A high-purity extraction method for fecal human-derived DNA based on secondary magnetic bead purification

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to a method for high-purity extraction of human fecal DNA based on secondary magnetic bead purification. The method involves first performing preliminary lysis of the fecal supernatant, then using coarse magnetic beads to bind nucleic acids in the sample. After preliminary rinsing and elution, the coarse magnetic beads and nucleic acids are eluted into a first elution buffer. Next, using the first elution buffer as the sample, gentle lysis is performed, followed by the use of fine magnetic beads to bind nucleic acids in the sample. Further rinsing and elution yield high-purity fecal human DNA. The obtained high-purity DNA exhibits low inhibition and high purity, and can be directly used in downstream detection experiments.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a method for high-purity extraction of human DNA from feces based on secondary magnetic bead purification. Background Technology

[0002] Extraction of human DNA from fecal samples has significant applications in early colorectal cancer screening and research on gut microbiome-host interactions. However, fecal samples are complex, containing not only human cells and exfoliated DNA, but also large amounts of bacteria, food residues, polysaccharides, humic acid, and bile salts. These components not only interfere with DNA extraction efficiency but also remain as PCR inhibitors in the final product, severely affecting the accuracy and stability of downstream quantitative PCR and methylation conversion tests. Currently, while fecal DNA extraction kits based on magnetic beads are widely used, they mostly employ a single binding-rinsing-elution process, making it difficult to completely remove residual inhibitors.

[0003] To address the aforementioned issues, existing technologies have attempted to improve purity by adding pretreatment steps or employing specific capture magnetic beads. For example, some patents (such as CN119061001A) add a purification solution after lysis to precipitate some impurities; other technologies (such as CN116042601A) use sequence-specific probe magnetic beads to selectively enrich target human DNA fragments from total nucleic acids. However, pretreatment purification methods are difficult to completely remove residual soluble inhibitors in the elution products, while specific capture methods have limitations such as complex operation, strong dependence on the target sequence, and high cost, making them unsuitable for applications requiring the preservation of all human DNA information.

[0004] Therefore, how to further reduce the residual inhibitors in the final product while ensuring DNA yield, so that it can directly meet the requirements of downstream experiments with high template purity, such as methylation transformation, remains an urgent technical problem to be solved in the current fecal human DNA extraction technology. Summary of the Invention

[0005] Based on this, the present invention proposes a method for high-purity extraction of human DNA from feces based on secondary magnetic bead purification, in order to provide a simple, universal and efficient solution to the above problems.

[0006] The technical solution of this invention to solve the technical problem is as follows: This invention provides a method for high-purity extraction of human DNA from feces based on secondary magnetic bead purification, comprising the following steps: 1) Aspirate fecal supernatant, use crude extraction lysis buffer to perform preliminary lysis of human cells in fecal supernatant, add proteinase K and RNase, then add crude extraction magnetic beads and binding buffer to allow the crude extraction magnetic beads to bind to nucleic acids in the sample, then use crude extraction rinsing buffer to rinse the crude extraction magnetic beads briefly, and then elute the nucleic acids into the first elution buffer. 2) Using the first elution buffer obtained in step 1) as the refined sample, the sample was bound using refined magnetic beads and refined lysis binding buffer, purified using refined wash buffer 1 and refined wash buffer 2, and eluted using the second elution buffer; high-purity fecal human DNA was obtained. The obtained DNA can be directly used for downstream experiments such as methylation, library construction, and sequencing.

[0007] In step 1), The crude lysis buffer is primarily used to lyse human cells in fecal supernatant without violently lysing other microbial cells in the sample. Therefore, it requires a relatively mild lysis capacity, i.e., a low-concentration dissociation salt buffer. The crude lysis buffer is selected from any one or more of the following components: sodium citrate, Tris-HCl, EDTA, etc. Preferably, the crude lysis buffer is 0.5M sodium citrate, 0.1M Tris-HCl, or 0.1M EDTA.

[0008] Proteinase K is used to digest proteins in buffer systems, and a proteinase K concentration of 20% by mass is preferred.

[0009] RNase is used to degrade RNA released into the buffer system after cell rupture, and RNase with a concentration of 10 μg / μL is preferred.

[0010] The coarse extraction magnetic beads are carboxyl-modified supercis microspheres designed to bind nucleic acids released into the buffer solution after crude extraction lysis. Promega magnetic beads are preferred. Coarse and fine extraction magnetic beads are two different sizes; larger carboxyl-modified beads are used for coarse extraction, while purified beads with complete fragment recovery are recommended for fine extraction.

[0011] The binding solution is used to promote the binding of human DNA in the sample to the crude magnetic beads; isopropanol is preferred.

[0012] The crude extraction rinsing solution is a high-concentration ethanol solution, preferably 70% ethanol.

[0013] The first eluent can be sterile water. The first eluent will be used as the sample for the final extraction after the crude extraction; therefore, the eluent volume should not be too small, ideally 1 mL.

[0014] In step 2), Compared to coarse magnetic beads, refined magnetic beads are used to recover cellular DNA from refined samples. Human DNA fragments are longer, and refined magnetic beads are more suitable for these longer fragments. Kangwei Century's CMPure magnetic beads (catalog number: CW3171) are preferred.

[0015] The lysis buffer used in the refining process has a relatively strong lysis capacity. The refining lysis buffer is a buffer solution containing a high concentration of ionizing salt and isopropanol, selected from any one or more of the following components: guanidine isothiocyanate, Tris-HCl, sodium chloride, and Triton X-100. Preferably, 10M guanidine isothiocyanate, 0.1M Tris-HCl, 0.5M sodium chloride, and 0.2% (w / w) Triton X-100 are used.

[0016] The rinsing solution 1 contains certain salt ions and ethanol, selected from any one or more of the following components: guanidine hydrochloride, Tris-HCl, EDTA, ethanol, etc. Preferably, 4M guanidine hydrochloride, 0.1M Tris-HCl, 0.5M EDTA, and 40% ethanol by mass fraction are used.

[0017] The rinsing solution 2 mainly contains high concentrations of ethanol, preferably 70% ethanol.

[0018] The second eluent can be sterile water.

[0019] This invention provides a high-purity extraction method for human DNA from feces based on secondary magnetic bead purification, applicable to fecal samples preserved in a preservation solution. This method can be automated when used with a corresponding extraction instrument.

[0020] In a preferred embodiment of the present invention, the method for high-purity extraction of fecal human DNA based on secondary magnetic bead purification specifically includes the following steps: 1) First, aspirate approximately 3 mL of fecal supernatant. If the sample is a frozen fecal sample, wait until it reaches room temperature, add a small amount of sterile water, mix well, and then aspirate approximately 3 mL of the supernatant to form the experimental sample. Next, add 500 μL of 0.5 M sodium citrate to the sample and vortex to mix. Then, add 60 μL of 20% proteinase K and 10 μL of 10 μg / μL RNase, mix thoroughly, and incubate at 60°C for 20 minutes, vortexing periodically during incubation. In the above lysis process, sodium citrate acts as a mild lysis salt to lyse only the human cells in the sample, rather than violently lysing other microbial cells. The subsequent addition of proteinase K and RNase prevents direct contact between the enzymes and the salt, thus preventing enzyme inactivation. Proteinase K reacts at 60°C, and RNase also functions at this temperature. After lysis, add 2 mL of isopropanol and 150 μL of crude magnetic beads to the mixture and vortex to mix for 10 minutes. In the above binding process, isopropanol promotes the binding of human DNA to the crude magnetic beads. Subsequently, the DNA-bound crude magnetic beads are initially rinsed with 70% ethanol, as 70% ethanol can dissolve a large portion of organic impurities and can preliminarily wash away inhibitors and other substances bound to the surface of the crude magnetic beads. After the above rinsing, the crude magnetic beads are eluted into 1 mL of enzyme-free water to form the first eluent.

[0021] 2) Using the first elution buffer as the refined sample, a strong lysis binding buffer and refined magnetic beads are added and vortexed for 10 minutes to prevent incompletely lysed human cells from remaining in the crude nucleic acid and to promote the binding of the refined magnetic beads to the DNA in the sample. Purification is then performed using refined wash buffer 1 and refined wash buffer 2, followed by elution with the second elution buffer. The refined lysis binding buffer consists of: 10M guanidine isothiocyanate, 0.1M Tris-HCl, 0.5M sodium chloride, and 0.2% Triton 100. Refined wash buffer 1 consists of: 4M guanidine hydrochloride, 0.1M Tris-HCl, 0.5M EDTA, and 40% ethanol. Refined wash buffer 2 is 70% ethanol; the second elution buffer is sterile water. After binding, the refined magnetic beads are rinsed and eluted. The resulting nucleic acid can be directly used for downstream detection experiments.

[0022] The method of this invention has the following technical advantages: This invention proposes a high-purity extraction method for human fecal DNA based on secondary magnetic bead purification. Specifically, the fecal supernatant is first subjected to preliminary lysis. Crude magnetic beads are used to bind the nucleic acids in the sample. After preliminary rinsing and elution, the crude magnetic beads and nucleic acids are eluted into a first elution buffer. Then, using the first elution buffer as the sample, gentle lysis is performed. Fine magnetic beads are then used to bind the nucleic acids in the sample. After further rinsing and elution, high-purity human fecal DNA is obtained. The obtained high-purity DNA has low inhibition and high purity, and can be directly used for downstream detection experiments. Attached Figure Description

[0023] Figure 1 The state of nucleic acid elution plates obtained using a nucleic acid extractor for different extraction schemes.

[0024] Figure 2 This refers to the state of nucleic acids obtained through manual extraction after replacing the refined magnetic beads. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0026] In the embodiments of the present invention: For crude extraction, Promega's nucleic acid extraction magnetic beads were used.

[0027] The refined magnetic beads used are CMPure magnetic beads from Kangwei Century (item number: CW3171).

[0028] Both proteinase K and RNase are from Jiangsu Kangwei Century Biotechnology Co., Ltd.

[0029] R magnetic beads are from Taizhou Yanyikang Biotechnology Co., Ltd.

[0030] Example 1 In this embodiment, the method for high-purity extraction of human fecal DNA based on secondary magnetic bead purification includes the following steps: 1) Draw 3 mL of fecal supernatant from the fecal storage tube containing the preservation solution into a 15 mL centrifuge tube; 2) Add 0.5 mL of 0.5 M sodium citrate (i.e. crude lysis buffer), 60 μ L of 20% proteinase K and 10 μ L of 10 μg / μL RNase to the centrifuge tube from the previous step, and incubate in a 60 °C water bath for 20 minutes, vortexing every 6 minutes to mix. 3) After incubation, add 2 mL of isopropanol (i.e., binding solution) and 150 μL of crude magnetic beads to the tube, and shake to mix for 10 minutes. 4) Place the centrifuge tube on a magnetic rack to attract the magnet, remove the solution from the tube, and then remove the centrifuge tube; 5) Add 4 mL of 70% ethanol (i.e. crude extract rinsing solution) to the tube, vortex to mix for 1 minute, place on a magnetic rack to remove the solution from the tube, and remove the centrifuge tube. 6) Without drying, add 1 mL of sterile water (i.e., the first elution buffer) directly into the centrifuge tube, vortex and mix for 10 minutes; place on a magnetic rack to attract magnets, and transfer the first elution buffer into a new centrifuge tube; 7) Add 2.5 mL of the purification lysis binding solution and 60 μL of purification magnetic beads to the new centrifuge tube from the previous step, and vortex at room temperature for 10 minutes to mix. The composition of the purification lysis binding solution is as follows: 10 M guanidine isothiocyanate, 0.1 M Tris-HCl, 0.5 M sodium chloride and 0.2% Triton 100 by mass.

[0031] 8) After mixing, place the centrifuge tube on a magnetic rack to attract the magnet, remove the solution from the tube, and then remove the centrifuge tube. 9) Add 4 mL of the purification and rinsing solution 1 to the tube, vortex and mix for 1 minute, place it on a magnetic rack to remove the solution in the tube, and remove the centrifuge tube; the components of the purification and rinsing solution 1 are as follows: 4M guanidine hydrochloride, 0.1M Tris-HCl, 0.5M EDTA and 40% ethanol by mass.

[0032] 10) Add 4 mL of 70% ethanol (i.e., refined rinsing solution 2) to the tube, vortex and mix for 1 minute, place it on a magnetic rack to remove the solution from the tube, and remove the centrifuge tube. 11) Dry for 5 minutes to allow the solution on the surface of the refined magnetic beads to evaporate; 12) Add 120 μL of sterile water (i.e., the second elution buffer) to dissolve the magnetic beads, vortex for 10 minutes, use a magnetic rack to attract the magnet, transfer the elution buffer, and finally obtain high-purity fecal human DNA.

[0033] Example 2 Using the extraction protocol of Example 1, human DNA was extracted from fecal samples of six volunteers (all feces were preserved in Kangwei Century CWY041 preservation solution). Considering that fecal samples contain both microbial DNA and human DNA, and that the content of human DNA in the total DNA of the sample is less than 0.01%, the nucleic acid concentration cannot accurately reflect the content of human DNA in the extracted product. Therefore, in this example, to determine the content of human DNA, the human internal reference gene β-actin was amplified using real-time PCR, and the Ct value was compared to reflect the content of template (human DNA) and inhibitor in the nucleic acid.

[0034] Upstream primer: GCGCCGTTCCGAAAGTT (SEQ ID NO: 1) Downstream primer: CGGCGGATCGGCAAA (SEQ ID NO: 2) Amplification was performed using the Kangwei Century qPCR detection kit (catalog number CW0957), and the amplification system was referenced (CN114107289B). The results are shown in Table 1: Table 1 .

[0035] Based on the results of this experiment, it can be concluded that human fecal DNA can be obtained using this invention.

[0036] Example 3 The experimental procedure in Example 1 was simplified to the following operating parameters. The Kangwei Century CWE240 extractor was used to extract the sample in Example 2 and the difference between the extracted sample and the manual extraction was compared.

[0037] The automated extraction of board positions is shown in Table 2: Table 2 .

[0038] The program's runtime parameters are shown in Table 3: Table 3 .

[0039] Note: At pause 1, add 150 μL of crude extraction magnetic beads and 2 mL of isopropanol. At pause 2, add 2.5 mL of fine extraction lysis binding buffer.

[0040] The samples from Example 2 were extracted using a Kangwei Century CWE240 extractor, and the results are shown in Table 4: Table 4 .

[0041] Based on the results of this experiment, it can be concluded that the automated adaptation using the experimental scheme provided by this invention produces a product whose Ct value during fluorescence amplification is comparable to that obtained through manual operation.

[0042] Comparative Example 1: Human feces contain intestinal microbial cells and exfoliated human cells, with intestinal microorganisms making up the vast majority and exfoliated human cells accounting for less than 0.01% of the total, resulting in a relatively low concentration of human DNA. In the current stage of biomedical development, human DNA in feces can be used for methylation, thereby enabling early screening for diseases such as gastric cancer and colorectal cancer. However, there are currently few magnetic bead-based kits specifically designed for extracting human DNA from feces; most kits tend to extract total DNA from fecal samples and cannot effectively separate the low concentration of human DNA. Currently, the most common commercially available nucleic acid extraction kit for fecal sample DNA is MP's MagBeads Fast DNA Kit for Soil. This kit includes a unique de-inhibition solution to remove inhibitors from fecal samples. Considering that competing products lose some DNA (including human DNA and microbial DNA) during the de-inhibition step, a comparative experiment was conducted using a non-de-inhibition approach from competing products. Sixteen fecal samples were used to perform automated nucleic acid extraction using the present invention (secondary magnetic bead purification), MP magnetic bead extraction kit (original MP), and MP magnetic bead extraction kit (without inhibition) with corresponding extraction instruments. The extraction effect was compared by comprehensively considering the state, concentration, purity, and fluorescence amplification of the elution buffer.

[0043] The state of the eluent is as follows Figure 1 As shown in Tables 5 and 6 below: Table 5. Concentration of total nucleic acid (human DNA + microbial DNA) extracted from fecal samples .

[0044] Table 6. Total nucleic acid purity of extracted fecal samples .

[0045] The results of amplifying the human internal control are shown in Table 7: Table 7 .

[0046] Interpreting the above experimental results: The difference is evident in the appearance of the nucleic acids. The nucleic acids obtained in this invention are consistent with the original MP, and are clearer than the MP without inhibition method. Although the total fecal DNA content extracted by this invention is lower than that of the original MP and MP without inhibition, the nucleic acid purity of this invention is the best. However, the average Ct value of the human internal control amplified by this invention is lower than that of the original MP and MP without inhibition methods, indicating that the human DNA content in the total nucleic acid obtained by this invention is higher than that of the control method.

[0047] Comparative Example 2: Using the extraction scheme of Example 1, the refined magnetic beads were replaced with R magnetic beads.

[0048] The experimental samples were provided by eight volunteers from Jiangsu Kangwei Century Biotechnology Co., Ltd.

[0049] The experimental method involves dividing the same sample into two equal parts. One part of the sample is extracted manually as described in Example 1. The other part is extracted using the same procedure as described in this invention, except that the fine extraction beads are replaced with R-beads. The amplification method for all samples is the same as in Example 2.

[0050] Results: After replacing the fine extraction magnetic beads with R magnetic beads for extraction, the obtained nucleic acid state was as follows: Figure 2 As shown. From Figure 2 It can be seen that the nucleic acid obtained by the extraction method of the present invention is clear and transparent, while the nucleic acid obtained by using R magnetic beads as fine extraction magnetic beads has color (impurities).

[0051] The amplification results are shown in Table 8: Table 8 .

[0052] Based on the results of this experiment, it can be concluded that after amplifying the human internal control, the average amplification Ct value of this invention is approximately 1.7 lower than that of the replaced refined magnetic bead method. This indicates that the nucleic acid obtained by the method of this invention contains relatively less inhibitor and has a higher nucleic acid concentration.

[0053] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for high-purity extraction of human fecal DNA based on secondary magnetic bead purification, characterized in that, The method includes the following steps: 1) Aspirate fecal supernatant, use crude extraction lysis buffer to perform preliminary lysis of human cells in fecal supernatant, add proteinase K and RNase, then add crude extraction magnetic beads and binding buffer to allow the crude extraction magnetic beads to bind to nucleic acids in the sample, then use crude extraction rinsing buffer to rinse the crude extraction magnetic beads briefly, and then elute the nucleic acids into the first elution buffer. 2) Using the first elution buffer obtained in step 1) as the refined sample, the refined magnetic beads and refined lysis binding buffer were used for binding, the refined washing buffer 1 and refined washing buffer 2 were used for purification, and the second elution buffer was used for elution; high-purity fecal human DNA was obtained.

2. The method according to claim 1, characterized in that, In step 1), the crude extract lysis solution is selected from any one or more of the following components: sodium citrate, Tris-HCl, and EDTA.

3. The method according to claim 1, characterized in that, In step 1), the crude magnetic beads are carboxyl-modified supercis microspheres.

4. The method according to claim 1, characterized in that, In step 1), the binding solution is isopropanol.

5. The method according to claim 1, characterized in that, In step 1), the crude extraction rinsing solution is a high-concentration ethanol solution.

6. The method according to claim 1, characterized in that, The first and second eluents are sterile water.

7. The method according to claim 1, characterized in that, In step 2), the refined magnetic beads used are CMPure magnetic beads from Kangwei Century.

8. The method according to claim 1, characterized in that, In step 2), the refining and pyrolysis binding solution is selected from any one or more of the following components: guanidine isothiocyanate, Tris-HCl, sodium chloride, and Tritonx 100.

9. The method according to claim 1, characterized in that, In step 2), the rinsing solution 1 is selected from any one or more of the following components: guanidine hydrochloride, Tris-HCl, EDTA, and ethanol; the rinsing solution 2 mainly includes high-concentration ethanol.

10. The method according to claim 1, characterized in that, In step 1), The crude extract lysis buffer consisted of 0.5 M sodium citrate, 0.1 M Tris-HCl, or 0.1 M EDTA. The proteinase K was 20% by mass. The RNase was RNase with a concentration of 10 μg / μL; The binding solution is isopropanol; The crude extraction washing solution is 70% ethanol; The first elution buffer is sterile water, 1 mL in volume; In step 2), The components of the purification and pyrolysis binding solution are 10M guanidine isothiocyanate, 0.1M Tris-HCl, 0.5M sodium chloride and 0.2% Triton 100 by mass. The components of the refined rinsing solution 1 are 4M guanidine hydrochloride, 0.1M Tris-HCl, 0.5M EDTA and 40% ethanol by mass. The rinsing solution 2 is 70% ethanol; The second eluent is sterile water.

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