A plasma cell-free DNA extraction kit, extraction method and application

Abstract

This invention belongs to the field of biotechnology and discloses a plasma cell-free DNA extraction kit, extraction method, and applications. The kit includes magnetic beads, lysis buffer, proteinase K, binding buffer, washing buffer, and elution buffer. The extraction method involves mixing the plasma sample, lysis buffer, proteinase K, binding buffer, and magnetic beads. The lysed plasma cell-free DNA (cfDNA) is incubated with the magnetic beads to form a magnetic bead-cfDNA complex. Under a magnetic field, the complex is washed to remove various impurities, and the plasma cfDNA is obtained after elution. The method of this invention is simple and rapid, with short sample lysis time and stable quality. Replacing isopropanol with anhydrous ethanol during lysis results in high nucleic acid recovery, making it suitable for plasma cfDNA extraction. The purity is suitable for subsequent sequencing and PCR / Q-PCR. The magnetic bead method is beneficial for large-scale use and has significant implications for research and clinical applications.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a plasma free DNA extraction kit, an extraction method and application. BACKGROUND

[0002] Nucleic acid is a kind of biological macromolecule, which is a carrier of genetic information and widely exists in all animal and plant cells and microorganisms. Nucleic acid in the body often combines with protein to form nucleoprotein. Nucleic acid plays a decisive role in the growth and heredity of organisms and has an important relationship with the occurrence of tumors. Therefore, the research on nucleic acid is one of the important topics in modern medical research.

[0003] Nucleic acid is usually combined with protein in the human body, and high-quality nucleic acid is the basis for subsequent research. Therefore, it is necessary to ensure sufficient extraction and the integrity of the primary structure of nucleic acid in the extraction process, and prevent the pollution of biological macromolecules such as protein. At present, the separation technology of nucleic acid mainly includes:

[0004] Phenol / chloroform extraction method: this method is the most widely used and classical nucleic acid separation and purification technology. First, the sample material is lysed, and then mixed with phenol and chloroform. Phenol is a denaturing agent for protein, and repeated extraction is used to denature protein. Sodium dodecyl sulfate (SDS) can lyse the cell membrane, and in the presence of proteinase K and EDTA, the protein or polypeptide or small peptide molecules are digested to denature and degrade the nucleoprotein, so that the DNA is separated from the nucleoprotein. DNA is easily dissolved in water and not dissolved in organic solvents. The surface of protein molecules has hydrophilic groups, which can also easily hydrate, and a layer of hydration layer is formed on the surface, so that the protein molecules can smoothly enter the aqueous solution to form a stable colloidal solution. When the organic solution exists, the colloidal stability of the protein is destroyed, and the protein is denatured and precipitated. After centrifugation, three layers are formed from top to bottom, the upper layer is the water phase layer containing nucleic acid, the middle layer is the emulsion layer mainly containing protein, and the lower layer is the phenol chloroform organic solvent. The protein and nucleic acid can be effectively separated by absorbing the upper water phase. However, the disadvantage of this method is that the reagent used is a toxic reagent.

[0005] Salt precipitation method: after the tissue and cells are lysed in the lysis solution, high salt (NaCl, NH4Cl, KCl) is added to denature the protein to form insoluble substances, so that the impurities and nucleic acid can be separated. The disadvantage of this method is that the impurities are not completely removed.

[0006] Glass bead method: under the condition of high-lye salt (guanidine hydrochloride, NaCl, guanidine isothiocyanate), glass beads are used to adsorb nucleic acid, and under the condition of low-lye salt, nucleic acid is eluted, so as to separate nucleic acid from protein, sugar and lipid impurities. The disadvantage of this method is that there will be residual glass beads.

[0007] Silica gel column method: This method uses a special silica matrix material to efficiently and specifically adsorb DNA and RNA under specific high-salt, low-pH conditions, and release DNA and RNA under low-salt, high-pH conditions. The centrifuge column contains synthetic resin resin, which has the function of adsorbing DNA, thereby achieving the separation of nucleic acids from impurities such as proteins. Its disadvantages are that it requires a large number of samples, consumes a lot of materials, and is not suitable for extracting nucleic acids from rare samples.

[0008] Magnetic bead method: Currently widely used in DNA extraction and purification experiments. The basic structure of magnetic beads generally consists of three layers: an inner layer of polystyrene, a second layer encapsulating the magnetic material iron(III) oxide (Fe3O4), and an outermost layer of a polymer material modified with functional groups. Utilizing nanoscale magnetic beads with surface-labeled functional groups, nucleic acids are adsorbed, while impurities such as proteins are not. The magnetic beads can aggregate and disperse under a magnetic field, thereby achieving the separation of nucleic acids from impurities. The magnetic beads used in this method are generally hydroxyl-based magnetic beads. This magnetic bead system includes: magnetic beads, polyethylene glycol (PEG), and salt ions, etc., at a certain concentration of PEG and salt ions (such as Na+). + In this environment, the hydration layer of DNA molecules is removed, disrupting the colloidal thermodynamic stability of DNA and altering its conformation. A large number of negatively charged phosphate groups are exposed, which, through Na+... + The formation of an "electric bridge" with hydroxyl groups allows DNA to be adsorbed onto the surface of hydroxyl-modified polymeric magnetic beads (i.e., solid-phase carriers), forming a "nucleic acid-magnetic bead complex." At the same time, the magnetic beads are superparamagnetic and can be adsorbed by an external magnetic field. This process is reversible, and under appropriate conditions, DNA molecules can be eluted and recovered.

[0009] The human circulatory system constantly carries blood cells. After somatic cells and blood cells rupture and degrade, they release their cytoplasm contents, including proteins, exosomes, RNA, and DNA fragments carrying various genetic information, known as cfDNA (cell-free DNA). These DNA fragments contain abnormalities such as mutations, deletions, insertions, rearrangements, copy number abnormalities, and methylation, especially ctDNA (circulating tumor DNA) fragments from the genome. This is known as liquid biopsy, which is less invasive and allows for multiple sample collections to observe dynamic changes. In peripheral blood, genetically abnormal ctDNA constitutes only a very small portion of all DNA, approximately 0.1% to 1%, and is highly fragmented (usually tens to over one hundred base pairs), making extraction difficult and reducing the sensitivity and specificity of clinical testing. Current DNA extraction methods yield low levels of ctDNA.

[0010] Currently available kits are easy to use, but they extract low levels of ctDNA, have a generally low extraction rate, and are expensive. Therefore, there is a need for a kit that offers high extraction efficiency and a cost-effectiveness ratio, improving extraction efficiency while reducing extraction costs, thus facilitating subsequent research and clinical applications. Summary of the Invention

[0011] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a plasma cell-free DNA extraction kit, which has high extraction efficiency, is simple and rapid, and has low cost.

[0012] The present invention also proposes a method for extracting cell-free DNA from blood plasma.

[0013] The present invention also proposes the application of the plasma cell-free DNA extraction kit or the plasma cell-free DNA extraction method in the preparation of plasma methylation markers.

[0014] According to one aspect of the present invention, a plasma cell-free DNA extraction kit is provided, comprising magnetic beads, lysis buffer, proteinase K, and binding buffer; wherein the lysis buffer comprises Tris-HCl, ethylenediaminetetraacetic acid (EDTA), polysorbate-20 (Tween20), and polyethylene glycol octylphenyl ether (Triron X-100).

[0015] In some embodiments of the present invention, the lysis buffer comprises: 1-2 M Tris-HCl with a pH of 8.0-9.0, 0.5-1 M ethylenediaminetetraacetic acid with a pH of 8.0-9.0, polysorbate-20 with a mass concentration of 1-30%, polyethylene glycol octylphenyl ether with a mass concentration of 0.1-3%, and anhydrous ethanol with a mass concentration of 30-40%.

[0016] In some embodiments of the present invention, the total volume of the lysis solution is 50 mL.

[0017] In some embodiments of the present invention, the magnetic beads are used to adsorb DNA and participate in the reaction in a suspension state.

[0018] In some embodiments of the present invention, the magnetic beads include any one of hydroxyl magnetic beads, amino magnetic beads, and silica gel membrane magnetic beads.

[0019] In some preferred embodiments of the present invention, the magnetic beads are selected from the hydroxyl magnetic beads.

[0020] In some embodiments of the present invention, the magnetic beads are added as a magnetic bead suspension with a volume of 50–100 μL.

[0021] In some embodiments of the present invention, the proteinase K is used to degrade proteins and inactivate nucleases (DNase and RNase).

[0022] In some embodiments of the present invention, the concentration of proteinase K is 20-21 mg / mL.

[0023] In some preferred embodiments of the present invention, the concentration of proteinase K is 20 mg / mL.

[0024] In some embodiments of the present invention, the volume of the proteinase K is 50–100 μL.

[0025] In some embodiments of the present invention, the binding solution comprises: 5-7.5M guanidine hydrochloride or 3M guanidine isothiocyanate, and anhydrous ethanol with a mass concentration of 30-45%.

[0026] In some embodiments of the present invention, the plasma cell-free DNA extraction kit further includes a washing solution and an elution solution.

[0027] In some embodiments of the present invention, the cleaning fluid includes a first cleaning fluid and a second cleaning fluid.

[0028] In some embodiments of the present invention, the first cleaning solution comprises: 0.1-10M guanidine isothiocyanate, 0.1-1M Tris-HCl with a pH of 8.0-9.0, 10-500mM ethylenediaminetetraacetic acid (EDTA), 7-9M sodium perchlorate, and anhydrous ethanol or isopropanol with a mass concentration of 20-40%.

[0029] In some embodiments of the present invention, the second cleaning solution comprises: 1-5000 mM Tris-HCl and anhydrous ethanol with a mass concentration of 70-80%.

[0030] In some embodiments of the invention, the elution buffer is used to elute DNA from a plasma sample from the magnetic beads.

[0031] In some embodiments of the present invention, the eluent comprises: 1-15 mM Tris-HCl and 1-15 mM TE buffer.

[0032] In some embodiments of the present invention, the pH of the eluent is 8.0 to 9.0.

[0033] In some embodiments of the present invention, the TE buffer is a buffer solution prepared from Tris and EDTA.

[0034] According to a second aspect of the present invention, a method for extracting cell-free DNA from plasma is provided, comprising the following steps using the aforementioned cell-free DNA extraction kit:

[0035] S1: Mix the plasma sample with the lysis buffer and proteinase K to obtain the first mixture;

[0036] S2: Add magnetic beads and binding liquid to the first mixture to obtain a second mixture;

[0037] S3: Magnetic adsorption is applied to the second mixture to extract the plasma-free DNA.

[0038] In some embodiments of the present invention, in step S1, the plasma sample is mixed with the lysis buffer and the proteinase K, and then incubated at 45-60°C for 20-30 min to obtain the first mixture.

[0039] In some embodiments of the present invention, the lysis buffer comprises: 1-2 M Tris-HCl with a pH of 8.0-9.0, 0.5-1 M ethylenediaminetetraacetic acid with a pH of 8.0-9.0, polysorbate-20 with a mass concentration of 1-30%, polyethylene glycol octylphenyl ether with a mass concentration of 0.1-3%, and anhydrous ethanol with a mass concentration of 30-40%.

[0040] In some embodiments of the present invention, the concentration of proteinase K is 20-21 mg / mL.

[0041] In some embodiments of the present invention, the concentration of proteinase K is 20 mg / mL.

[0042] In some embodiments of the present invention, the volume of the proteinase K is 50–100 μL.

[0043] In some embodiments of the present invention, the magnetic beads in step S2 are added in the form of a magnetic bead suspension, the volume of which is 50-100 μL.

[0044] In some embodiments of the present invention, the binding solution comprises: 5-7.5M guanidine hydrochloride or 3M guanidine isothiocyanate, and anhydrous ethanol with a mass concentration of 30-45%.

[0045] In some embodiments of the present invention, in addition to adding the magnetic beads and the binding solution, step S2 also includes adding vector RNA or Poly A and anhydrous ethanol.

[0046] In some embodiments of the present invention, the second mixture includes a magnetic bead-DNA complex.

[0047] In some embodiments of the present invention, after the second mixture is magnetically adsorbed in step S3, a first precipitate containing the magnetic bead-DNA complex is collected; the first precipitate is washed, magnetically adsorbed, and a second precipitate containing the magnetic bead-DNA complex is collected; the second precipitate is washed, magnetically adsorbed, and a third precipitate containing the magnetic bead-DNA complex is collected.

[0048] In some embodiments of the present invention, step S3 further includes washing the third precipitate, magnetically adsorbing and collecting a fourth precipitate containing the magnetic bead-DNA complex; washing the fourth precipitate, magnetically adsorbing and collecting a fifth precipitate containing the magnetic bead-DNA complex; magnetically adsorbing the fifth precipitate, collecting the magnetic bead-DNA complex, and drying the magnetic bead-DNA complex.

[0049] In some embodiments of the present invention, step S3 further includes adding an elution buffer, incubating the magnetic bead-DNA complex, magnetically adsorbing the magnetic beads, collecting the elution buffer, and obtaining the plasma free DNA.

[0050] In some embodiments of the present invention, the first precipitate and the second precipitate are cleaned using a first cleaning solution; the first cleaning solution comprises: 0.1-1M guanidine isothiocyanate, 0.1-1M Tris-HCl with a pH of 8.0-9.0, 10-500mM ethylenediaminetetraacetic acid, 7-9M sodium perchlorate, and anhydrous ethanol or isopropanol with a mass concentration of 20-40%.

[0051] In some embodiments of the present invention, the third and fourth precipitates are cleaned using a second cleaning solution; the second cleaning solution comprises: 1-5000 mM Tris-HCl and anhydrous ethanol with a mass concentration of 70-80%.

[0052] In some embodiments of the present invention, the eluent comprises: 1-15 mM Tris-HCl and 1-15 mM TE buffer.

[0053] In some embodiments of the present invention, the pH of the eluent is 8.0 to 9.0.

[0054] In some embodiments of the present invention, the elution buffer and the magnetic bead-DNA complex are incubated at 50-65°C for 5-10 minutes.

[0055] According to a third aspect of the present invention, the application of the plasma cell-free DNA extraction kit or the plasma cell-free DNA extraction method in the preparation of plasma methylation markers is proposed.

[0056] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved:

[0057] This invention utilizes a plasma cell-free DNA extraction kit. First, plasma sample, lysis buffer, and proteinase K are added and thoroughly mixed. The mixture is then lysed at 45–60°C. After treatment with the lysis buffer, the cfDNA in the plasma sample mixes with a magnetic bead suspension to form a magnetic bead-cfDNA complex. Then, through washing and elution processes, various impurities in the magnetic bead-cfDNA complex are removed to obtain cfDNA. The advantages of this method are: 1. It is simple, rapid, quick, and produces stable quality; 2. Anhydrous ethanol replaces isopropanol, facilitating laboratory operation, improving the working environment, and reducing costs; 3. Due to the high adsorption rate and recovery rate of nucleic acids by magnetic beads, it is beneficial for the extraction of trace amounts of ctDNA (circulating tumor DNA) from plasma; 4. The extracted DNA has high purity and is suitable for subsequent sequencing and PCR / Q-PCR applications; 5. The entire method is performed under a magnetic field, thus the magnetic bead method for cfDNA extraction is suitable for large-scale use. This method has significant implications for both research and clinical applications. Detailed Implementation

[0058] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0059] This invention provides a plasma cell-free DNA extraction kit, comprising:

[0060] Lysis buffer for pretreatment of plasma samples: 1–2 M Tris-HCl, pH 8.0–9.0; 0.5–1 M EDTA, pH 8.0–9.0; 1–30% polysorbate-20; 0.1–3% polyethylene glycol octylphenyl ether; 30–40% anhydrous ethanol; add water to make up to a total volume of 50 mL.

[0061] Proteinase K, used to degrade proteins and inactivate nucleases, has a concentration of 20 mg / mL.

[0062] Magnetic beads: Hydroxyl magnetic beads, added in the form of a magnetic bead suspension, with an addition volume of 50-100 μL;

[0063] The binding solution used to adsorb DNA from plasma samples is: 5-7.5M guanidine hydrochloride or 3M guanidine isothiocyanate, and anhydrous ethanol with a mass concentration of 30-45%.

[0064] The first cleaning solution used for washing consists of: 0.1–10 M guanidine isothiocyanate, 0.1–1 M Tris-HCl with a pH of 8.0–9.0, 10–500 mM ethylenediaminetetraacetic acid (EDTA), 7–9 M sodium perchlorate, and anhydrous ethanol or isopropanol with a mass concentration of 20–40%.

[0065] The second cleaning solution used for washing: 1-5000mM Tris-HCl, and anhydrous ethanol with a mass concentration of 70-80%;

[0066] Elution buffer for elution: 1–15 mM Tris-HCl, 1–15 mM TE buffer, pH 8.0–9.0.

[0067] This invention also provides a method for extracting cell-free DNA from plasma, using the above-mentioned kit for DNA extraction, comprising the following steps:

[0068] (1) In a nuclease-free centrifuge tube, add 3 mL of plasma sample, 50–100 μL of proteinase K, and 1–2 mL of lysis buffer in sequence. Vortex for 30 seconds and then briefly centrifuge to obtain the first mixture.

[0069] (2) After incubating the first mixture at 45-60°C for 20-30 min, add 2-4 mL of binding solution, 5-10 μL of Poly A, 4-6 mL of anhydrous ethanol, and 50-100 μL of magnetic bead suspension to obtain a second mixture containing magnetic bead-DNA complex.

[0070] (3) After briefly centrifuging the second mixture, place it on a magnetic rack for magnetic adsorption for 2 min, remove the liquid from the centrifuge tube, and collect the first precipitate containing the magnetic bead-DNA complex; transfer the first precipitate to a new centrifuge tube, add 1 mL of the first washing solution, vortex for 10 s, and briefly centrifuge to obtain the third mixture; place the centrifuge tube containing the third mixture on a magnetic rack for magnetic adsorption for 2 min, remove the liquid from the centrifuge tube, and collect the second precipitate containing the magnetic bead-DNA complex; add 1 mL of the first washing solution to the second precipitate, vortex for 10 s, and briefly centrifuge to obtain the fourth mixture; place the centrifuge tube containing the fourth mixture on a magnetic rack for magnetic adsorption for 2 min, remove the liquid from the centrifuge tube, and collect the third precipitate containing the magnetic bead-DNA complex;

[0071] Add 500–750 μL of the second washing solution to the third precipitate, vortex for 10 seconds, and then briefly centrifuge to obtain the fifth mixture. Place the centrifuge tube containing the fifth mixture on a magnetic rack for magnetic adsorption for 2 minutes, discard the liquid in the centrifuge tube, and collect the fourth precipitate containing the magnetic bead-DNA complex. Add 500–750 μL of the second washing solution to the fourth precipitate, vortex for 10 seconds, and then briefly centrifuge to obtain the sixth mixture. Place the centrifuge tube containing the sixth mixture on a magnetic rack for magnetic adsorption for 2 minutes, discard the liquid in the centrifuge tube, and collect the fifth precipitate containing the magnetic bead-DNA complex. Vortex the fifth precipitate, briefly centrifuge, place it on a magnetic rack for magnetic adsorption for 1 minute, discard the residual liquid in the centrifuge tube, collect the magnetic bead-DNA complex, and dry the magnetic bead-DNA complex at 45–55 °C for 10–15 minutes.

[0072] Add 25–50 μL of elution buffer to the dried magnetic bead-DNA complex, vortex for 30 seconds to mix thoroughly to obtain the seventh mixture, and incubate the seventh mixture at 50–65 °C for 5–10 min; after incubation, vortex the seventh mixture and centrifuge briefly, place it on a magnetic rack for magnetic adsorption for 2 min, collect the elution buffer, and transfer it to a new nuclease-free centrifuge tube to obtain cell-free plasma DNA.

[0073] The above-mentioned plasma cell-free DNA extraction kit and method can be used to prepare and detect methylation markers of tumor genes.

[0074] Example 1

[0075] This embodiment compares the DNA extraction efficiency with and without the addition of anhydrous ethanol or isopropanol to the lysis buffer.

[0076] In this embodiment, a plasma extraction procedure was simulated. 60 ng of ultrasonic DNA was mixed with 3 mL of plasma to simulate plasma extraction. At the same time, anhydrous ethanol / isopropanol was added to the lysis buffer, with or without it. The extraction efficiency was calculated and compared with the results.

[0077] In this embodiment, the lysis buffer includes: 1-2M Tris-HCl with a pH of 8.0-9.0, 0.5-1M ethylenediaminetetraacetic acid with a pH of 8.0-9.0, polysorbate-20 with a mass concentration of 1-30%, polyethylene glycol octylphenyl ether with a mass concentration of 0.1-3%, with no added or anhydrous ethanol or isopropanol added; water is added to make up the total volume to 50 mL;

[0078] The binding solution comprises: 5-7.5M guanidine hydrochloride or 3M guanidine isothiocyanate dissolved in 1L of water, and anhydrous ethanol with a mass concentration of 30-45%.

[0079] Magnetic beads: Hydroxyl magnetic beads, added in the form of a magnetic bead suspension, with an addition volume of 50-100 μL;

[0080] The first cleaning solution includes: 0.1-10M guanidine isothiocyanate, 0.1-1M Tris-HCl with a pH of 8.0-9.0, 10-500mM ethylenediaminetetraacetic acid (EDTA), 7-9M sodium perchlorate, and anhydrous ethanol or isopropanol with a mass concentration of 20-40%.

[0081] The second cleaning solution includes: 1-5000 mM Tris-HCl and anhydrous ethanol with a mass concentration of 70-80%.

[0082] The elution buffer consists of 1–15 mM Tris-HCl, 1–15 mM TE buffer, and pH 8.0–9.0.

[0083] The method for extracting cell-free DNA from plasma includes the following steps:

[0084] (1) Take three 15mL centrifuge tubes and add 3mL of plasma to be processed to each tube. The three centrifuge tubes are numbered 1 (without anhydrous ethanol or isopropanol), 2 (with isopropanol), and 3 (with anhydrous ethanol).

[0085] (2) Add 50-100 μL of proteinase K and 1-3 mL of lysis buffer to the centrifuge tubes respectively. Incubate the mixture in a water bath at 45-60°C for 20-30 min. Then add 2-4 mL of binding buffer, 5-10 μL of Poly A, 0 or 4.5 mL of anhydrous ethanol / isopropanol and 50-100 μL of magnetic bead suspension. Vortex for 30 s.

[0086] (3) Place the 15mL centrifuge tube on the magnetic rack and magnetically adsorb for 5 minutes. Discard the supernatant. Remove the centrifuge tube from the magnetic rack, add 1000μL of the first cleaning solution, vortex for 10 seconds and then briefly centrifuge. Transfer all the liquid in the centrifuge tube to a 1.5mL centrifuge tube.

[0087] (4) Place the centrifuge tube on a magnetic rack for 2 minutes, transfer all the supernatant to the 15 mL centrifuge tube, and wash off the magnetic beads on the wall of the 15 mL centrifuge tube as much as possible. Vortex the 15 mL centrifuge tube for 10 seconds and then centrifuge briefly. Transfer the liquid in the centrifuge tube to a 1.5 mL centrifuge tube.

[0088] (5) Place the 1.5 mL centrifuge tube on a magnetic rack for 2 min and discard the supernatant; remove the 1.5 mL centrifuge tube from the magnetic rack, add 500-750 μL of the second washing solution, vortex for 10 s and then briefly centrifuge, place the centrifuge tube on a magnetic rack for 2 min and discard the supernatant; repeat the addition of the second washing solution once; after briefly centrifuging the centrifuge tube, place it on a magnetic rack for 1 min, remove as much of the residual supernatant as possible, and dry the centrifuge tube at 45-55℃ for 10-15 min;

[0089] (6) Add 25 μL of elution buffer to a centrifuge tube, vortex for 30 seconds to mix evenly, and incubate at 50-65°C for 5-10 minutes. After incubation, vortex the mixture briefly, place it on a magnetic rack for magnetic adsorption for 2 minutes, collect the elution buffer and transfer it to a new nuclease-free centrifuge tube to obtain plasma free DNA.

[0090] The concentration of DNA extracted by the above method was determined using ThermoFisher Qubit 4 and HS dsDNA quantification kit, and the recovery rates were calculated and compared. The results are shown in Table 1.

[0091] Table 1

[0092]

[0093] As shown in Table 1, in this method, the DNA extraction efficiency after adding isopropanol or anhydrous ethanol to the lysis buffer was 2.86 times and 3.64 times higher than that without adding isopropanol or anhydrous ethanol, respectively. It can be seen that the extraction efficiency after adding isopropanol or anhydrous ethanol to the lysis buffer is better than that without adding isopropanol or anhydrous ethanol, and the extraction efficiency is higher when adding anhydrous ethanol.

[0094] Example 2

[0095] This example compares the DNA extraction efficiency after adding different amounts of anhydrous ethanol to the lysis buffer.

[0096] In this embodiment, a plasma extraction procedure was simulated. 60 ng of ultrasonic DNA was mixed with 3 mL of plasma to simulate plasma extraction. At the same time, different volumes of anhydrous ethanol were added to the lysis buffer. The extraction efficiency was calculated and compared with the results.

[0097] In this embodiment, the lysis buffer includes: 1–2 M Tris-HCl with a pH of 8.0–9.0, 0.5–1 M ethylenediaminetetraacetic acid with a pH of 8.0–9.0, polysorbate-20 with a mass concentration of 1–30%, polyethylene glycol octylphenyl ether with a mass concentration of 0.1–3%, and different volumes of anhydrous ethanol; water is added to make up the total volume to 50 mL. The remaining reagents are the same as in Example 1.

[0098] The method for extracting cell-free DNA from plasma in this embodiment is the same as in Embodiment 1.

[0099] The concentration of DNA extracted by the above method was determined using ThermoFisher Qubit 4 and HS dsDNA quantification kit, and the recovery rates were calculated and compared. The results are shown in Table 2.

[0100] Table 2

[0101]

[0102] As shown in Table 2, the extraction efficiencies of adding different volumes of anhydrous ethanol to the lysis buffer in this method, with 4.5 mL as a reference, were 4.05 times, 2.58 times, 2.31 times, and 3.17 times that of 4.5 mL, respectively. This indicates that the extraction efficiency is optimal when 5.0 mL of anhydrous ethanol is added to the lysis buffer.

[0103] Example 3

[0104] This example compares the DNA extraction efficiency after adding isopropanol and anhydrous ethanol to the lysis buffer.

[0105] In this embodiment, a plasma extraction process was simulated. 15 ng or 60 ng of ultrasonic DNA was mixed with 3 mL of PBS or 3 mL of plasma to simulate plasma extraction. At the same time, isopropanol or anhydrous ethanol was added to the lysis buffer, and the extraction efficiency was calculated and compared.

[0106] In this embodiment, the lysis buffer includes: 1–2 M Tris-HCl with a pH of 8.0–9.0, 0.5–1 M ethylenediaminetetraacetic acid with a pH of 8.0–9.0, polysorbate-20 with a mass concentration of 1–30%, polyethylene glycol octylphenyl ether with a mass concentration of 0.1–3%, and isopropanol or anhydrous ethanol; water is added to make up the total volume to 50 mL. The remaining reagents are the same as in Example 1.

[0107] The method for extracting cell-free DNA from plasma in this embodiment is the same as in Embodiment 1.

[0108] The concentration of DNA extracted by the above method was determined using ThermoFisher Qubit 4 and HS dsDNA quantification kit, and the recovery rates were calculated and compared. The results are shown in Table 3.

[0109] Table 3

[0110]

[0111] As shown in Table 3, compared with the DNA extraction efficiency of adding isopropanol, the extraction efficiencies of adding anhydrous ethanol were 1.125 times, 1.098 times, 1.081 times, and 1.062 times that of adding isopropanol, respectively. This indicates that the extraction efficiency of adding anhydrous ethanol to the lysis buffer is superior to that of adding isopropanol.

[0112] Example 4

[0113] This embodiment compares the extraction efficiency of DNA extracted using magnetic beads from different companies.

[0114] In this embodiment, a simulated plasma extraction process was performed. 15 ng of ultrasonic DNA was mixed with 3 mL of PBS or 3 mL of plasma to simulate plasma extraction. The extraction efficiency was compared with the results obtained from plasma DNA extraction using magnetic bead extraction kits from Company A and Company B. Specifically, Company A's magnetic bead extraction kit (catalog number DP710-01, Tiangen Biotech Co., Ltd., Beijing) is for large-volume plasma cell-free DNA extraction using magnetic beads; Company B's magnetic bead extraction kit (BSC95S1, Bori Technology Co., Ltd., Hangzhou) is for plasma cell-free DNA extraction.

[0115] In this embodiment, the lysis buffer includes: 1–2 M Tris-HCl with a pH of 8.0–9.0, 0.5–1 M ethylenediaminetetraacetic acid with a pH of 8.0–9.0, polysorbate-20 with a mass concentration of 1–30%, polyethylene glycol octylphenyl ether with a mass concentration of 0.1–3%, and anhydrous ethanol with a mass concentration of 30–40%; water is added to make up the total volume to 50 mL. The remaining reagents are the same as in Example 1.

[0116] The method for extracting cell-free DNA from plasma in this embodiment is the same as in Embodiment 1.

[0117] The concentration of DNA extracted using the ThermoFisher Qubit 4 and HS dsDNA quantification kit was determined, and the recovery rates were calculated and compared. The extraction rate was calculated using the formula: (b-b0) / a, where b is the total amount of DNA extracted using this method or a kit from Company A or Company B, b0 is the total amount of DNA extracted from the blank control, and a is the total amount of DNA before extraction. For example, the extraction rate in item 1 was calculated as: (13.4-1.3) / 15 = 80.7%. The results are shown in Table 4.

[0118] Table 4

[0119]

[0120] As shown in Table 4, the DNA extraction efficiencies using this method are 80.7% and 78.7%, respectively, while the extraction efficiencies using commercially available magnetic bead extraction kits from companies A and B are 42.7% and 40%, and 52.7% and 52%, respectively. Therefore, the extraction efficiency of this method is superior to that of other commercially available magnetic bead extraction kits.

[0121] Example 5

[0122] This embodiment compares the extraction efficiency of DNA extracted using column extraction methods from different companies.

[0123] In this embodiment, plasma samples from different patient sources were used to extract cfDNA from 3 mL of patient plasma samples. Simultaneously, the extraction efficiency was compared with the results obtained using a column-based kit from Company C (D3182-03S, Meiji Technology Co., Ltd., Guangzhou) as a control.

[0124] In this embodiment, the lysis buffer includes: 1–2 M Tris-HCl with a pH of 8.0–9.0, 0.5–1 M ethylenediaminetetraacetic acid with a pH of 8.0–9.0, polysorbate-20 with a mass concentration of 1–30%, polyethylene glycol octylphenyl ether with a mass concentration of 0.1–3%, and anhydrous ethanol with a mass concentration of 30–40%; water is added to make up the total volume to 50 mL. The remaining reagents are the same as in Example 1.

[0125] The method for extracting cell-free DNA from plasma in this embodiment is the same as in Embodiment 1.

[0126] The concentration of DNA extracted by the above method was determined using ThermoFisher Qubit 4 and HS dsDNA quantification kit, and the recovery rates were calculated and compared. The results are shown in Table 5.

[0127] Table 5

[0128]

[0129] As shown in Table 5, compared with the extraction efficiency of Company C's column-based extraction kit, the extraction efficiencies of this method are 2.91 times, 2.34 times, 2.85 times, and 2.54 times that of Company C's column-based extraction method, respectively. Therefore, the extraction efficiency of this method is superior to that of other column-based extraction kits available on the market.

[0130] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A plasma cell-free DNA extraction kit, characterized by, The magnetic beads, the lysis solution, the proteinase K and the binding solution are included.

2. The plasma cell-free DNA extraction kit of claim 1, wherein, The concentration of the proteinase K is 20-21 mg / mL.

3. The plasma cell-free DNA extraction kit of claim 1, wherein, The magnetic beads are selected from hydroxyl magnetic beads, and the magnetic beads are added in the form of magnetic bead suspension with a volume of 50-100 µL.

4. The plasma cell-free DNA extraction kit of claim 1, wherein, The binding solution includes 5-7.5 M guanidine hydrochloride or 3 M guanidine isothiocyanate and 30-45% anhydrous ethanol by mass concentration.

5. The plasma cell-free DNA extraction kit of claim 1, wherein, The plasma free DNA extraction kit further includes a washing solution and an elution solution. The washing solution includes a first washing solution and a second washing solution. The first washing solution includes 0.1-10 M guanidine isothiocyanate, 0.1-1 M Tris-HCl with a pH of 8.0-9.0, 10-500 mM ethylenediaminetetraacetic acid, 7-9 M sodium perchlorate, 20-40% anhydrous ethanol or isopropanol by mass concentration. The second washing solution includes 1-5000 mM Tris-HCl and 70-80% anhydrous ethanol by mass concentration.

6. The plasma cell-free DNA extraction kit of claim 5, wherein, The elution solution includes 1-15 mM Tris-HCl and 1-15 mM TE buffer.

7. A method for extracting plasma cell-free DNA, characterized by, The pH of the elution solution is 8.0-9.

0. The plasma free DNA extraction kit is used for extraction, and the method includes the following steps: S1: mixing a plasma sample with a lysis solution and a proteinase K to obtain a first mixed solution; S2: adding magnetic beads and a binding solution to the first mixed solution to obtain a second mixed solution; 8. The extraction method of claim 7, wherein, S3: performing magnetic adsorption on the second mixed solution to extract the plasma free DNA.

9. The extraction method according to claim 7, characterized in that, After the plasma sample is mixed with the lysis solution and the proteinase K in step S1, the first mixed solution is obtained by incubation at 45-60 °C for 20-30 min.

10. The extraction method of claim 7, wherein, In step S2, in addition to the magnetic beads and the binding solution, carrier RNA or Poly A and anhydrous ethanol are also added.

11. The extraction method of claim 10, wherein, After the magnetic adsorption on the second mixed solution in step S3, a first precipitate containing magnetic bead-DNA complexes is collected; the first precipitate is washed, and magnetic adsorption is performed to collect a second precipitate containing magnetic bead-DNA complexes; the second precipitate is washed, and magnetic adsorption is performed to collect a third precipitate containing magnetic bead-DNA complexes.

12. The extraction method of claim 7, wherein, Step S3 further includes washing the third precipitate, performing magnetic adsorption, collecting a fourth precipitate containing magnetic bead-DNA complexes, washing the fourth precipitate, performing magnetic adsorption, collecting a fifth precipitate containing magnetic bead-DNA complexes, performing magnetic adsorption on the fifth precipitate, collecting magnetic bead-DNA complexes, and drying the magnetic bead-DNA complexes. Step S3 further includes adding an elution solution, incubating the elution solution with the magnetic bead-DNA complexes, performing magnetic adsorption, and collecting the elution solution, thereby obtaining the plasma free DNA.

13. The extraction method of claim 12, wherein, Incubate the eluent and the magnetic bead-DNA complex at 50-65℃ for 5-10 min.

14. The extraction method of claim 10, wherein, Wash the first precipitate and the second precipitate with the first washing solution.

15. The extraction method of claim 10, wherein, Wash the third precipitate and the fourth precipitate with the second washing solution.

16. Use of the plasma cell-free DNA extraction kit of any one of claims 1-6 or the plasma cell-free DNA extraction method of any one of claims 7-15 in the preparation of plasma methylation markers.

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