 10 min; for example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or any time within the range of 5 - 10 min.

[0103] Those skilled in the art can understand that after the bisulfite conversion, in order to obtain a purified or isolated DNA product, it is often necessary to appropriately carry out the purification and separation steps of the target DNA product. For example, the target DNA product is separated and purified by combining with magnetic adsorption technology; for example, it is treated with appropriate reagents (such as a wash solution, such as an elution solution, or any combination thereof).

[0104] In some embodiments, the bisulfite conversion method further includes:

[0105] Lyzing and binding the conversion product with any of the above lysis binding solutions to obtain a second lysis binding product;

[0106] Rinsing the second lysis binding product with the wash solution to obtain a second rinsing product;

[0107] Eluting the second rinsing product with the elution solution to obtain the bisulfite - converted DNA.

[0108] In some embodiments, when performing sulfite conversion, the time for lysing and binding the conversion product with any of the above lysis-binding solutions is 10 - 15 min; for example, it can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, or any time within the range of 10 - 15 min. In some embodiments, when performing sulfite conversion, when rinsing the second lysis-binding product with the rinsing solution, the elution time is 1 - 5 min. In some embodiments, when performing sulfite conversion, when eluting the second rinsing product with the elution solution, the elution temperature is 23 - 56 °C, for example, it can be 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 32 °C, 35 °C, 38 °C, 40 °C, 42 °C, 45 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, or any temperature within the range of 23 - 56 °C; the elution time is 5 - 10 min, for example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or any time within the range of 5 - 10 min.

[0109] In some embodiments, the method for obtaining the purified DNA comprises the following steps:

[0110] Mix the lysis-binding solution with any of the above and a plasma sample, and then perform lysis and binding to obtain a first lysis-binding product;

[0111] Rinse the first lysis-binding product with the rinsing solution to obtain a first rinsing product;

[0112] And elute the first rinsing product with the elution solution to obtain the purified DNA.

[0113] In some embodiments, when eluting the first rinsing product with the elution solution, the elution temperature is 23 - 56 °C, for example, it can be 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 32 °C, 35 °C, 38 °C, 40 °C, 42 °C, 45 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, or any temperature within the range of 23 - 56 °C; the elution time is 5 - 10 min, for example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or any time within the range of 5 - 10 min.

[0114] Of course, the above purification and separation method is not restrictive. Using other methods well-known to those skilled in the art to obtain the purified or separated target DNA is also a technical solution within the scope of the claims of this application. Detailed implementation manners

[0115] Specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the present application are shown in the 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 so that the present application can be more thoroughly understood and the scope of the present application can be fully conveyed to those skilled in the art.

[0116] Example 1.

[0117] Lysis binding solution, the components and their contents are: 4M guanidine hydrochloride, 4M sodium iodide, 30 mM HEPES, 3 wt% sodium deoxycholate, 4 mM phenylmethylsulfonyl fluoride, 4 wt% glycerol, 20 wt% polyoxyethylene lauryl ether, and 8 wt% 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate sodium salt.

[0118] Wash solution, the components and their contents are: 4M guanidine hydrochloride, 30 mM HEPES, 0.8M sodium chloride, and 10 wt% PEG6000.

[0119] Sulfite solution, the components and their contents are: 8.5M sodium bisulfite, 3 wt% DMSO, 0.6M trehalose, and 4M urea.

[0120] Elution solution, the components and their contents are: 5 mM ethylenediaminetetraacetic acid, 50 mM Tris-HCl.

[0121] The magnetic beads are purchased from Tianjin Huachen Beier Biotechnology Co., Ltd. Its magnetic core is an iron-cobalt-nickel alloy Fe-Co-NiOX, and the particle size distribution is between 100-1000 nm. The surface layer of the magnetic core is coated with at least a carbon layer and an inorganic silica layer from the inside to the outside, and polyethylene glycol is covalently grafted on the outermost layer.

[0122] The kit of the present application includes the lysis binding solution, wash solution, sulfite solution, elution solution, and magnetic beads listed above.

[0123] Method for extracting free DNA from plasma:

[0124] Using ACTB as an internal reference, it detects the content of cfDNA extracted from healthy human plasma samples and the conversion efficiency. The process includes the following steps: using the magnetic bead method plasma free DNA extraction kit (product number: DP720) of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract and purify free DNA, and then using the EZ DNA Methylation Lighting TM Kit (product number: D5030) of Zymo Research Corporation, Germany to transform and purify free DNA. All operations are carried out according to the instructions provided by the kit.

[0125] 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 min (usually 10 - 15 min), place on a magnetic rack, and discard the supernatant.

[0126] 2) Washing and purification: Take out the centrifuge tube, add 1.5 mL of washing solution, shake and mix, transfer the washing solution and magnetic beads to a 2 mL centrifuge tube, place on a magnetic rack, perform magnetic separation, and aspirate and discard the waste liquid.

[0127] 3) Elution: Add 80 μL (usually 50 - 100 μL) of elution solution, place in a thermostatic shaker, and elute at 37 °C for 8 min (usually elute at 23 - 56 °C for 5 - 10 min).

[0128] Take 2 μL of the template for 2100 detection of DNA fragment distribution and genomic DNA residue. Take 1 μL of the template for Qubit double-stranded DNA detection. The remaining template liquid is used for subsequent transformation.

[0129] Method for bisulfite conversion of free DNA in plasma:

[0130] 1) Bisulfite conversion: Remove the centrifuge tube from the magnetic rack, add 150 μL (usually 100 - 200 μL) of bisulfite solution, place in a thermostatic shaker, and incubate at 90 °C for 8 min (usually incubate at 80 - 95 °C for 5 - 10 min).

[0131] 2) Binding: Take out the centrifuge tube, add 1 mL of lysis and binding solution, add 20 μL of magnetic beads, invert and mix several times, shake and mix for 15 min (usually mix for 10 - 15 min).

[0132] 3) Washing and purification: Place the centrifuge tube on a magnetic rack, perform magnetic separation, carefully aspirate the supernatant into a new centrifuge tube, add 650 μL (usually add 500 - 800 μL) of washing solution, shake in a thermostatic shaker for 5 min (usually shake for 1 - 5 min), perform magnetic separation, and aspirate and discard the waste liquid;

[0133] 4) Elution: Add 80 μL (usually add 50 - 100 μL) of elution solution, place in a thermostatic shaker, and elute at 37 °C for 8 min (usually elute at 23 - 56 °C for 5 - 10 min);

[0134] 5) Place the centrifuge tube on a magnetic rack, perform magnetic separation, carefully aspirate the supernatant into a new centrifuge tube, and store at -20 °C for later use.

[0135] Effect verification:

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

[0137] Primers and probe for ACTB:

[0138] ACTB_F: 5’-ACATTTCCACCTCCCACCC-3’

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

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

[0141] The product size is 65bp.

[0142] Among them, the PCR amplification system adopted in this embodiment is shown in Table 1.

[0143] Table 1

[0144] Volume (μl) Final concentration Taq DNA Polymerase (Biochain) 1 / 4.2× buffer (Biochain) 6 1× ACTB_F (10 μM) 0.5 200 nM ACTB_R (10 μM) 0.5 200 nM ACTB_P (10 μM) 0.5 200 nM cfDNA 10 / <![CDATA[H 2 O]]> 6.5 / Total 25.0 /

[0145] The PCR amplification program is: 95°C, 3 min; (93°C, 30 s; 59.7°C, 30 s; - read fluorescence signal) 40 cycles; 40°C, 5 s. The threshold is set at 240.

[0146] (1) Determination of DNA fragment distribution and genomic DNA residue

[0147] Use the Agilent Technologies 2100 analyzer to perform fragment distribution determination and genomic DNA residue determination on the extracted cell-free DNA.

[0148] Detect the ACTB gene according to the reaction system and reaction program of Example 1 above. The DNA fragment distribution results of the extracted DNA are as Figure 1 . The DNA fragment distribution results of the reference reagent (that is, the scheme of extracting and purifying cell-free DNA using the magnetic bead method plasma cell-free DNA extraction kit (product number: DP720) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and then transforming and purifying cell-free DNA with the EZ DNA Methylation Lighting TM Kit (product number: D5030) of Zymo Research Corporation, Germany) are as Figure 2 .

[0149] From Figure 1 and Figure 2As can be seen from the results, the results of the 2100 analyzer indicate that the cfDNA obtained by the rapid extraction of the present invention is cfDNA, and there is no genomic residue.

[0150] (2) Detection of extraction recovery rate and concentration

[0151] The cfDNA concentration of plasma samples was detected using the Thermo 1×dsDNA HS Working Solution. The results of the method in Example 1 were compared with the method using a reference reagent (i.e., using the magnetic bead method plasma free DNA extraction kit (product number: DP720) of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract and purify free DNA, and then using the EZDNA Methylation Lighting TM Kit (product number: D5030) of Zymo Research Corporation, Germany, to transform and purify free DNA). The results are shown in Table 2.

[0152] Table 2

[0153]

[0154] The concentration test using the Qubit reagent showed that the yield of the method of the present invention was 1.18 times higher than that of the reference reagent, with high extraction efficiency and simple operation.

[0155] (3) Results of fluorescence quantitative detection

[0156] The average Ct value of the ACTB gene in plasma free DNA was detected. The specific results are shown in Table 3.

[0157] Table 3

[0158] Test sample Example 1 Reference kit Plasma 1 27.73 29.27 Plasma 2 27.89 29.19 Plasma 3 27.92 29.10 Average value 27.85 29.19

[0159] Note: The smaller the average value of the detected Ct value, the higher the starting concentration, indicating a higher overall extraction recovery rate.

[0160] Referring to Table 3 and Figure 3 as shown, it was found that the average Ct value of the free DNA of the ACTB gene after extraction and transformation was 27.85, and the average Ct value of the free DNA of the reference kit was 29.19. The extraction and transformation method of the present invention was about 2 Ct values earlier than that of the reference reagent, and the extraction yield and transformation efficiency were significantly higher than those of the reference reagent results.

[0161] Comparative Example 4

[0162] The difference between this comparative example and Example 1 is that the sulfite reagent does not contain DMSO; other reagents and the extraction method of free DNA in plasma are the same as those in Example 1.

[0163] Comparative Example 5

[0164] The difference between this comparative example and Example 1 is that the sulfite reagent does not contain urea; other reagents and the method for extracting free DNA from plasma are the same as in Example 1.

[0165] Refer to the above method to detect the average Ct value of the ACTB gene in plasma free DNA, and the specific results are shown in Table 4.

[0166] Table 4

[0167] Test sample Comparative example 4 Comparative example 5 Plasma 1 29.56 30.26 Plasma 2 29.63 30.19 Plasma 3 29.72 30.23 Average value 29.64 30.23

[0168] Examples 1-5 and Comparative Examples 1-19

[0169] The differences between the following examples (or comparative examples) and Example 1 are that the components and ratios in the sulfite conversion solution are different. Other reagents and their compositions, as well as the extraction method, all refer to Example 1, as shown in Table 5 specifically. Further explanation: When the components in the sulfite conversion solution are different, only the urea is completely replaced by a urea substitute, and / or, the DMSO is completely replaced by a DMSO substitute; and when the ratio in the sulfite conversion solution is different, only the addition amounts of urea and / or its substitute, and / or, DMSO and / or its substitute are different. Among them, the urea substitute can be any one of sucrose, gelatin, and polyvinylpyrrolidone; among them, the substitute for DMSO is any one of hydroquinone, glucomannan, and water-soluble vitamin E.

[0170]

[0171] Use the above implementation schemes to detect the cfDNA concentration in plasma samples respectively (for comparative research with the free DNA magnetic bead method extraction kit of Tiangen Biochemical Technology Co., Ltd.), and the results are shown in Table 5.

[0172] It can be seen from the data results in Table 5 that the urea and DMSO added in the sulfite conversion solution can cooperate with each other to significantly increase the extraction concentration of the final target DNA. However, when urea is replaced with other substances, such as sucrose (Comparative Example 1), gelatin (Comparative Example 2), and polyvinylpyrrolidone (Comparative Example 3), they cannot achieve good results when used together with DMSO. And when DMSO is replaced with other substances, such as hydroquinone (Comparative Example 10), glucomannan (Comparative Example 11), and water-soluble vitamin E (Comparative Example 12), they cannot achieve good results when used together with urea.

[0173] It is also found from Examples 1-5 and Comparative Examples 6-9, Comparative Examples 13-19 that: when the urea content in the sulfite conversion solution is 4-7 M and the DMSO content is 2-13 wt%, a better synergistic effect is achieved.

[0174] The description of the present disclosure has been presented for purposes of illustration and description, and is not intended to be exhaustive or to limit the present disclosure to the forms disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments were chosen and described in order to best explain the principles of the present disclosure and its practical application, and to enable others of ordinary skill in the art to understand the present disclosure and design various embodiments with various modifications suited to particular uses.

Claims

1. A sulfite conversion solution comprising: sulfite, DMSO and urea; in, In the sulfite conversion solution, the content of DMSO is 2-13 wt %, and the content of urea is 4-7 M.

2. The sulfite conversion solution according to claim 1, wherein the content of the sulfite in the sulfite conversion solution is 7-10 M.

3. The sulfite conversion solution according to claim 1, further comprising trehalose; Preferably, in the sulfite conversion solution, the content of trehalose is 0.2-1M.

4. A DNA methylation conversion and extraction kit, comprising the sulfite conversion solution according to any one of claims 1 to 3; Preferably, the kit further comprises: a lysis and binding solution, a rinse solution and an elution solution.

5. The kit according to claim 4, wherein the lysis and binding solution comprises a chaotropic agent, a buffer, a detergent, desphenylmethylsulfonyl fluoride, an osmotic pressure balancing agent and a surfactant; Preferably, in the lysis and binding solution, the content of the chaotropic agent is 5-11M; and / or, in the lysis and binding solution, the content of the buffer is 10-50 mM; and / or, in the lysis and binding solution, the content of the detergent is 1-5wt%; and / or, in the lysis and binding solution, the content of dephenylmethylsulfonyl fluoride is 3-5 mM; And / or, in the lysis and binding solution, the content of the osmotic pressure balancing agent is 3-5wt%; and / or, in the lysis and binding solution, the content of the surfactant is 15-35wt%; More preferably, the chaotropic agent is selected from any one or more combinations of guanidine chaotropic agents and inorganic salt chaotropic agents; preferably, the guanidine chaotropic agent is selected from any one or more combinations of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate and guanidine hydrochloride; preferably, the inorganic salt chaotropic agent is selected from any one or more combinations of sodium iodide, sodium perchlorate and sodium bromide; And / or, the buffer is selected from any one or more combinations of Tris sulfuric acid, Tris hydrochloric acid and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES); And / or, the detergent is selected from any one or more combinations of sodium deoxycholate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate and sodium cholate; And / or, the osmotic pressure balancing agent is selected from any one or more combinations of glycerol, sodium citrate, mannitol and sorbitol; And / or, the surfactant is selected from any one or more combinations of lauryl alcohol polyoxyethylene ether, 3-[3-(cholamidopropyl)dimethylamino]propane sulfonic acid sodium salt, sodium dodecyl sulfonate, polyethylene glycol octylphenyl ether, sodium alkylbenzene sulfonate and N-lauroyl sarcosine.

6. The kit according to claim 4 or 5, wherein the rinsing solution comprises a chaotropic agent, a buffer, a sodium salt and polyethylene glycol; Preferably, in the rinse solution, the content of the chaotropic agent is 2-6M; and / or, in the rinse solution, the content of the buffer is 10-50 mM; and / or, in the rinse liquid, the content of the sodium salt is 0.5-1M; and / or, in the rinse liquid, the content of the polyethylene glycol is 2-20wt%; More preferably, the chaotropic agent is selected from any one or more combinations of guanidine chaotropic agents and inorganic salt chaotropic agents; preferably, the guanidine chaotropic agent is selected from any one or more combinations of guanidine thiocyanate, guanidine isothiocyanate, guanidine isocyanate and guanidine hydrochloride; preferably, the inorganic salt chaotropic agent is selected from any one or more combinations of sodium iodide, sodium perchlorate and sodium bromide; And / or, the buffer is selected from any one or more combinations of Tris sulfuric acid, Tris hydrochloric acid and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES); And / or, the sodium salt is selected from any one or more combinations of sodium chloride, potassium chloride, calcium chloride, sodium sulfate and potassium sulfate.

7. The kit according to any one of claims 4 to 6, wherein the eluent comprises a chelating agent and a buffer; Preferably, in the eluent, the content of the chelating agent is 0.01-10 mM; and / or, in the eluent, the content of the buffer is 1-100 mM; More 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, the buffer is selected from any one or more combinations of Tris sulfuric acid, Tris hydrochloric acid and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES).

8. A method for sulfite conversion of DNA, the conversion method comprising: A step of contacting and mixing the sulfite conversion solution according to any one of claims 1 to 3 with purified DNA to obtain a conversion product; Preferably, the contact mixing conditions include: contact mixing at a temperature of 80-95°C; And / or, the contact mixing time is 5-10 minutes.

9. The sulfite conversion method according to claim 8, further comprising: The transformation product is cleaved and combined using the cleavage and combination solution according to any one of claims 4 to 7 to obtain a second cleavage and combination product; The second cleavage and binding product is rinsed with the rinse solution to obtain a second rinse product; The second rinse product is eluted with the elution solution to obtain the DNA after sulfite conversion.

10. The sulfite conversion method according to claim 8, wherein the method for obtaining the purified DNA comprises the following steps: The cleavage and binding solution according to any one of claims 4 to 7 is mixed with a plasma sample and then cleaved and bound to obtain a first cleavage and binding product; Using the rinse solution to rinse the first cleavage and binding product to obtain a first rinse product; And, the first washing product is eluted with the elution solution to obtain purified DNA.

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