CfDNA extraction kit and extraction method

Abstract

The invention provides a cfDNA extraction kit and an extraction method. The cfDNA extraction kit provided by the invention comprises the first binding solution, the second binding solution and the magnetic microspheres, and by limiting the components and contents of the first binding solution and the second binding solution, the cfDNA extraction kit can be promoted to generate a good matching effect with the magnetic microspheres, so that the bottleneck of a traditional single-step paramagnetic particle method is broken through by regulating and controlling the binding environment of the magnetic microspheres in stages, and the detection sensitivity of the cfDNA extraction kit is improved. The extraction efficiency and the extraction purity of the cfDNA are effectively improved, and the method has the advantages of simplicity and convenience in operation and high repeatability. Reliable technical support is provided for liquid biopsy, and the method is suitable for application scenes such as tumor liquid biopsy and noninvasive prenatal diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a cfDNA extraction kit and an extraction method. Background Art

[0002] Cell-free DNA (cfDNA) refers to DNA fragments that are free from cells and range in length from 100 to 200 base pairs. It is primarily released into the extracellular environment of the human body through pathways such as apoptosis and necrosis, and is often found in physiological extracellular environments such as blood, lymph, milk, urine, and amniotic fluid. Currently, cfDNA testing is a common form of liquid biopsy on the market and has been widely used in various aspects such as tumor detection, guidance evaluation, and prognosis assessment. However, cfDNA is low in content and small in fragment size, making its extraction difficult. Losses are also prone to occur during the extraction process, resulting in low detection sensitivity, which to some extent limits its application in clinical diagnosis.

[0003] Currently, cfDNA extraction methods can be mainly divided into traditional phenol / chloroform extraction, silica gel membrane adsorption column method and magnetic bead extraction method. Among them, magnetic bead extraction method has the advantages of simple operation, short time consumption, easy automation and high-throughput processing compared to the first two extraction methods. It can directly extract cfDNA from crude samples such as plasma, reducing the pretreatment steps, and its shear force on DNA is small, which helps to maintain the integrity of cfDNA. However, existing magnetic bead extraction kits are mainly single-step extraction methods, and their extraction efficiency is often poor. The extracted cfDNA is generally of low purity, resulting in insufficient sensitivity in downstream testing. Therefore, it is urgent to develop a cfDNA extraction kit with high extraction efficiency and high purity of the obtained cfDNA. Summary of the Invention

[0004] The present invention provides a cfDNA extraction kit that can effectively improve the purity and content of cfDNA.

[0005] The present invention provides a cfDNA extraction method that is simple to operate and suitable for high-throughput processing.

[0006] The present invention provides a cfDNA extraction kit, which includes a first binding solution, a second binding solution and magnetic microspheres;

[0007] The magnetic microspheres include a first magnetic microsphere and a second magnetic microsphere; the first magnetic microsphere and the second magnetic microsphere are core-shell structures, including a ferroferric oxide core and a silicon dioxide shell layer covering at least a portion of the surface of the core, and the silicon dioxide shell layer is modified with a carboxyl group;

[0008] 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;

[0009] 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.

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

[0011] The concentration of 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 buffer contains 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 wash solution consists of 70-90% (v / v) ethanol and 0.1-0.5 M sodium acetate at pH 5.0-6.0.

[0016] The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises an eluent;

[0017] The eluent consisted of TE buffer, pH 7.9-8.1.

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

[0019] In the cfDNA extraction kit as described above, 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 hydrochloride 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.

[0021] The present invention provides a cfDNA extraction method, wherein the method is performed using the above-mentioned cfDNA extraction kit and comprises the following steps:

[0022] Lysing the body fluid sample to obtain a lysate; mixing the lysate, the first binding solution, and the first magnetic microspheres to obtain first magnetic microspheres carrying impurities; and removing the first magnetic microspheres carrying impurities to obtain a supernatant;

[0023] 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.

[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 and the second magnetic microspheres is (35-45):1; and / or,

[0026] The volume ratio of the first binding liquid to the second binding liquid 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 invention provides a cfDNA extraction kit comprising a first binding fluid, a second binding fluid, and magnetic microspheres. By defining the components and contents of the first and second binding fluids, a good match between them and the magnetic microspheres can be achieved. Furthermore, by regulating the magnetic microsphere binding environment in stages, the kit overcomes the bottleneck of the traditional single-step magnetic bead method, effectively improving the extraction efficiency and purity of cfDNA while balancing ease of operation and high reproducibility. This invention provides reliable technical support for liquid biopsy and is suitable for applications such as tumor liquid biopsy and non-invasive prenatal diagnosis. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific embodiments listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present invention.

[0030] Magnetic bead extraction of cell-free DNA (cfDNA) is currently one of the mainstream cfDNA extraction technologies, particularly suitable for liquid biopsy samples such as plasma and serum. Its core principle is to utilize surface-functionalized magnetic microspheres to selectively adsorb cfDNA under specific buffer conditions, followed by magnetic field separation for purification. However, cfDNA fragments are generally 100-200 bp in length. Such short fragments carry less negative charge and bind weakly to magnetic microspheres. This results in low binding efficiency of existing magnetic bead methods for cfDNA fragments in the 100-200 bp range, which in turn affects the extraction efficiency and purity of cfDNA. Furthermore, magnetic microspheres also suffer from nonspecific adsorption of long genomic DNA fragments, further complicating the extraction of cfDNA using magnetic bead methods.

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

[0032] In the present invention, the magnetic microspheres include a first magnetic microsphere and a second magnetic microsphere. The first and second magnetic microspheres have the same structure but different uses. The first and second magnetic microspheres have a core-shell structure, comprising a ferrosoferric oxide core and a silica shell coating at least a portion of the core surface, wherein the silica shell is modified with carboxyl groups.

[0033] During the cfDNA extraction process, magnetic microspheres can be used in conjunction with the first binding liquid or the second binding liquid to provide two different uses: (1) Capturing impurities: With 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 that capture impurities can be easily separated from the solution; (2) Capturing cfDNA: With 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 that capture cfDNA can be easily separated from the solution. The settings of the first binding liquid and the second binding liquid can control the objects captured by the magnetic microspheres, thereby achieving staged separation of impurities and cfDNA.

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

[0035] First, under the acidic conditions (pH 4-5) provided by the first binding solution, the carboxyl groups (-COOH) modified on the surface of the first magnetic microspheres are protonated, reducing the negative surface charge (-COO⁻) of the first magnetic microspheres. Although the net negative charge density of the surface decreases, the overall charge remains predominantly negative. Under acidic conditions, amino groups (-NH₂) carried by impurities such as proteins and lipids are protonated to form positively charged ammonium groups (-NH⁺), contributing to the overall positive charge of the impurities. cfDNA is composed of nucleotides, whose phosphate backbone carries a negative charge (-PO₄³⁻). Therefore, even under acidic conditions, its net negative charge density decreases slightly, but the overall charge remains predominantly negative. Consequently, positively charged impurities preferentially electrostatically attract the remaining negative charge on the surface of the first magnetic microspheres, while negatively charged cfDNA electrostatically repel the negative charge on the surface of the first magnetic microspheres.

[0036] Secondly, the first binding solution contains a low concentration (0.5-1.5 M) of guanidine hydrochloride. This low concentration of guanidine hydrochloride reduces the ionic strength of the system, thereby reducing the competitive adsorption of impurity molecules by salt ions on the surface of the first magnetic microspheres. At this point, hydrophobic interactions and hydrogen bonding between impurity molecules (such as proteins) and the first magnetic microspheres dominate, making them more readily adsorbed on the surface than cfDNA. Furthermore, the low concentration of guanidine hydrochloride effectively prevents structural damage to impurities, preserving their native conformation and thus facilitating their binding to the first magnetic microspheres through surface functional groups. For example, the hydrophobic regions of proteins are more exposed at low guanidine hydrochloride concentrations, enhancing their hydrophobic interactions with the magnetic bead surface and leading to preferential adsorption.

[0037] Finally, the first binding solution contains polyethylene glycol 8000. Polyethylene glycol 8000 is a water-soluble macromolecular polymer that, through osmotic pressure, attracts water molecules from the surfaces of impurities such as proteins and lipids, compressing the impurities' hydration layer and bringing them closer to the surface of the magnetic microspheres. This strengthens the hydrophobic interaction and electrostatic attraction between the impurities and the first magnetic microspheres, promoting their preferential adsorption to the first magnetic microspheres. The macromolecular structure of polyethylene glycol 8000 also creates steric hindrance in the solution, reducing the nonspecific contact between short, negatively charged cfDNA fragments and the remaining negative charges on the surface of the first magnetic microspheres. This prevents cfDNA from being encapsulated and precipitated by impurities, ensuring stable retention of cfDNA in the supernatant and facilitating its specific capture by the second binding solution. Therefore, polyethylene glycol 8000 works synergistically with the guanidine hydrochloride in the first binding solution to regulate the interfacial behavior between impurities and cfDNA, achieving a staged separation effect characterized by preferential impurity adsorption and efficient cfDNA retention.

[0038] In short, the acidic environment enhances the electrostatic attraction between impurities and the first magnetic microspheres by regulating their charge state, while the low chaotropic salt concentration combined with polyethylene glycol 8000 promotes impurity adsorption by reducing ionic interference and enhancing hydrophobic interactions. This synergistic effect allows impurities (such as proteins and lipids) to preferentially bind to the surface of the first magnetic microspheres, while cfDNA, due to charge repulsion and its higher solubility in low-salt conditions, remains in the supernatant, paving the way for specific cfDNA capture in the subsequent second step.

[0039] In the present invention, the second binding solution can effectively promote the adsorption of cfDNA onto the surface of the second magnetic microsphere. 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.

[0040] On the one hand, the second binding solution provides a neutral environment (pH 6.5-7.5). Under this neutral environment, the carboxyl groups (-COOH) modified on the surface of the second magnetic microspheres are deprotonated and converted to negatively charged carboxyl ions (-COO⁻), increasing the negative charge density on the surface of the second magnetic microspheres. Although the phosphate backbone of cfDNA still carries a negative charge (-PO⁻), the isopropanol in the second binding solution partially neutralizes the negative charge of the cfDNA, reducing its solubility. At the same time, β-cyclodextrin reduces competition from lipid impurities for the hydrophobic sites on the second magnetic microspheres. Therefore, the increased negative charge density on the surface of the second magnetic microspheres under a neutral environment synergizes with the charge neutralization of cfDNA by isopropanol, facilitating cfDNA binding to the second magnetic microspheres through hydrophobic interactions and ionic bridges, thereby achieving efficient capture of cfDNA. This, combined with the preferential adsorption of impurities in the acidic environment of the first binding solution, creates a phased regulation, ultimately improving the extraction efficiency and purity of cfDNA.

[0041] On the other hand, the high concentration (2-4 M) of guanidine hydrochloride in the second binding solution disrupts the binding of cfDNA to residual impurities, enhances the interaction between cfDNA and the second magnetic microspheres, and inhibits nuclease activity. Isopropyl alcohol (IPA) neutralizes the negative charge of cfDNA, reducing its solubility, thereby promoting its precipitation and binding to the hydrophobic regions on the surface of the second magnetic microspheres. It also dissolves impurities such as salts and small molecules, thereby improving cfDNA purity. β-cyclodextrin preferentially forms inclusion complexes with lipid impurities, reducing their competitive adsorption to the hydrophobic regions on the surface of the second magnetic microspheres. Through dual hydrophobic interactions, isopropyl alcohol and β-cyclodextrin exert a synergistic effect, jointly driving the adsorption of cfDNA to the second magnetic microspheres in a more ordered conformation, enhancing the orderliness of cfDNA adsorption and improving capture specificity. Guanidine hydrochloride enhances the interaction between cfDNA and the second magnetic microspheres, and has a synergistic effect with the charge neutralization effect of isopropanol and the reduction of lipid competition by β-cyclodextrin, jointly achieving efficient and specific capture of cfDNA and improving extraction efficiency and purity.

[0042] Therefore, the cfDNA extraction kit provided by the present invention can promote a good match between the first and second binding solutions by limiting their components and contents. Furthermore, by regulating the magnetic microsphere binding environment in stages, it overcomes the bottleneck of the traditional single-step magnetic bead method, effectively improving the extraction efficiency and purity of cfDNA while combining ease of operation with high reproducibility. This invention provides reliable technical support for liquid biopsy and is suitable for applications such as tumor liquid biopsy and non-invasive prenatal diagnosis.

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

[0044] Proteinase K is a broad-spectrum serine protease that efficiently degrades histones, nucleoproteins, and other proteins (such as hemoglobin and albumin) bound to cfDNA, thereby releasing cfDNA. Proteinase K also prevents cfDNA degradation during extraction by degrading nucleases (such as DNAase), thereby ensuring the integrity of cfDNA.

[0045] Experimental results indicate that a Proteinase K concentration of 0.1-2 mg / mL in the cfDNA extraction kit is suitable for most body fluid samples, effectively degrading proteins and improving cfDNA extraction efficiency. This concentration also avoids excess enzyme activity and increased costs associated with high concentrations. For example, the Proteinase K concentration 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 between any two of these ranges.

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

[0047] The lysis buffer containing the above components can effectively disrupt any cellular structures present in body fluid samples, releasing cfDNA while simultaneously inhibiting nuclease activity to prevent cfDNA degradation. 5-7 M guanidine hydrochloride acts as a strong chaotropic agent, disrupting protein hydrogen bonds and hydrophobic interactions, denaturing histones, nucleoproteins, and nucleases in body fluid samples, and releasing cfDNA. 0.5-1.5% (v / v) Triton X-100 acts as a non-ionic detergent, disrupting the cell membrane phospholipid bilayer and, together with guanidine hydrochloride, promoting cell lysis while dissolving lipid impurities.

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

[0049] On the one hand, 70-90% (v / v) ethanol can reduce the solubility of cfDNA, promoting its precipitation and thus facilitating its binding to the second magnetic microspheres. It also dissolves water, salt, protein, and other organic impurities in the solution, helping to enhance the washing effect. Sodium acetate at pH 5.0-6.0 and 0.1-0.5 M dissociates into acetate ions and sodium ions in the system. Sodium ions neutralize the negative charge on the phosphate backbone of cfDNA, weakening the electrostatic repulsion between cfDNA molecules and making it easier for them to bind to functional groups (such as carboxyl groups) on the surface of the second magnetic microspheres, thereby reducing cfDNA loss during the washing process and increasing its content.

[0050] On the other hand, ethanol and sodium acetate have a synergistic effect. Ethanol can reduce the dielectric constant of the solution, promoting the dehydration and precipitation of cfDNA. The presence of sodium acetate further reduces the solubility of cfDNA. At the same time, it binds to residual salt ions (such as guanidine salts) through ionic bonds, removing them during the washing step. This reduces residual impurities, prevents the inhibitory effect of salt ions on downstream detection (such as PCR and sequencing), and improves experimental reliability.

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

[0052] TE buffer is composed of tris(hydroxymethyl)aminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA). The weak alkalinity of TE buffer (pH 7.9-8.1) can disrupt the ionic bond between cfDNA and the surface of the second magnetic microspheres, promoting the detachment of cfDNA from the second magnetic microspheres. EDTA also chelates metal ions, preventing nuclease degradation of cfDNA.

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

[0054] In the present invention, in the first binding stage, impurities are mainly adsorbed on the hydrophobic region of the silica shell of the first magnetic microsphere 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 microsphere through hydrophobic interaction.

[0055] Controlling the carboxyl group density of the first and second magnetic microspheres optimizes the exposure of the hydrophobic regions. Because carboxyl groups are modified on the silica shell surface, their density determines the hydrophilic-hydrophobic balance of the shell surface. When the carboxyl group density is too low (<1 μmol / m²), the hydrophobic regions of the shell are overexposed, leading to increased nonspecific adsorption (e.g., of long DNA and RNA fragments). When the carboxyl group density is too high (>5 μmol / m²), the carboxyl groups cover the hydrophobic regions of the shell, weakening the hydrophobic binding of cfDNA to the second magnetic microsphere surface. When the carboxyl group density of the first and second magnetic microspheres is between 1 and 5 μmol / m², the hydrophobic regions of the shell are optimally exposed, allowing only short cfDNA fragments (where hydrophobic bases are concentrated) to bind while repelling long fragments (where the hydrophobic regions are dispersed and difficult to anchor).

[0056] Controlling the carboxyl group density of the first and second magnetic microspheres can also regulate the binding strength of the ionic bridge. The carboxyl group density determines the number of binding sites for the ionic bridge. If the carboxyl group density is too low, there will be insufficient ionic bridge sites, and cfDNA will be easily lost during washing. If the carboxyl group density is too high, the ionic bridge will be too strong, requiring higher pH or temperature for elution, which can easily damage the integrity of the cfDNA.

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

[0058] Experiments have shown that when the carboxyl group density of the first and second magnetic microspheres is 1-5 μmol / m², the first magnetic microspheres can effectively bind impurities under the conditions of the first binding solution, and the second magnetic microspheres can effectively bind 100-200 bp cfDNA under the conditions of the second binding solution.

[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 exposed binding sites, and the higher the cfDNA adsorption efficiency: large-particle magnetic microspheres have a small specific surface area and a lower adsorption capacity, but the surface binding sites are more sparsely distributed, which can reduce nonspecific 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. The particle size directly affects their response speed in the magnetic field. The magnetic response of small-particle magnetic microspheres is slower and it may take longer to be completely adsorbed by the magnetic field. Large-particle magnetic microspheres have a higher content of magnetic substances, settle faster in the magnetic field, and have a short separation time, making them suitable for high-throughput automated operations.

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

[0063] Taking the above factors into consideration, the present invention has experimentally found that the particle size of the first magnetic microspheres and the second magnetic microspheres is 0.5-1.5 μm, which has an appropriate particle size and a moderate specific surface area, can take into account both extraction efficiency and purity requirements, and is convenient and quick to operate.

[0064] In the above technical solution, the first magnetic microspheres and the second magnetic microspheres are suspended in a phosphate buffered saline solution or a tris hydrochloride 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] Suspending the first and second magnetic microspheres in a buffer solution maintains the dispersion stability of the microspheres and prevents their aggregation. Phosphate-buffered saline (PBS), containing sodium chloride, potassium chloride, and phosphate, provides physiological ionic strength, preventing electrostatic aggregation of the microspheres. Tris-HCl buffer stabilizes the surface charge of the microspheres by adjusting their pH, ensuring their activity while suspended.

[0066] When the concentration of the first magnetic microspheres and the second magnetic microspheres is 5-15 mg / mL, both adsorption efficiency and operational convenience can be taken into account; if the concentration of the magnetic microspheres is too low, incomplete capture of cfDNA may result; if the concentration of the magnetic microspheres is too high, the risk of magnetic microsphere agglomeration may increase, affecting separation efficiency.

[0067] A second aspect of the present invention provides a cfDNA extraction method, which is performed using the above-mentioned cfDNA extraction kit and comprises the following steps:

[0068] Lysing the body fluid sample to obtain a lysate; mixing the lysate, the first binding solution, and the first magnetic microspheres to obtain first magnetic microspheres carrying impurities; and removing the first magnetic microspheres carrying impurities 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 buffer and incubated at 55-65°C for 20-40 minutes to obtain a lysate. The first binding buffer and the first magnetic microsphere suspension are then added to the lysate. The mixture is vortexed and allowed to stand at room temperature for 3-10 minutes to encourage adsorption of impurities to the surface of the first magnetic microspheres, resulting in first magnetic microspheres carrying impurities. The impurities are removed by magnetic separation, and the supernatant is retained. Subsequently, the second binding buffer and the second magnetic microsphere suspension are added to the supernatant. The mixture is vortexed and allowed to stand at room temperature for 3-10 minutes to encourage adsorption of cfDNA to the surface of the second magnetic microspheres, resulting in second magnetic microspheres carrying cfDNA. Magnetic separation is then performed to obtain second magnetic microspheres carrying cfDNA. Washing buffer is then added to the second magnetic microspheres carrying cfDNA, vortexed for 5-20 seconds, allowed to stand at room temperature for 0.5-5 minutes, and magnetic separation is performed to discard the supernatant. The washing process is repeated once, and the second magnetic microspheres are air-dried at room temperature. Finally, add the elution solution to the dried second magnetic microspheres, gently blow to mix, and incubate at 50-70°C for 5-20 min to promote the dissociation of cfDNA from the second magnetic microspheres; remove the second magnetic microspheres by magnetic separation, transfer the supernatant to a new centrifuge tube, and the cfDNA sample can be obtained.

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

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

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

[0074] In one embodiment of the present invention, 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 eluent can be 50 μL, which helps to improve the cfDNA extraction efficiency and extraction purity.

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

[0076] The technical solutions of this application are further explained below with reference to specific examples. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. All reagents used, unless otherwise specified, were commercially available or publicly available.

[0077] Example 1:

[0078] This embodiment provides a cfDNA extraction kit, including proteinase 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 eluent;

[0079] The concentration of proteinase K was 0.5 mg / mL;

[0080] The lysis buffer was prepared with 6 M guanidine hydrochloride and 1% (v / v) Triton X-100;

[0081] The pH of the first binding solution was 4.5, and it was prepared with 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, comprising a ferroferric oxide (Fe3O4) core and a silica (SiO2) shell coated on the surface of the core. The silica shell is modified with carboxyl groups with a carboxyl group density of 3 μmol / m². The magnetic microspheres are suspended in phosphate buffered saline (PBS) at a pH of 7.4 to prepare a magnetic microsphere suspension with a concentration of 10 mg / mL (divided into a first magnetic microsphere suspension and a second magnetic microsphere suspension).

[0084] The washing solution was prepared from 80% (v / v) ethanol, pH 5.5, and 0.3 M sodium acetate;

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

[0086] This embodiment also provides a method for extracting cfDNA using the above kit, comprising the following steps:

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

[0088] (2) Impurity removal: Add 2 mL of the first binding solution and 50 μL of the first magnetic microsphere suspension to the above lysate, vortex and mix thoroughly, and then let it stand at room temperature (25°C) for 5 min to promote the adsorption of impurities on the surface of the first magnetic microspheres to obtain the first magnetic microspheres carrying impurities; remove the first magnetic microspheres carrying impurities by magnetic separation, and retain the supernatant;

[0089] (3) Capture: Add 2 mL of the second binding solution and 50 μL of the second magnetic microsphere suspension to the above supernatant, vortex and mix well, and then let it stand at room temperature (25°C) for 5 min to promote the adsorption of cfDNA on the surface of the second magnetic microspheres to obtain second magnetic microspheres carrying cfDNA; obtain second magnetic microspheres carrying cfDNA by magnetic separation;

[0090] (4) Washing: Add 500 μL of washing solution to the second magnetic microspheres carrying cfDNA, vortex for 10 s, let stand at room temperature (25°C) for 1 min, and discard the supernatant by magnetic separation; repeat the washing once and dry the second magnetic microspheres at room temperature (25°C);

[0091] (5) Elution: Add 50 μL of elution buffer to the dried second magnetic microspheres, gently blow to mix, and incubate at 60°C for 10 min to promote the dissociation of cfDNA from the second magnetic microspheres; remove the second magnetic microspheres by magnetic separation, transfer the supernatant to a new centrifuge tube, and obtain the cfDNA sample.

[0092] Comparative Example 1:

[0093] This comparative example uses a magnetic bead-based free DNA large-scale extraction kit with the product number IVD5435 produced by Guangzhou Meiji Biotechnology Co., Ltd. to extract cfDNA. The extraction kit includes proteinase K, buffer SDS, binding solution MLK, magnetic bead solution MPF, washing solution MAW1, washing solution MAW2 and eluent EB. The extraction process includes the following steps:

[0094] (1) Lysis: First transfer 100 μL of proteinase K to a 15 mL centrifuge tube, then transfer 2 mL of plasma sample to the centrifuge tube, then add 100 μL of Buffer SDS to the centrifuge tube, invert several times to mix, incubate at 55°C for 30 min, invert several times during the process, and place at room temperature (25°C) for 5 min to allow the lysate to return to room temperature;

[0095] (2) Binding: Add 3.8 mL of binding solution MLK and 150 μL of magnetic beads MPF to the above lysate, mix by inversion at room temperature (25°C) for 10 min, transfer to a magnetic stand and let it stand for 5 min to allow cfDNA to adsorb to the magnetic beads, aspirate and discard the solution, centrifuge briefly, and then aspirate and discard the residual liquid;

[0096] (3) Washing: Add 1.0 mL of washing solution MAW1 to the above magnetic beads, vortex for 5 seconds, invert 10-15 times, transfer to a magnetic stand for adsorption for 1 minute, and discard the solution; repeat the washing with washing solution MAW1; add 1.0 mL of washing solution MW2, vortex for 5 seconds, invert 10-15 times, transfer to a magnetic stand for adsorption for 1 minute, and discard the solution; repeat the washing with washing solution MAW2; after a brief centrifugation, discard the residual liquid; dry the magnetic beads in a metal bath at 37°C for 10-15 minutes to completely dry them;

[0097] (4) Elution: Add 50 μL of elution buffer EB to the dried magnetic beads, incubate at 37°C with shaking for 5 min to fully dissolve the cfDNA, transfer to a magnetic stand and adsorb for 1 min, transfer the supernatant containing cfDNA to a new centrifuge tube, centrifuge the original centrifuge tube briefly, and collect the residual liquid in a new centrifuge tube to obtain the cfDNA sample.

[0098] Comparative Example 2:

[0099] This comparative example provides a cfDNA extraction kit, including proteinase 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 eluent. The components and contents thereof can be referred to in Example 1, with the only difference being that the second binding solution does not contain β-cyclodextrin.

[0100] This comparative example also provides a method for extracting cfDNA using the above-mentioned kit. The specific steps can be referred to Example 1 to obtain a cfDNA sample.

[0101] Comparative Example 3:

[0102] This comparative example provides a cfDNA extraction kit, including proteinase 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 eluent. The components and contents thereof can be referred to in Example 1, with the only difference being that the second binding solution does not contain isopropanol.

[0103] This comparative example also provides a method for extracting cfDNA using the above-mentioned kit. The specific steps can be referred to Example 1 to obtain a cfDNA sample.

[0104] Test Case

[0105] (1) The absorbance of the cfDNA samples at 260 nm (A260) and at 280 nm (A280) was tested using a NanoDrop UV spectrophotometer. The purity of the cfDNA samples was evaluated by the A260 / A280 ratio. The results are shown in Table 1.

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

[0107] Table 1

[0108]

[0109] Table 2

[0110]

[0111] According to the results in Tables 1 and 2, it can be found that, after clinical sample testing (n=5), the purity of cfDNA extracted using the cfDNA extraction kit and cfDNA extraction method provided by the present invention (A260 / A280=1.75-1.85) was significantly better than that of the single-step magnetic bead method of Comparative Example 1 (A260 / A280=1.65-1.67), and its 100-200 bp cfDNA content (16.9-18.8 ng) was also significantly improved compared with the single-step magnetic bead method of Comparative Example 1 (10.9-12.6). This shows that the present invention can break through the bottleneck of the traditional single-step magnetic bead method by regulating the binding environment of the magnetic microspheres in stages, and has both simple operation and high repeatability (CV≤5%), effectively improving the efficiency and purity of cfDNA extraction, providing 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 in Tables 1 and 2, it can be found that guanidine hydrochloride, isopropyl alcohol and β-cyclodextrin in the second binding solution of the present invention have a synergistic effect. Only when the three are used together can the efficiency and purity of cfDNA extraction be significantly improved.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cfDNA extraction kit, characterized in that: comprising 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 are core-shell structures, including a ferroferric oxide core and a silica shell layer covering at least a portion of the core surface, and the silica shell layer is modified with carboxyl groups; The first binding solution has a pH of 4-5 and comprises 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) isopropyl alcohol and 5-15 g / L β-cyclodextrin.

2. The cfDNA extraction kit according to claim 1, characterized in that The cfDNA extraction kit also includes proteinase K; The concentration of the proteinase K is 0.1-2 mg / mL.

3. The cfDNA extraction kit according to claim 1 or 2, characterized in that The cfDNA extraction kit also includes a lysis solution; The lysis solution includes 5-7 M guanidine hydrochloride and 0.5-1.5% (v / v) Triton X-100.

4. The cfDNA extraction kit according to claim 1 or 2, characterized in that The cfDNA extraction kit also includes a washing solution; The washing solution includes 70-90% (v / v) ethanol and pH 5.0-6.0, 0.1-0.5 M sodium acetate.

5. The cfDNA extraction kit according to claim 1 or 2, characterized in that The cfDNA extraction kit further includes an eluent; The eluent includes TE buffer at pH 7.9-8.

1.

6. The cfDNA extraction kit according to claim 1 or 2, characterized in that The carboxyl group density of the first magnetic microspheres and the second magnetic microspheres is 1-5 μmol / m².

7. The cfDNA extraction kit according to claim 6, characterized in that The particle size of the first magnetic microspheres and the second magnetic microspheres is 0.5-1.5 μm.

8. The cfDNA extraction kit according to claim 1 or 2, characterized in that The first magnetic microspheres and the second magnetic microspheres are suspended in a phosphate buffered saline solution or a tris hydrochloride 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.

9. A cfDNA extraction method, characterized in that: The method is performed using the cfDNA extraction kit according to any one of claims 1 to 8, comprising the following steps: Lysing the body fluid sample to obtain a lysate; mixing the lysate, the first binding solution, and the first magnetic microspheres to obtain first magnetic microspheres carrying impurities; removing the first magnetic microspheres carrying impurities to obtain a supernatant; The supernatant, the second binding liquid and the second magnetic microspheres are mixed to obtain second magnetic microspheres carrying cfDNA, and the cfDNA is obtained after washing and elution.

10. The cfDNA extraction method according to claim 9, characterized in that The volume ratio of the first binding solution 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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