A cfDNA extraction kit and extraction method

Abstract

The application provides a cfDNA extraction kit and an extraction method. The cfDNA extraction kit provided by the application comprises a first binding liquid, a second binding liquid and magnetic microspheres. By limiting the components and contents of the first binding liquid and the second binding liquid, a good matching effect can be achieved between the first binding liquid, the second binding liquid and the magnetic microspheres. Then, the magnetic microsphere binding environment is regulated in stages, the bottleneck of the traditional single-step magnetic bead method is broken, the extraction efficiency and the extraction purity of the cfDNA are effectively improved, and the operation is simple and highly repeatable. The application provides reliable technical support for liquid biopsy, and is suitable for application scenarios such as tumor liquid biopsy and non-invasive prenatal diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a cfDNA extraction kit and extraction method. BACKGROUND

[0002] Cell-free DNA (cfDNA) refers to DNA fragments free in the extracellular environment, with a length of 100 to 200 base pairs, which is mainly released into the extracellular environment of the human body through apoptosis, necrosis and other pathways, and is often present in blood, lymph, milk, urine and amniotic fluid and other physiological extracellular environments. At present, cfDNA detection is a common form of liquid biopsy on the market, which has been widely used in tumor detection, guidance evaluation, prognosis evaluation and other aspects. However, cfDNA has low content and small fragments, and is difficult to extract, and the extraction process is prone to deletion, resulting in low detection sensitivity, which to some extent limits its application in clinical diagnosis.

[0003] At present, the cfDNA extraction method can be mainly divided into traditional phenol / trichloromethane extraction, silica gel membrane adsorption column method and magnetic bead extraction method. Among them, the magnetic bead extraction method has the advantages of simple operation, short time consumption, easy automation and high-throughput processing compared with the first two extraction methods, which can directly extract cfDNA from crude samples such as blood plasma, reduces the pretreatment step, and has small shearing force on DNA, which helps to maintain the integrity of cfDNA. However, the existing magnetic bead extraction kit is mainly a single-step extraction method, and its extraction efficiency is often poor, and the purity of the extracted cfDNA is generally low, which leads to insufficient sensitivity of downstream detection. Therefore, it is urgent to develop a cfDNA extraction kit with high extraction efficiency and high purity of cfDNA. SUMMARY

[0004] The present application provides a cfDNA extraction kit, which can effectively improve the purity and content of cfDNA.

[0005] The present application provides a cfDNA extraction method, which is simple and suitable for high-throughput processing.

[0006] The present application provides a cfDNA extraction kit, which comprises a first binding solution, a second binding solution and magnetic microspheres.

[0007] The magnetic microspheres comprise first magnetic microspheres and second magnetic microspheres; the first magnetic microspheres and the second magnetic microspheres have a core-shell structure, comprising a ferroferric oxide core and a silica shell layer coated on at least part of the core surface, and the silica shell layer is modified with carboxyl groups;

[0008] The pH of the first binding solution is 4-5, which comprises 0.5-1.5 M guanidine hydrochloride and 5-15% (w / v) polyethylene glycol 8000;

[0009] The pH of the second binding solution is 6.5-7.5, and the second binding solution comprises 2-4 M guanidine hydrochloride, 10-30% (v / v) isopropanol, and 5-15 g / L β-cyclodextrin.

[0010] The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises proteinase K.

[0011] The concentration of the proteinase K is 0.1-2 mg / mL.

[0012] The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises a lysis solution.

[0013] The lysis solution comprises 5-7 M guanidine hydrochloride and 0.5-1.5% (v / v) triton X-100.

[0014] The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises a washing solution.

[0015] The washing solution comprises 70-90% (v / v) ethanol and 0.1-0.5 M sodium acetate with a pH of 5.0-6.0.

[0016] The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises an elution solution.

[0017] The elution solution comprises a TE buffer with a pH of 7.9-8.1.

[0018] The cfDNA extraction kit as described above, wherein the carboxyl density of the first magnetic microspheres and the second magnetic microspheres is 1-5 μmol / m².

[0019] The cfDNA extraction kit as described above, wherein the particle size of the first magnetic microspheres and the second magnetic microspheres is 0.5-1.5 μm.

[0020] The cfDNA extraction kit as described above, wherein the first magnetic microspheres and the second magnetic microspheres are suspended in a phosphate buffered saline solution or a tris-hydroxymethyl aminomethane hydrochloride buffer with a pH of 7.0-8.0, and the concentration of the first magnetic microspheres and the second magnetic microspheres is 5-15 mg / mL.

[0021] The present application provides a cfDNA extraction method, wherein the cfDNA extraction kit described above is used, and the method comprises the following steps:

[0022] The body fluid sample is lysed to obtain a lysate; the lysate, the first binding solution, and the first magnetic microspheres are mixed to obtain the first magnetic microspheres carrying impurities; and the first magnetic microspheres carrying impurities are removed to obtain a supernatant.

[0023] Mixing the supernatant, the second binding solution and the second magnetic microspheres to obtain the second magnetic microspheres carrying the cfDNA, and washing and eluting the second magnetic microspheres to obtain the cfDNA.

[0024] The cfDNA extraction method as described above, wherein the volume ratio of the first binding solution to the first magnetic microspheres is (35-45): 1; and / or,

[0025] The volume ratio of the second binding solution to the second magnetic microspheres is (35-45): 1; and / or,

[0026] The volume ratio of the first binding solution to the second binding solution is (1-2):(1-2); and / or,

[0027] The body fluid sample is selected from at least one of blood, lymph, milk, urine and amniotic fluid.

[0028] The present application provides a cfDNA extraction kit, comprising a first binding solution, a second binding solution and magnetic microspheres. By limiting the components and contents of the first binding solution and the second binding solution, a good matching effect with the magnetic microspheres can be achieved. Then, by regulating the magnetic microsphere binding environment in stages, the bottleneck of the traditional single-step magnetic bead method is broken, and the extraction efficiency and purity of cfDNA are effectively improved. The present application also has the advantages of simple operation and high repeatability. The present application provides reliable technical support for liquid biopsy, and is suitable for tumor liquid biopsy and non-invasive prenatal diagnosis and other application scenarios. DETAILED DESCRIPTION

[0029] In order for those skilled in the art to better understand the scheme of the present application, the present application will be further described in detail below. The following specific embodiments are only used to describe the principles and characteristics of the present application, and the examples are used to explain the present application, but not to limit the scope of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0030] The magnetic bead method for extracting cell-free DNA (cfDNA) is one of the mainstream cfDNA extraction technologies at present, and is especially suitable for liquid biopsy samples such as plasma and serum. The core principle is to use surface functionalized magnetic microspheres to selectively adsorb cfDNA under specific buffer conditions, and then to realize purification through magnetic field separation. However, the fragment length of cfDNA is generally 100-200 bp, and such short fragments carry less negative charge, so their binding force with magnetic microspheres is weak, which causes the existing magnetic bead method to have low binding efficiency for cfDNA with a fragment length of 100-200 bp, thereby affecting the extraction efficiency and purity of cfDNA. At the same time, the magnetic microspheres also have the problem of non-specific adsorption of long fragment genomic DNA, which further increases the difficulty of extracting cfDNA by the magnetic bead method.

[0031] To solve the above problems, the first aspect of the present application provides a cfDNA extraction kit, comprising a first binding liquid, a second binding liquid and magnetic microspheres.

[0032] In the present application, the magnetic microspheres include first magnetic microspheres and second magnetic microspheres, and the first magnetic microspheres and the second magnetic microspheres are magnetic microspheres with the same structure, but different uses. Among them, the first magnetic microspheres and the second magnetic microspheres are core-shell structures, including a ferroferric oxide core and a silica shell layer coated on at least part of the core surface, and the silica shell layer is modified with carboxyl groups.

[0033] In the cfDNA extraction process, the magnetic microspheres in combination with the first binding liquid or the second binding liquid can provide two different purposes: (1) capturing impurities: under the cooperation of the first binding liquid, the first magnetic microspheres can capture impurities, and then under the action of an external magnetic field, the first magnetic microspheres capturing impurities can be easily separated from the solution; (2) capturing cfDNA: under the cooperation of the second binding liquid, the second magnetic microspheres can capture cfDNA, and then under the action of an external magnetic field, the second magnetic microspheres capturing cfDNA can be easily separated from the solution. Through the setting of the first binding liquid and the second binding liquid, the objects captured by the magnetic microspheres can be regulated, so as to realize the staged separation of impurities and cfDNA.

[0034] In the present application, the first binding liquid can drive proteins, lipids and other impurities to be adsorbed on the surface of the first magnetic microspheres. The pH of the first binding liquid is 4.0-5.0, and it includes 0.5-1.5 M guanidine hydrochloride and 5-15% (w / v) polyethylene glycol 8000.

[0035] Firstly, under the acidic condition (pH 4-5) provided by the first binding solution, the carboxyl groups (-COOH) on the surface of the first magnetic microspheres will be protonated, resulting in a decrease in the negative charge (-COO-) on the surface of the first magnetic microspheres. Although the net negative charge density of the first magnetic microspheres decreases, the overall negative charge is still dominant. Under acidic conditions, the amino groups (-NH2) carried by impurities such as proteins and lipids will be protonated to form positively charged ammonium groups (-NH3+), causing the overall positive charge of the impurities. cfDNA is composed of nucleotides, and its phosphate backbone carries a negative charge (-PO4 3-), so even under acidic conditions, the net negative charge density decreases slightly, but the overall negative charge is still dominant. Therefore, positively charged impurities will be preferentially attracted to the remaining negative charge on the surface of the first magnetic microspheres, while negatively charged cfDNA will be repelled by the negative charge on the surface of the first magnetic beads.

[0036] Secondly, the first binding solution contains a low concentration (0.5-1.5 M) of guanidine hydrochloride. Low concentrations of guanidine hydrochloride can reduce the ionic strength of the system, thereby reducing the competitive adsorption of salt ions on the surface of the first magnetic microspheres. At this time, the interaction between impurity molecules (such as proteins) and the surface of the first magnetic microspheres, such as hydrophobic interaction and hydrogen bonding, dominates, and is more easily adsorbed on the surface of the first magnetic microspheres compared to cfDNA. At the same time, low concentrations of guanidine hydrochloride can effectively prevent the destruction of impurity structures, allowing them to maintain their natural overall conformation, thereby making it easier for them to bind to the first magnetic microspheres through surface functional groups. For example, the hydrophobic regions of proteins are more easily exposed under low concentrations of guanidine hydrochloride, and their hydrophobic interaction with the surface of the magnetic beads is enhanced, thereby preferentially adsorbing.

[0037] Finally, the first binding solution contains polyethylene glycol 8000. Polyethylene glycol 8000 is a water-soluble macromolecular polymer that can remove water molecules from the surface of proteins, lipids, and other impurities through the osmotic pressure effect, compressing the hydration layer of the impurities, making it easier for the impurity molecules to approach the surface of the magnetic microspheres, thereby strengthening the hydrophobic interaction and electrostatic attraction between the impurities and the first magnetic microspheres, and promoting the preferential adsorption of impurities on the first magnetic microspheres. The macromolecular structure of polyethylene glycol 8000 also forms steric hindrance in the solution, reducing the non-specific contact between negatively charged short fragment cfDNA and the remaining negative charge on the surface of the first magnetic microspheres, while avoiding the precipitation of cfDNA being wrapped by impurities, ensuring that cfDNA is stably retained in the supernatant, creating conditions for the subsequent specific capture of cfDNA by the second binding solution. Therefore, polyethylene glycol 8000 and guanidine hydrochloride in the first binding solution work together to regulate the interfacial behavior of impurities and cfDNA, achieving the effect of phased separation of preferential adsorption of impurities and efficient retention of cfDNA.

[0038] In short, the acidic environment enhances the electrostatic attraction between the impurities and the first magnetic microspheres by adjusting the charge state, while the low concentration of chaotropic salt combined with polyethylene glycol 8000 promotes the adsorption of impurities by reducing ion interference and enhancing hydrophobic interaction. Under the synergistic effect of the two, impurities (such as proteins and lipids) are preferentially bound to the surface of the first magnetic microspheres, while cfDNA remains in the supernatant due to charge repulsion and high solubility under low salt conditions, laying the foundation for the subsequent specific capture of cfDNA in the second step.

[0039] In the present application, the second binding solution can effectively promote the adsorption of cfDNA on the surface of the second magnetic microspheres. The pH of the second binding solution is 6.5-7.5, including 2-4 M guanidine hydrochloride, 10-30% (v / v) isopropanol, and 5-15 g / L β-cyclodextrin.

[0040] On the one hand, the second binding solution provides a neutral (pH 6.5-7.5) environment, in which the carboxyl groups (-COOH) on the surface of the second magnetic microspheres are deprotonated and converted into negatively charged carboxyl ions (-COO⁻), increasing the negative charge density on the surface of the second magnetic microspheres. At this time, although the phosphate backbone of cfDNA is still negatively charged (-PO4³⁻), the isopropanol in the second binding solution can neutralize part of the negative charge of cfDNA, reducing its solubility, while the β-cyclodextrin reduces the competition of lipid impurities for the hydrophobic sites of the second magnetic microspheres. Therefore, the increase in negative charge density on the surface of the second magnetic microspheres in the neutral environment, combined with the charge neutralization state of cfDNA under the action of isopropanol, makes it easier for cfDNA to bind to the second magnetic microspheres through hydrophobic interaction, ion bridge, etc., thereby achieving efficient capture of cfDNA. This is a phased regulation that preferentially adsorbs impurities in the acidic environment of the first binding solution, ultimately improving the extraction efficiency and purity of cfDNA.

[0041] On the other hand, the high concentration (2-4 M) guanidine hydrochloride in the second binding solution can destroy the binding of cfDNA and residual impurities, enhance the interaction of cfDNA with the second magnetic microspheres, and inhibit the activity of nucleases. Isopropyl alcohol (IPA) can neutralize the negative charge of cfDNA, reduce the solubility of cfDNA, thereby promoting the precipitation of cfDNA and promoting its binding to the hydrophobic region on the surface of the second magnetic microspheres; it can also dissolve impurities such as salts and small molecules, thereby improving the purity of cfDNA. β-cyclodextrin can form inclusion compounds with lipid impurities, thereby reducing the competitive adsorption of lipid impurities on the hydrophobic region on the surface of the second magnetic microspheres. Isopropyl alcohol and β-cyclodextrin have a synergistic effect through double hydrophobic interaction, which drives cfDNA to adsorb on the surface of the second magnetic microspheres in a more ordered conformation, enhances the order of cfDNA adsorption, and improves the capture specificity. Guanidine hydrochloride enhances the interaction of cfDNA with the second magnetic microspheres, and has a synergistic effect with the charge neutralization of isopropyl alcohol and the reduction of lipid competition of β-cyclodextrin, which together achieve efficient and specific capture of cfDNA and improve the extraction efficiency and purity.

[0042] Therefore, the cfDNA extraction kit provided by the present application can promote good matching effect with magnetic microspheres by limiting the components and contents of the first binding solution and the second binding solution, and then regulate the binding environment of magnetic microspheres in stages, break through the bottleneck of traditional single-step magnetic bead method, effectively improve the extraction efficiency and purity of cfDNA, and have the advantages of simple operation and high repeatability. The present application provides reliable technical support for liquid biopsy, and is suitable for tumor liquid biopsy and non-invasive prenatal diagnosis and other application scenarios.

[0043] In the above technical solution, the cfDNA extraction kit further comprises proteinase K, and the concentration of the proteinase K is 0.1-2 mg / mL.

[0044] Proteinase K is a broad-spectrum serine protease that can efficiently degrade histones, nuclear proteins and other proteins (such as hemoglobin and albumin) bound to cfDNA, thereby releasing cfDNA. In addition, proteinase K can also avoid the degradation of cfDNA during the extraction process by degrading nucleases (such as DNAase), thereby ensuring the integrity of cfDNA.

[0045] It is found through experiments that when the concentration of proteinase K in the cfDNA extraction kit is 0.1-2 mg / mL, it is suitable for most body fluid samples and can exert a better protein degradation effect, further improve the cfDNA extraction efficiency, and also avoid excessive enzyme activity and cost increase due to too high concentration. For example, the concentration of proteinase K can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, or any value within the range of any two of the above values.

[0046] In the above technical solution, the cfDNA extraction kit further comprises a lysis solution, and the lysis solution comprises 5-7 M guanidine hydrochloride and 0.5-1.5% (v / v) Triton X-100.

[0047] The lysis solution containing the above components can efficiently destroy the cell structure that may exist in the body fluid sample, release cfDNA, and inhibit nuclease activity to avoid cfDNA degradation. Among them, 5-7 M guanidine hydrochloride can destroy the hydrogen bond and hydrophobic interaction of proteins, denature histones, nuclear proteins and nucleases in the body fluid sample, and release cfDNA; 0.5-1.5% (v / v) Triton X-100 can destroy the phospholipid bilayer of the cell membrane as a non-ionic detergent, and promote cell lysis together with guanidine hydrochloride, while dissolving lipid impurities.

[0048] In the above technical solution, the cfDNA extraction kit further comprises a washing solution, and the washing solution comprises 70-90% (v / v) ethanol and 0.1-0.5 M sodium acetate with a pH of 5.0-6.0.

[0049] On the one hand, 70-90% (v / v) ethanol can reduce the solubility of cfDNA, promote the precipitation of cfDNA, and thus promote the combination of cfDNA with the second magnetic microspheres; it can also dissolve water, salt, proteins and other organic impurities in the solution, which helps to enhance the washing effect. 0.1-0.5 M sodium acetate with a pH of 5.0-6.0 is dissociated into acetate ions and sodium ions in the system, and the sodium ions neutralize the negative charges on the phosphate backbone of cfDNA, weaken the electrostatic repulsion between cfDNA molecules, and make them more easily combine with the functional groups (such as carboxyl groups) on the surface of the second magnetic microspheres, thereby reducing the loss of cfDNA during the washing process and improving its content.

[0050] On the other hand, ethanol has a synergistic effect with sodium acetate. Ethanol can reduce the dielectric constant of the solution, causing cfDNA to dehydrate and precipitate, and the presence of sodium acetate can further reduce the solubility of cfDNA, while binding residual salt ions (such as guanidine salt) through ionic bonds, allowing them to be removed with the washing step, thereby reducing impurity residues and avoiding the inhibitory effect of salt ions on downstream detection (such as PCR, sequencing), and improving experimental reliability.

[0051] In the above technical solution, the cfDNA extraction kit further comprises an eluent, and the eluent comprises a TE buffer with a pH of 7.9-8.1.

[0052] The TE buffer is a buffer composed of Tris(hydroxymethyl)aminomethane (Tris) and Ethylene Diamine Tetraacetic Acid (EDTA). The weak alkaline nature (pH 7.9-8.1) of the TE buffer can break the ionic bonds between cfDNA and the surface of the second magnetic microspheres, causing cfDNA to fall off the surface of the second magnetic microspheres, and the EDTA in it can also chelate metal ions to prevent nuclease degradation of cfDNA.

[0053] In the above technical solution, the carboxyl density of the first magnetic microspheres and the second magnetic microspheres is 1-5 μmol / m².

[0054] In the present application, in the first binding stage, impurities are mainly adsorbed on the hydrophobic region of the silica shell of the first magnetic microspheres through hydrophobic interaction; in the second binding stage, cfDNA is also mainly adsorbed on the hydrophobic region of the silica shell of the second magnetic microspheres through hydrophobic interaction.

[0055] Controlling the carboxyl density of the first magnetic microspheres and the second magnetic microspheres can optimize the degree of exposure of the hydrophobic region. Since the carboxyl group is modified on the surface of the silica shell, its density determines the balance between hydrophilic and hydrophobic levels on the surface of the shell; when the carboxyl density is too low (<1 μmol / m²), the hydrophobic region of the shell is excessively exposed, leading to an increase in non-specific adsorption (such as long fragment DNA, RNA); when the carboxyl density is too high (>5 μmol / m²), the carboxyl group covers the hydrophobic region of the shell, weakening the hydrophobic binding of cfDNA to the surface of the second magnetic microspheres; when the carboxyl density of the first magnetic microspheres and the second magnetic microspheres is 1-5 μmol / m², the carboxyl density just allows the hydrophobic region of the shell to be moderately exposed, allowing only short fragment cfDNA (hydrophobic base concentration) to bind, and excluding long fragments (hydrophobic region dispersed, difficult to anchor).

[0056] Controlling the carboxyl density of the first magnetic microspheres and the second magnetic microspheres can also regulate the binding strength of the ionic bridge. The carboxyl density determines the number of ionic bridge binding sites. If the carboxyl density is too low: the ionic bridge sites are insufficient, and the cfDNA is easy to be lost in the washing; if the carboxyl density is too high: the ionic bridge is too strong, and a higher pH or temperature is needed for elution, which is easy to damage the integrity of the cfDNA.

[0057] Controlling the carboxyl density of the first magnetic microspheres and the second magnetic microspheres can also indirectly affect impurity competition. Carboxyl is negatively charged under neutral conditions, and can reduce the hydrophobic competition of negatively charged impurities (such as RNA) through electrostatic repulsion.

[0058] Experiments have found that when the carboxyl density of the first magnetic microspheres and the second magnetic microspheres is 1-5 μmol / m², the first magnetic microspheres can well play a role in binding impurities under the condition of the first binding liquid, and the second magnetic microspheres can well play a role in binding 100-200 bp cfDNA under the condition of the second binding liquid.

[0059] In the above technical solution, the particle size of the first magnetic microspheres and the second magnetic microspheres is 0.5-1.5 μm.

[0060] The smaller the particle size, the larger the specific surface area of the magnetic microspheres, the more binding sites can be exposed, and the higher the adsorption efficiency of cfDNA: large-particle-size magnetic microspheres have small specific surface area and low adsorption capacity, but the surface binding sites are more sparse, which can reduce non-specific adsorption and is suitable for scenarios with higher purity requirements.

[0061] The separation of the first magnetic microspheres and the second magnetic microspheres depends on the external magnetic field, and the particle size directly affects the response speed in the magnetic field. The magnetic response of small-particle-size magnetic microspheres is slower, and it may take a longer time to be completely adsorbed by the magnetic field; large-particle-size magnetic microspheres have higher content of magnetic substances and settle faster in the magnetic field, so the separation time is short and is suitable for high-throughput automatic operation.

[0062] In addition, small-particle-size magnetic microspheres have better dispersibility and can fully contact the sample, reducing the adsorption deviation caused by uneven local concentration; large-particle-size microspheres have poor dispersibility and are easy to aggregate due to gravity settlement, which may lead to insufficient local binding sites.

[0063] Taking the above factors into account, experiments have found that the particle size of the first magnetic microspheres and the second magnetic microspheres is 0.5-1.5 μm, which is appropriate in size and has moderate specific surface area, and can balance the extraction efficiency and purity requirements, and is convenient and fast to operate.

[0064] In the technical solution, the first magnetic microspheres and the second magnetic microspheres are suspended in a phosphate buffered saline solution or a Tris-HCl buffer solution with a pH of 7.0-8.0, and the concentration of the first magnetic microspheres and the second magnetic microspheres is 5-15 mg / mL.

[0065] The suspension of the first magnetic microspheres and the second magnetic microspheres in the buffer solution can maintain the dispersion stability of the magnetic microspheres and avoid the aggregation of the magnetic microspheres. The phosphate buffered saline solution (PBS) contains sodium chloride, potassium chloride, phosphate and other substances that can provide physiological ionic strength to prevent the aggregation of the magnetic microspheres due to electrostatic interaction. The Tris-HCl buffer solution can stabilize the surface charge of the magnetic microspheres by adjusting the pH to ensure that the magnetic microspheres remain active in the suspended state.

[0066] When the concentration of the first magnetic microspheres and the second magnetic microspheres is 5-15 mg / mL, the adsorption efficiency and the operation convenience can be considered; if the concentration of the magnetic microspheres is too low, the cfDNA capture may not be complete; if the concentration of the magnetic microspheres is too high, the risk of aggregation of the magnetic microspheres may be increased, affecting the separation efficiency.

[0067] The second aspect of the present application provides a cfDNA extraction method, which is performed using the cfDNA extraction kit described above, and includes the following steps:

[0068] The body fluid sample is lysed to obtain a lysate; the lysate, the first binding solution and the first magnetic microspheres are mixed to obtain the first magnetic microspheres carrying impurities; the first magnetic microspheres carrying impurities are removed to obtain a supernatant;

[0069] The supernatant, the second binding solution and the second magnetic microspheres are mixed to obtain the second magnetic microspheres carrying cfDNA, and the cfDNA is obtained after washing and elution.

[0070] Specifically, the plasma sample can be mixed with proteinase K and lysis solution, and incubated at 55-65℃ for 20-40 min to obtain a lysate. Then, the first binding solution and the first magnetic microsphere suspension are added to the lysate, and after vortex mixing, the mixture is allowed to stand at room temperature for 3-10 min to facilitate the adsorption of impurities on the surface of the first magnetic microspheres, thereby obtaining the first magnetic microspheres carrying impurities. The first magnetic microspheres carrying impurities are removed by magnetic separation, and the supernatant is retained. Subsequently, the second binding solution and the second magnetic microsphere suspension are added to the supernatant, and after vortex mixing, the mixture is allowed to stand at room temperature for 3-10 min to facilitate the adsorption of cfDNA on the surface of the second magnetic microspheres, thereby obtaining the second magnetic microspheres carrying cfDNA. The second magnetic microspheres carrying cfDNA are obtained by magnetic separation. Then, the second magnetic microspheres carrying cfDNA are added with the washing solution, vortexed for 5-20 s, and allowed to stand at room temperature for 0.5-5 min, and the supernatant is discarded by magnetic separation. The washing is repeated once, and the second magnetic microspheres are air-dried at room temperature. Finally, the air-dried second magnetic microspheres are added with the elution solution, and the mixture is gently mixed and incubated at 50-70℃ for 5-20 min to facilitate the dissociation of cfDNA from the second magnetic microspheres. The second magnetic microspheres are removed by magnetic separation, and the supernatant is transferred to a new centrifuge tube, thereby obtaining the cfDNA sample.

[0071] Further, the volume ratio of the first binding solution to the first magnetic microspheres is (35-45): 1. For example, the first binding solution can be 2 mL, and the magnetic microsphere suspension can be 50 μL.

[0072] Further, the volume ratio of the second binding solution to the second magnetic microspheres is (35-45): 1. For example, the second binding solution can be 2 mL, and the magnetic microsphere suspension can be 50 μL.

[0073] Further, the volume ratio of the first binding solution to the second binding solution is (1-2):(1-2). For example, the first binding solution can be 2 mL, and the second binding solution can be 2 mL.

[0074] In an embodiment of the present application, the plasma sample can be 2 mL, the proteinase K can be 50 μL, the lysis solution can be 2 mL, the first binding solution can be 2 mL, the second binding solution can be 2 mL, the first magnetic microsphere suspension can be 50 μL, the second magnetic microsphere suspension can be 50 μL, the washing solution can be 500 μL, and the elution solution can be 50 μL, which helps to improve the extraction efficiency and purity of cfDNA.

[0075] Further, the body fluid sample is selected from at least one of blood, lymph, milk, urine, and amniotic fluid.

[0076] Hereinafter, the technical solutions of the present application will be further explained and described in combination with specific examples. The experimental methods not specified in the following examples are generally carried out under conventional conditions or under the conditions recommended by the manufacturers. The reagents used, if not specifically stated, are commercially available or can be obtained from public channels.

[0077] Example 1

[0078] The present example provides a cfDNA extraction kit, comprising protease K, a lysis solution, a first binding solution, a second binding solution, a first magnetic microsphere suspension, a second magnetic microsphere suspension, a washing solution, and an elution solution.

[0079] The concentration of the protease K is 0.5 mg / mL.

[0080] The lysis solution is prepared from 6 M guanidine hydrochloride and 1% (v / v) Triton X-100.

[0081] The first binding solution has a pH of 4.5 and is prepared from 1 M guanidine hydrochloride and 10% (w / v) polyethylene glycol 8000.

[0082] The second binding solution has a pH of 7.0 and is prepared from 3 M guanidine hydrochloride, 20% (v / v) isopropanol, and 10 g / L β-cyclodextrin.

[0083] The magnetic microspheres are carboxylated magnetic microspheres with a particle size of 0.5-1.5 μm. The carboxylated magnetic microspheres have a core-shell structure, including a Fe3O4 core and a SiO2 shell layer coated on the surface of the core. The SiO2 shell layer is modified with carboxyl groups, and the carboxyl group density is 3 μmol / m². The magnetic microspheres are suspended in a phosphate buffered saline solution (PBS) with a pH of 7.4 to prepare a magnetic microsphere suspension (divided into a first magnetic microsphere suspension and a second magnetic microsphere suspension) with a concentration of 10 mg / mL.

[0084] The washing solution is prepared from 80% (v / v) ethanol and 0.3 M sodium acetate with a pH of 5.5.

[0085] The elution solution is a TE (Tris-EDTA) buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0±0.1).

[0086] The present example also provides a method for extracting cfDNA using the above-mentioned kit, comprising the following steps:

[0087] (1) Lysis: Mix 2 mL of a plasma sample with 50 μL of protease K and 2 mL of a lysis solution, and incubate at 60°C for 30 min to obtain a lysate.

[0088] (2) Impurity removal: 2 mL of the first binding solution and 50 μL of the first magnetic microsphere suspension were added to the above lysate, which was mixed uniformly by vortex oscillation and then left to stand at room temperature (25°C) for 5 min to promote the adsorption of impurities onto the surface of the first magnetic microspheres, thereby obtaining first magnetic microspheres carrying impurities; the first magnetic microspheres carrying impurities were removed by magnetic separation, and the supernatant was retained;

[0089] (3) Capture: 2 mL of the second binding solution and 50 μL of the second magnetic microsphere suspension were added to the above supernatant, which was mixed uniformly by vortex oscillation and then left to stand at room temperature (25°C) for 5 min to promote the adsorption of cfDNA onto the surface of the second magnetic microspheres, thereby obtaining second magnetic microspheres carrying cfDNA; the second magnetic microspheres carrying cfDNA were obtained by magnetic separation;

[0090] (4) Washing: 500 μL of the washing solution was added to the above second magnetic microspheres carrying cfDNA, which was vortexed for 10 s and left to stand at room temperature (25°C) for 1 min, and the supernatant was discarded by magnetic separation; the washing was repeated once, and the second magnetic microspheres were air-dried at room temperature (25°C);

[0091] (5) Elution: 50 μL of the elution solution was added to the above air-dried second magnetic microspheres, which was mixed uniformly by gentle blowing, and then incubated at 60°C for 10 min to promote the dissociation of cfDNA from the second magnetic microspheres; the second magnetic microspheres were removed by magnetic separation, and the supernatant was transferred to a new centrifuge tube to obtain a cfDNA sample.

[0092] Comparative Example 1

[0093] This comparative example used a magnetic bead method free DNA mass extraction kit produced by Guangzhou Meiji Biotechnology Co., Ltd. with the product number IVD5435 to extract cfDNA. The extraction kit included protease K, Buffer SDS, binding solution MLK, magnetic bead solution MPF, washing solution MAW1, washing solution MAW2, and elution solution EB. The extraction process included the following steps:

[0094] (1) Lysis: 100 μL of protease K was first transferred to a 15 mL centrifuge tube, 2 mL of the plasma sample was then transferred to the centrifuge tube, and then 100 μL of Buffer SDS was added to the centrifuge tube, which was mixed uniformly by inversion several times, incubated at 55°C for 30 min, inverted several times during the incubation, and left to stand at room temperature (25°C) for 5 min to restore the lysate to room temperature;

[0095] (2) Binding: 3.8 mL of binding solution MLK and 150 μL of magnetic beads MPF were added to the above lysis solution, mixed well by inverting at room temperature (25°C) for 10 min, transferred to a magnetic stand and allowed to stand for 5 min to adsorb the cfDNA to the magnetic beads, the solution was aspirated, and after brief centrifugation, the residual liquid was aspirated;

[0096] (3) Washing: 1.0 mL of washing solution MAW1 was added to the above magnetic beads, vortexed for 5 s, inverted for 10-15 times, transferred to a magnetic stand and adsorbed for 1 min, and the solution was aspirated; the washing solution MAW1 was used to repeat the washing once; then 1.0 mL of washing solution MW2 was added, vortexed for 5 s, inverted for 10-15 times, transferred to a magnetic stand and adsorbed for 1 min, and the solution was aspirated; the washing solution MAW2 was used to repeat the washing once; after brief centrifugation, the residual liquid was aspirated; and the magnetic beads were completely dried in a 37°C metal bath for 10-15 min;

[0097] (4) Elution: 50 μL of elution solution EB was added to the above dried magnetic beads, and the cfDNA was fully dissolved by incubating at 37°C for 5 min, then transferred to a magnetic stand and adsorbed for 1 min, the supernatant containing cfDNA was transferred to a new centrifuge tube, the original centrifuge tube was briefly centrifuged, and the residual liquid was collected into the new centrifuge tube to obtain the cfDNA sample.

[0098] Comparative Example 2:

[0099] This comparative example provides a cfDNA extraction kit, which comprises protease K, lysis solution, first binding solution, second binding solution, first magnetic microsphere suspension, second magnetic microsphere suspension, washing solution, and elution solution. The components and contents thereof can refer to those of Example 1, and the only difference is that the second binding solution does not contain β-cyclodextrin.

[0100] This comparative example also provides a method for extracting cfDNA using the above kit, and the specific steps can refer to those of Example 1 to obtain a cfDNA sample.

[0101] Comparative Example 3:

[0102] This comparative example provides a cfDNA extraction kit, which comprises protease K, lysis solution, first binding solution, second binding solution, first magnetic microsphere suspension, second magnetic microsphere suspension, washing solution, and elution solution. The components and contents thereof can refer to those of Example 1, and the only difference is that the second binding solution does not contain isopropyl alcohol.

[0103] This comparative example also provides a method for extracting cfDNA using the above kit, and the specific steps can refer to those of Example 1 to obtain a cfDNA sample.

[0104] Test Example

[0105] (1) The absorbance of the cfDNA sample at 260 nm (A260) and the absorbance at 280 nm (A280) were tested by using the ultraviolet spectrophotometer NanoDrop, and the purity of the cfDNA sample was evaluated by the ratio of A260 / A280, and the results can be seen in Table 1.

[0106] (2) The content of cfDNA of 100-200 bp (unit: ng) in the cfDNA sample was tested by using the automatic nucleic acid protein analysis system Qsep100 according to the capillary electrophoresis method, and the results can be seen in Table 2.

[0107] Table 1

[0108]

[0109] Table 2

[0110]

[0111] According to the results of Table 1 and Table 2, it can be found that through clinical sample testing (n=5), the purity of cfDNA (A260 / A280=1.75-1.85) extracted by using the cfDNA extraction kit and the cfDNA extraction method provided by the application is significantly better than that of the single-step magnetic bead method of Comparative Example 1 (A260 / A280=1.65-1.67), and the content of cfDNA of 100-200 bp (16.9-18.8 ng) is also obviously improved than that of the single-step magnetic bead method of Comparative Example 1 (10.9-12.6), which indicates that by regulating the magnetic microsphere binding environment in stages, the bottleneck of the traditional single-step magnetic bead method can be broken through, and the operation simplicity and high repeatability (CV≤5%) are combined, the cfDNA extraction efficiency and purity are effectively improved, which provides reliable technical support for liquid biopsy, and is suitable for application scenarios such as tumor liquid biopsy and non-invasive prenatal diagnosis.

[0112] In addition, according to the results of Table 1 and Table 2, it can also be found that the guanidine hydrochloride, isopropyl alcohol and β-cyclodextrin in the second binding solution of the application have a synergistic effect, and only when the three are used together, the cfDNA extraction efficiency and purity can be obviously improved.

[0113] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the application, but not to limit them; although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the application.

Claims

1. A cfDNA extraction kit, characterized in that, It includes a first binding liquid, a second binding liquid, and magnetic microspheres; The magnetic microspheres include a first magnetic microsphere and a second magnetic microsphere; the first magnetic microsphere and the second magnetic microsphere have a core-shell structure, including a magnetite core and a silica shell covering at least part of the core surface, wherein the silica shell is modified with carboxyl groups; The first binding solution has a pH of 4-5 and includes 0.5-1.5 M guanidine hydrochloride and 5-15% (w / v) polyethylene glycol 8000; The second binding solution has a pH of 6.5-7.5 and includes 2-4 M guanidine hydrochloride, 10-30% (v / v) isopropanol and 5-15 g / L β-cyclodextrin; The cfDNA extraction kit also includes proteinase K; the concentration of proteinase K is 0.1-2 mg / mL; The cfDNA extraction kit also includes a lysis buffer; the lysis buffer comprises 5-7 M guanidine hydrochloride and 0.5-1.5% (v / v) Triton X-100; The cfDNA extraction kit also includes a washing solution; the washing solution comprises 70-90% (v / v) ethanol and 0.1-0.5 M sodium acetate at pH 5.0-6.

0. The cfDNA extraction kit also includes an elution buffer; the elution buffer includes a TE buffer with a pH of 7.9-8.

1. The carboxyl group density of the first magnetic microsphere and the second magnetic microsphere is 1-5 μmol / m²; The particle size of the first magnetic microsphere and the second magnetic microsphere is 0.5-1.5 μm; The first magnetic microsphere and the second magnetic microsphere are suspended in a phosphate buffer solution or a tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH of 7.0-8.0, and the concentration of the first magnetic microsphere and the second magnetic microsphere is 5-15 mg / mL.

2. A method for extracting cfDNA, characterized in that, The cfDNA extraction kit according to claim 1 is used, comprising the following steps: The body fluid sample is lysed to obtain lysate; the lysate, the first binding solution, and the first magnetic microspheres are mixed to obtain a first magnetic microsphere carrying impurities; the first magnetic microsphere carrying impurities is removed to obtain a supernatant; The supernatant, the second binding solution, and the second magnetic microspheres were mixed to obtain the second magnetic microspheres carrying cfDNA. After washing and elution, the cfDNA was obtained.

3. The cfDNA extraction method according to claim 2, characterized in that, The volume ratio of the first binding liquid to the first magnetic microspheres is (35-45):1; and / or, The volume ratio of the second binding liquid to the second magnetic microspheres is (35-45):1; and / or, The volume ratio of the first binding liquid to the second binding liquid is (1-2):(1-2); and / or, The body fluid sample is selected from at least one of blood, lymph, milk, urine, and amniotic fluid.

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