A method for extracting and transforming large-volume fecal DNA

Abstract

The present invention discloses a method for extracting and transforming large-volume fecal DNA, using a lysis solution and an enzyme to directly lyse a large-volume fecal sample, removing impurities and interferences from the lysed fecal sample by an impurity remover, and then precipitating DNA by an isopropanol method, thereby simplifying the extraction steps and solving the problem of more impurities and less host nucleic acid when preparing fecal DNA. Subsequently, under the condition of adding a protective solution, the extracted DNA is transformed using high temperature and bisulfite to protect the DNA and reduce DNA damage. Finally, a binding solution is added to the transformed sample, and Bis-DNA is prepared using a magnetic bead method. The present invention combines DNA extraction and transformation, greatly reducing the experimental steps and reducing the experimental time.

Description

Technical Field

[0001] The present invention specifically relates to a method for extracting and transforming large-volume fecal DNA. Background Art

[0002] Feces contains a lot of impurities, such as undigested food residues, polysaccharides, pigments, humic acid, bile acid, choline bile salts, intestinal microbiota, etc., which has always been a major challenge for extracting human nucleic acids from feces. On the one hand, humic acid has a strong inhibitory effect on PCR, and humic acid is combined with DNA and is difficult to remove; on the other hand, the host exfoliated cells in feces are only 1% of intestinal microorganisms, so how to obtain rare human nucleic acids as much as possible is also a problem. Since the nucleic acids in feces are easily degraded after being removed from the body, the current method of preserving feces on the market basically adopts the method of adding feces to the preservation solution. The ratio of feces to the preservation solution is about 1:1 to 1:10, which makes it more difficult to extract human DNA from feces.

[0003] In response to the above difficulties, the existing technical solutions are: Shanghai Ruiyi Biotechnology Co., Ltd.'s "Fecal Host DNA Methylation Detection" (China Patent Publication No. CN 108220286A) adopts the method of adding low concentration (25-500mM) of guanidine salt, vortexing at room temperature for 1-5min, and then centrifuging at 10000g for 5min, and then adding an impurity remover to remove impurities. Guangzhou Kangliming Biotechnology Co., Ltd. uses the "Human SDC2 Gene Methylation Detection Kit (Fluorescence PCR Method)" method, which is to centrifuge the feces at 5000rpm for 10min, then take 9mL of the supernatant and centrifuge it at 5000rpm for 10min, then take the supernatant and pass it through an SPE column to remove impurities, and then lyse and use probe magnetic beads to specifically adsorb the required nucleic acids. The existing technical solutions have more or less certain limitations in technical methods or clinical applications, such as harsh reaction conditions, complex operations, low yields of human nucleic acids, and high costs.

[0004] The technology for preparing fecal Bis-DNA on the market is relatively complicated. The time required to convert fecal DNA with bisulfite is relatively long. In order to adapt to automated instruments, the existing technology uses less feces (including preservation solution), so it is impossible to make reasonable applications of the collected feces, resulting in less human DNA in the prepared fecal nucleic acid. In addition, when bisulfite is used to convert unmethylated cytosine C in DNA into uracil U (methylated cytosine C is not affected), more than 90% of the DNA will be damaged, greatly affecting the integrity of the extracted host DNA. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for extracting and converting large-volume fecal DNA, thereby solving the problems in the above-mentioned background technology.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for extracting and converting large-volume fecal DNA is provided, comprising the following steps:

[0007] S1. Lysing the stool sample using a lysis solution and an enzyme, wherein the lysis solution includes sodium dodecyl sulfate;

[0008] S2. Remove the lysed fecal sample by using a decontamination agent to remove impurities:

[0009] S3, DNA precipitation using isopropanol method;

[0010] S4, adding a protective solution, transforming the extracted DNA using high temperature and bisulfite; the protective solution comprises 0.1-5%wt hydroquinone and 50-98%wt diethylene glycol dimethyl ether;

[0011] S5. Add binding solution to the transformed sample and prepare Bis-DNA using the magnetic bead method.

[0012] In a preferred embodiment of the present invention, step S1 specifically includes the following:

[0013] S11, place the sample tube containing feces on a multi-tube mixer, and vortex mix at 1000-2500 rpm for 5-10 minutes;

[0014] S12, transfer 1-3 mL of the vortex-mixed sample to a 5 mL centrifuge tube;

[0015] S13, adding 100-500 μL of lysis solution to the 5 mL centrifuge tube to which the sample was transferred, wherein the lysis solution comprises 1-30% wt sodium dodecyl sulfate;

[0016] S14. Add 10-100 μL of proteinase K to the 5 mL centrifuge tube in step 3, wherein the concentration of proteinase K is 10-50 mg / mL; vortex to mix, and incubate at 70° C. for 5-30 min.

[0017] In a preferred embodiment of the present invention, step S2 specifically includes the following:

[0018] The sample after incubation is taken out, quickly cooled to room temperature, 100-350 μL of impurity remover A and 100-500 μL of impurity remover B are added in sequence, vortexed and mixed, centrifuged at 10000-12000 rpm for 2-10 min, and the supernatant is taken; the impurity remover A comprises 0.2-10% wt of ammonium aluminum sulfate dodecahydrate and 0.1-5% wt of ammonium acetate; the impurity remover B comprises 5-60% wt of sodium acetate.

[0019] In a preferred embodiment of the present invention, step S3 specifically includes the following:

[0020] S31, take 1-3 mL of supernatant into a 5 mL centrifuge tube, add 0.5-2 mL of pre-cooled isopropanol, mix by inversion, and centrifuge at 10000-12000 rpm for 2-10 min;

[0021] S32, discard the supernatant, retain the precipitate, discard the residual liquid with a pipette, add 50-150 μL of an eluent, wherein the eluent includes 0.1-1% wt Tris and 0.01-1% wt ethylenediaminetetraacetic acid, and incubate at 70° C. for 2-10 min;

[0022] S33, take out and shake vigorously for 2 minutes, centrifuge at 10000-12000 rpm for 2-10 minutes, and take the supernatant;

[0023] In a preferred embodiment of the present invention, step S4 specifically includes the following:

[0024] S41, transfer 50-120 μL of supernatant to a new 2 mL centrifuge tube and add 30-80 μL of protection solution;

[0025] S42, add 80-150 μL of conversion solution, wherein the conversion solution includes 1-6 M sodium metabisulfite, vortex mix, centrifuge instantaneously, and place in a metal bath with the program of 98° C., 5-10 min; 80° C., 0.5-1.5 h; 20° C., 2 min.

[0026] In a preferred embodiment of the present invention, step S5 specifically includes the following:

[0027] S51. Take out the sample after transformation, add 50-150 μL of binding solution; the binding solution includes 2-6.5 M guanidine thiocyanate, 0.1-1% wt ethylenediaminetetraacetic acid, 1-5% wt TRIS-HCL (pH 6.5) solution, 100-300 μL anhydrous ethanol and 50 μL magnetic beads; the magnetic beads are carboxyl magnetic beads with a concentration of 25-50 mg / mL, vortex mix, incubate at room temperature for 5-20 minutes, and mix every 3-5 minutes.

[0028] S52: After the incubation, centrifuge the sample immediately and place it on a magnetic rack for 2-5 minutes. Wait for the solution to become clear and discard the supernatant.

[0029] S53, add 600 μL of cleaning solution A, wherein the cleaning solution A includes 1-45% wt guanidine hydrochloride, 1-5% wt sodium chloride, 1-10% wt Tris-HCL (pH 7.5) solution, 0.1-1% wt Proclin 300 solution, and 50-70% wt anhydrous ethanol, vortex mix for 20 seconds, and centrifuge instantaneously; place on a magnetic rack, wait for 1-2 minutes for the solution to clarify, and discard the supernatant;

[0030] S54, add 600 μL of cleaning solution B, wherein the cleaning solution B includes 0.1-1%wt sodium chloride, 5-30%wt Tris-HCL (pH7.5) solution, 0.1-1%wt Proclin 300 solution, and 50-80%wt anhydrous ethanol, vortex mix for 20 seconds, and centrifuge instantaneously; place on a magnetic rack, wait for 1-2 minutes for the solution to clarify, and discard the supernatant;

[0031] S55, repeat step S54;

[0032] S56. Use 200 μL and 10 μL pipettes to discard the remaining liquid, place the centrifuge tube containing the magnetic beads on a metal bath, and incubate at 60°C for 3 min.

[0033] S57, adding 20-100 μL of an eluent, wherein the eluent includes 0.1-1%wt Tris and 0.01-1%wt ethylenediaminetetraacetic acid, incubating at 60°C for 2-10 min, and mixing every 2-3 min;

[0034] S58. Place on a magnetic rack and wait for 1-10 minutes for the solution to become clear. Transfer the supernatant to a new centrifuge tube.

[0035] In a preferred embodiment of the present invention, S6, PCR amplification procedure is also included. The PCR reaction system includes 15uL SDC2 reaction solution A or SDC2 reaction solution B and 5uL template DNA. The PCR amplification procedure is as follows:

[0036] Table 1

[0037]

[0038] Compared with the background technology, this technical solution has the following advantages:

[0039] 1. The present invention creatively proposes a method for preparing Bis-DNA by combining isopropanol precipitation with the magnetic bead method. Preparing DNA by isopropanol precipitation saves time and solves the problem of unreasonable use of stool samples;

[0040] 2. The unique dehumidification system of the present invention can remove humic acid, bile acid, choline, bile salts, polysaccharides and other substances in feces that have strong inhibitory effects on PCR to the greatest extent;

[0041] 3. Directly lyse DNA, reduce the number of DNA sample processing steps, and retain as much host DNA as possible;

[0042] 4. Compared with the traditional Bis-DNA method, the present invention combines the extraction of DNA by isopropanol precipitation and the preparation of BisDNA by magnetic beads, which greatly reduces the experimental steps and reduces the experimental time. DETAILED DESCRIPTION

[0043] The present invention is specifically described below in conjunction with the examples: wherein Bis-DNA: DNA converted with bisulfite; CpG island: a CG-dense region in a gene promoter region.

[0044] Example

[0045] The embodiment provides a method for extracting and converting large-volume fecal DNA. 20 fecal samples are extracted. The specific extraction steps are as follows:

[0046] 1. The stool collection tube was fully thawed at room temperature and vortexed at 2500 rpm / m for 5 min;

[0047] 2. Take 3 mL of stool sample into a 10 mL centrifuge tube; add 350 μL of lysis buffer and 45 μL of proteinase K, mix by inverting for about 20 times, and incubate in a 70°C constant temperature water bath for 15 minutes;

[0048] 3. Take out a 10 mL centrifuge tube, quickly cool it to room temperature, add 660 μL of impurity remover A and 450 μL of impurity remover B, mix by inverting for about 20 times, and centrifuge at 11000 rpm for 2 min;

[0049] 4. Transfer 3 mL of supernatant to a new 10 mL centrifuge tube, add 1.6 mL of pre-cooled isopropanol, mix by inverting for about 20 times, and centrifuge at 11,000 rpm for 10 min;

[0050] 5. Discard the supernatant, aspirate and discard the residual liquid at the bottom of the tube, and avoid the precipitation during operation;

[0051] 6. Add 80 μL of eluent to the 10 mL centrifuge tube containing the precipitate;

[0052] 7. Incubate in a 70℃ constant temperature water bath for 5 min, take out and shake vigorously for 2 min, and resuspend the precipitate;

[0053] 8. Centrifuge at 11000 rpm for 2 min;

[0054] 9. Transfer 70 μL of supernatant to a 2 mL centrifuge tube;

[0055] 10. Add 95 μL of conversion solution and 40 μL of protection solution, vortex to mix, centrifuge briefly, and place in a metal bath with the program of 98°C, 10 min; 80°C, 1 h.

[0056] 11. Take out 100 μL of binding solution from the previous step, 200 μL of ethanol, and 50 μL of magnetic beads, vortex to mix, incubate at room temperature for 10 minutes, and mix every 5 minutes.

[0057] 12. Centrifuge briefly, place on a magnetic rack, wait for the solution to become clear (3-5 min), and remove the supernatant.

[0058] 13. Add 600 μL of cleaning solution 1, vortex mix for 20 seconds, centrifuge briefly, place on a magnetic rack, wait for the solution to clarify, and remove the supernatant.

[0059] 14. Add 600 μL of cleaning solution 2, vortex mix for 20 seconds, centrifuge briefly, place on a magnetic rack, wait for the solution to clarify, and remove the supernatant.

[0060] 15. Add 600 μL of cleaning solution 2, vortex mix for 20 seconds, centrifuge briefly, place on a magnetic rack, wait for the solution to clarify, and remove the supernatant.

[0061] 16. Use a 100μL pipette to remove any remaining liquid, and then use a 10μL pipette to completely remove any remaining liquid.

[0062] 17. Dry in a metal bath at 60°C for 3 minutes until the beads are matte.

[0063] 18. Add 50 μL of elution buffer, vortex to mix, incubate at 60°C for 5 min, and mix every 2 min.

[0064] 19. Vortex to mix, centrifuge briefly, place on a magnetic rack, wait for the solution to clarify, and transfer the supernatant to a new 1.5 mL centrifuge tube;

[0065] 20. Configure PCR reaction mix according to the table below;

[0066] Table 2

[0067] Components Volume (μL) Reaction solution A or reaction solution B 15 Template DNA 5 Total 20

[0068] 21. Set up the PCR run program according to the amplification program in the table below and perform PCR.

[0069] Table 3

[0070]

[0071] Test results:

[0072] Table 4

[0073]

[0074]

[0075] Comparative Example

[0076] In the comparative example, 20 stool samples were extracted using a commercial kit, and the specific extraction steps were as follows:

[0077] Preparation before the experiment:

[0078] 1. Prepare Buffer BCB: When using for the first time, add 5.8 mL of Buffer SDW and 280 μL of Buffer SBH to the Buffer BCB powder bottle, vortex intermittently for about 5 minutes to fully dissolve, and mark it.

[0079] Note: After preparation, Buffer BCB should be stored at 4℃ with the lid tightly closed. The validity period is 2 months.

[0080] 2. Prepare Buffer DW1: When using for the first time, add 8.5 mL of anhydrous ethanol to the Buffer DW1 bottle and mix thoroughly, and mark it.

[0081] 3. Prepare Buffer DW2: When using for the first time, add 12 mL of anhydrous ethanol to the Buffer DW2 bottle and mix thoroughly, and mark it.

[0082] Experimental procedures

[0083] 1. Repeatedly blow and aspirate the stool sample in the preservation solution, and transfer 7 mL of the homogenized suspension into a 10 mL screw-cap centrifuge tube (the sample should be taken out 30 minutes in advance and equilibrated to room temperature);

[0084] 2. Add 800uL①buffer SLS and ②100uL Proteinase K Solusion, mix well by inverting for about 20 times, and incubate in a 70℃ constant temperature water bath for 15min;

[0085] 3. Take out the screw-cap centrifuge tube, cool it at room temperature for 10 minutes, and add 1.5 mL of ③Buffer STD. Mix by inverting for about 20 times, centrifuge at 10000×rcf for 5 minutes, and transfer 5.5 mL of supernatant to a 10 mL capped centrifuge tube (avoiding the precipitate and floating materials. If the supernatant is less than 5.5 mL, use ⑤Buffer SDW to make up the difference);

[0086] 4. Add 1 mL of ④Buffer SDL to the 10 mL centrifuge tube with the cap containing the supernatant, and mix by inverting for about 20 times.

[0087] Incubate in a 70°C constant temperature water bath for 10 min;

[0088] 5. Take out a 10 mL centrifuge tube with a cap, cool it at room temperature for 10 min, and centrifuge it at 10000×rcf for 2 min;

[0089] 6. Transfer all the supernatant to a new 10 mL centrifuge tube with a snap cap, add 3.2 mL of pre-cooled isopropanol, mix by inverting for about 20 times, and centrifuge at 12000×rcf for 10 min;

[0090] 7. Discard the supernatant, and then use a 200uL pipette to discard the residual liquid at the bottom of the centrifuge tube, avoiding the precipitate (if the experiment needs to be suspended before transformation, the precipitate can be stored at -20±5℃, but not more than 12h);

[0091] 8. Add 300uL⑤BufferSDW to the 10mL centrifuge tube with the lid pressed containing the precipitate;

[0092] 9.70℃ constant temperature water bath for 5 minutes, take out and shake vigorously for 2 minutes, resuspend the precipitate;

[0093] 10. Centrifuge at 12000×rcf for 2 min;

[0094] 11. Pipette all the liquid into adsorption column A (adsorption column A is placed on a 2 mL centrifuge tube) and centrifuge at 12000×rcf for 3 min. Discard adsorption column A and keep the liquid in the 2 mL centrifuge tube.

[0095] 12. Add 420uL⑥Buffer BCB (make sure it is prepared according to the reagent preparation requirements) and 160uL⑦

[0096] Buffer DPB, vortex to mix for 5 seconds, and centrifuge;

[0097] 13. Turn on the circulating metal bath and set the following program:

[0098] 14.95℃5min; 60℃20min; 95℃5min; 60℃20min; 20℃5min

[0099] 15. After the thermal cycle metal bath is heated to 90°C, place a 2 mL centrifuge tube;

[0100] 16. After transformation, take out a 2 mL centrifuge tube, add 420 uL⑧Buffer CBD and 550 uL anhydrous ethanol in sequence, vortex and mix for 5 seconds, and centrifuge at 2000×ref for 1 minute;

[0101] 17. Pipette 600uL of liquid into adsorption column B (adsorption column B is placed on a 2mL collection tube) and centrifuge at 12000×rcf for 30s, then discard the liquid in the collection tube;

[0102] 18. Repeat step 9-conversion, pass the remaining liquid through the column twice, and pour out the liquid in the collection tube;

[0103] 19. Add 600uL⑨BufferDW1 to the adsorption column, centrifuge at 12000×rcf for 30s, and discard the liquid in the collection tube; 20. Add 600uL⑩BufferDW2 to the adsorption column, centrifuge at 12000×rcf for 30s, and discard the liquid in the collection tube; 21. Add 600uL anhydrous ethanol to the adsorption column, centrifuge at 12000×rcf for 30s;

[0104] 22. Use a new collection tube and centrifuge at 12000×rcf for 2 min;

[0105] 23. Place the adsorption column in a new 1.5 mL centrifuge tube and dry it in a 60°C constant temperature metal bath with the cover open for 3 min;

[0106] 24. Take out the centrifuge tube and adsorption column B, add 50uL⑤Buffer SDW to the membrane of adsorption column B, incubate in a 60℃ constant temperature metal bath with the lid closed for 3min, and centrifuge at 12000×rcf for 2min;

[0107] 25. Take out the centrifuge tube and adsorption column B, and drop 50uL⑤Buffer SDW onto the membrane of adsorption column B again, incubate in a 60℃ constant temperature metal bath with the lid closed for 3min, and centrifuge at 12000×rcf for 2min;

[0108] 26. Remove the centrifuge tube, collect the DNA and save it.

[0109] 27. Take out SDC2 reaction solution A, SDC2 reaction solution B and SDC2 positive control and thaw them at room temperature. Mix them well and centrifuge them quickly for 15 seconds for later use. Take out SDC2 mixed enzyme and centrifuge them quickly for 15 seconds for later use.

[0110] 28. Calculate the amount of reaction solution to be prepared based on the number of samples to be tested, negative controls, and SDC2 positive controls.

[0111] 29. Mix SDC2 reaction solution A and SDC2 reaction solution B with SDC2 mixed enzyme at a ratio of 0.3 μL of mixed enzyme to each tube, and centrifuge quickly for 15 seconds after mixing.

[0112] 30. After mixing the SDC2 enzyme mix, take 35.0 μL of each of the reaction solution A and the reaction solution B and add them into the PCR reaction tube.

[0113] 31. Take 5.0 μL of the stool sample DNA to be tested, negative control, and SDC2 positive control and add them to the PCR reaction tube containing the mixed solution. Carefully cover the tube, vortex to mix, and centrifuge for 1 minute.

[0114] Set up the PCR run program according to the amplification program in the table below and perform PCR:

[0115] Table 5

[0116]

[0117]

[0118] Test results:

[0119] Table 6

[0120]

[0121] Compare the performance differences between the test results of the embodiments and the comparative examples:

[0122] The performance of the two detection methods was compared by using commercial reagents and the reagent method of the present invention to detect the same sample in parallel. The main indicator of comparison is the difference in Ct values ​​detected at the same site of the same sample. The smaller the Ct value, the better the performance. The results are shown in the following table. The difference in Ct values ​​of the same site between the commercial kit and the present invention is positive, indicating that the detection results of the present invention are better.

[0123] Table 7

[0124]

[0125] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting and converting large-volume fecal DNA, characterized in that: The steps include: S1. Lysing the stool sample using a lysis solution and an enzyme, wherein the lysis solution includes sodium dodecyl sulfate; S2. Remove the lysed fecal sample by using a decontamination agent to remove impurities: S3, DNA precipitation using isopropanol method; S4, adding a protective solution, transforming the extracted DNA using high temperature and bisulfite; the protective solution comprises 0.1-5%wt hydroquinone and 50-98%wt diethylene glycol dimethyl ether; S5. Add binding solution to the transformed sample and prepare Bis-DNA using the magnetic bead method.

2. A method for extracting and converting large-volume fecal DNA according to claim 1, characterized in that: Step S1 specifically includes the following: S11, vortex the sample tube containing feces at 1000-2500 rpm for 5-10 minutes; S12, transfer the vortex-mixed sample to a centrifuge tube; S13, adding a lysate into the centrifuge tube, wherein the lysate comprises 1-30%wt sodium dodecyl sulfate; S14. Add proteinase K to the centrifuge tube of the lysed sample, wherein the concentration of proteinase K is 10-50 mg / mL; vortex to mix, and incubate at 70° C. for 5-30 min.

3. A method for extracting and converting large-volume fecal DNA according to claim 1, characterized in that: Step S2 specifically includes the following: The sample after incubation is taken out, quickly cooled to room temperature, impurity remover A and impurity remover B are added in sequence, vortexed and mixed, centrifuged at 10000-12000 rpm for 2-10 minutes, and the supernatant is taken; the impurity remover A comprises 0.2-10% wt of ammonium aluminum sulfate dodecahydrate and 0.1-5% wt of ammonium acetate; the impurity remover B comprises 5-60% wt of sodium acetate.

4. A method for extracting and converting large-volume fecal DNA according to claim 1, characterized in that: Step S3 specifically includes the following: S31, take the supernatant into a centrifuge tube, add pre-cooled isopropanol, mix by inversion, and centrifuge at 10000-12000 rpm for 2-10 min; S32, discard the supernatant, retain the precipitate, add 50-150 μL of an eluent, wherein the eluent comprises 0.1-1%wt Tris and 0.01-1%wt ethylenediaminetetraacetic acid, and incubate at 70°C for 2-10 min; S33, take out and shake vigorously for 2 minutes, centrifuge at 10000-12000rpm for 2-10 minutes, and take the supernatant.

5. The method for extracting and converting large-volume fecal DNA according to claim 1, characterized in that: Step S4 specifically includes the following: S41, transfer 50-120 μL of supernatant to a centrifuge tube and add 30-80 μL of protection solution; S42, add 80-150 μL of conversion solution, wherein the conversion solution includes 1-6 M sodium metabisulfite, vortex mix, centrifuge instantaneously, and place in a metal bath with the program of 98° C., 5-10 min; 80° C., 0.5-1.5 h; 20° C., 2 min.

6. The method for extracting and converting large-volume fecal DNA according to claim 1, characterized in that: Step S5 specifically includes the following: S51. Take out the sample after transformation, add 50-150 μL of binding solution; the binding solution includes 2-6.5 M guanidine thiocyanate, 0.1-1% wt ethylenediaminetetraacetic acid, 1-5% wt TRIS-HCL (pH 6.5) solution, 100-300 μL anhydrous ethanol and 50 μL magnetic beads; the magnetic beads are carboxyl magnetic beads with a concentration of 25-50 mg / mL, vortex mix, incubate at room temperature for 5-20 minutes, and mix every 3-5 minutes.

7. A method for extracting and converting large-volume fecal DNA according to claim 6, characterized in that: Step S5 also includes: S52: After the incubation, centrifuge the sample immediately and place it on a magnetic rack for 2-5 minutes. Wait for the solution to become clear and discard the supernatant. S53, add 600 μL of cleaning solution A, wherein the cleaning solution A includes 1-45% wt guanidine hydrochloride, 1-5% wt sodium chloride, 1-10% wt Tris-HCL (pH 7.5) solution, 0.1-1% wt Proclin 300 solution, and 50-70% wt anhydrous ethanol, vortex mix for 20 seconds, and centrifuge instantaneously; place on a magnetic rack, wait for 1-2 minutes for the solution to clarify, and discard the supernatant; S54, add 600 μL of cleaning solution B, wherein the cleaning solution B includes 0.1-1%wt sodium chloride, 5-30%wt Tris-HCL (pH7.5) solution, 0.1-1%wt Proclin 300 solution, and 50-80%wt anhydrous ethanol, vortex mix for 20 seconds, and centrifuge instantaneously; place on a magnetic rack, wait for 1-2 minutes for the solution to clarify, and discard the supernatant; S55, repeat step S54; S56, discard the residual liquid, place the centrifuge tube containing the magnetic beads on a metal bath, and incubate at 60°C for 3 min; S57, adding 20-100 μL of an eluent, wherein the eluent includes 0.1-1%wt Tris and 0.01-1%wt ethylenediaminetetraacetic acid, incubating at 60°C for 2-10 min, and mixing every 2-3 min; S58. Place on a magnetic rack and wait for 1-10 minutes for the solution to become clear. Transfer the supernatant to a new centrifuge tube.

8. The method for extracting and converting large-volume fecal DNA according to claim 1, characterized in that: Also included is S6, a PCR amplification procedure.

9. A method for extracting and converting large-volume fecal DNA according to claim 8, characterized in that: The PCR reaction system includes 15uL SDC2 reaction solution A or SDC2 reaction solution B and 5uL template DNA.

10. The method for extracting and converting large-volume fecal DNA according to claim 8, characterized in that: The PCR amplification procedure is as follows:

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