CN120591257B - CfDNA extraction kit and extraction method - Google Patents
CfDNA extraction kit and extraction methodInfo
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Abstract
本发明提供一种cfDNA提取试剂盒及提取方法。本发明提供的cfDNA提取试剂盒包括第一结合液、第二结合液和磁性微球，通过限定第一结合液和第二结合液的组分及含量，可以促使其与磁性微球产生良好的匹配效果，进而通过分阶段调控磁性微球结合环境，突破传统单步磁珠法的瓶颈，有效提高cfDNA的提取效率和提取纯度，并兼具操作简便性与高重复性。本发明为液体活检提供了可靠的技术支撑，适用于肿瘤液态活检及无创产前诊断等应用场景。This invention provides a cfDNA extraction kit and extraction method. The cfDNA extraction kit includes a first binding solution, a second binding solution, and magnetic microspheres. By limiting the components and contents of the first and second binding solutions, a good matching effect between them and the magnetic microspheres can be achieved. Furthermore, by controlling the binding environment of the magnetic microspheres in stages, the bottleneck of the traditional single-step magnetic bead method is overcome, effectively improving the extraction efficiency and purity of cfDNA, while also offering 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.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a cfDNA extraction kit and an extraction method.
Background
Cell free DNA (cfDNA) refers to a DNA fragment that is free from the outside of cells and has a length of 100 to 200 base pairs, and is released into the extracellular environment of the human body mainly through apoptosis, necrosis, etc., and is often present in the physiological extracellular environment of blood, lymph fluid, emulsion, urine, amniotic fluid, etc. Currently, cfDNA detection is a common liquid biopsy format in the market, and has been widely used in various aspects such as tumor detection, guidance evaluation, prognosis evaluation, etc. However, cfDNA content is low, fragments are small, extraction is difficult, deletion is easy to occur in the extraction process, detection sensitivity is low, and application of cfDNA in clinical diagnosis is limited to a certain extent.
At present, cfDNA extraction methods can be mainly classified into traditional phenol/chloroform extraction, silica gel membrane adsorption column methods and magnetic bead extraction methods. Compared with the former two extraction methods, the magnetic bead extraction method has the advantages of simple operation, short time consumption, easy automation and high-flux treatment, cfDNA can be directly extracted from crude samples such as blood plasma, pretreatment steps are reduced, and the shearing force of the cfDNA on the cfDNA is small, so that the integrity of the cfDNA is maintained. However, the existing magnetic bead extraction kit is mainly a single-step extraction method, the extraction efficiency is often poor, and the cfDNA obtained by extraction is generally low in purity, so that the sensitivity of downstream detection is insufficient. Therefore, development of a cfDNA extraction kit with high extraction efficiency and high purity of the obtained cfDNA is needed.
Disclosure of Invention
The invention provides a cfDNA extraction kit which can effectively improve the purity and content of cfDNA.
The invention provides a cfDNA extraction method which is simple and convenient to operate and suitable for high-throughput treatment.
The invention provides a cfDNA extraction kit, which comprises a first binding solution, a second binding solution and magnetic microspheres;
The magnetic microspheres comprise first magnetic microspheres and second magnetic microspheres, wherein the first magnetic microspheres and the second magnetic microspheres are of a core-shell structure and comprise a ferroferric oxide inner core and a silicon dioxide shell layer coated on at least part of the surface of the inner core, and carboxyl groups are modified on the silicon dioxide shell layer;
The pH of the first binding solution is 4-5, and comprises 0.5-1.5M guanidine hydrochloride and 5-15% (w/v) polyethylene glycol 8000;
the pH of the second binding solution is 6.5-7.5, and comprises 2-4M guanidine hydrochloride, 10-30% (v/v) isopropanol and 5-15 g/L beta-cyclodextrin.
A cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises proteinase K;
the concentration of proteinase K is 0.1-2 mg/mL.
The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises a lysate;
The lysate comprises 5-7M guanidine hydrochloride and 0.5-1.5% (v/v) triton X-100.
The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises a wash solution;
the washing liquid comprises 70-90% (v/v) ethanol and sodium acetate with pH of 5.0-6.0 and 0.1-0.5M.
A cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises an eluent;
the eluent comprises TE buffer with pH of 7.9-8.1.
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.
The cfDNA extraction kit as described above, wherein the particle size of the first magnetic microsphere and the second magnetic microsphere is 0.5-1.5 μm.
The cfDNA extraction kit as described above, wherein the first magnetic microsphere and the second magnetic microsphere are suspended in phosphate buffer solution or tris hydrochloride buffer solution with pH of 7.0-8.0, and the concentration of the first magnetic microsphere and the second magnetic microsphere is 5-15 mg/mL.
The invention provides a cfDNA extraction method, which is carried out by using the cfDNA extraction kit and comprises the following steps:
The body fluid sample is cracked to obtain a lysate, and the lysate, the first binding solution and the first magnetic microspheres are mixed to obtain first magnetic microspheres carrying impurities;
Mixing the supernatant, the second binding solution and the second magnetic microspheres to obtain the second magnetic microspheres carrying cfDNA, and washing and eluting to obtain cfDNA.
The cfDNA extraction method as described above, wherein 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 solution 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 at least one selected from blood, lymph, emulsion, urine, amniotic fluid.
The invention provides a cfDNA extraction kit which comprises a first binding solution, a second binding solution and magnetic microspheres, wherein the components and the contents of the first binding solution and the second binding solution are limited, so that the cfDNA extraction kit can promote the good matching effect with the magnetic microspheres, and further the bottleneck of the traditional single-step magnetic bead method is broken through by regulating the binding environment of the magnetic microspheres in stages, so that the cfDNA extraction efficiency and the cfDNA extraction purity are effectively improved, and the cfDNA extraction kit has simplicity and high repeatability in operation. The invention provides reliable technical support for liquid biopsy, and is suitable for application scenes such as liquid biopsy of tumors, noninvasive prenatal diagnosis and the like.
Detailed Description
The present invention will be described in further detail below for the purpose of better understanding of the aspects of the present invention by those skilled in the art. The following detailed description is merely illustrative of the principles and features of the present invention, and examples are set forth for the purpose of illustration only and are not intended to limit the scope of the invention. All other embodiments, which can be made by those skilled in the art based on the examples of the invention without making any inventive effort, are intended to be within the scope of the invention.
The magnetic bead method for extracting cell-free DNA (cfDNA) is one of the currently mainstream cfDNA extraction technologies, and is particularly suitable for liquid biopsy samples such as blood plasma, blood serum and the like. The core principle is that cfDNA is selectively adsorbed by using surface functionalized magnetic microspheres under a specific buffer condition, and then purification is realized by magnetic field separation. However, the length of the cfDNA fragments is generally 100-200 bp, and the short fragments carry fewer negative charges, and have weaker binding force with the magnetic microspheres, so that the existing magnetic bead method has lower binding efficiency on the cfDNA fragments with the length of 100-200 bp, and further the extraction efficiency and purity of the cfDNA are affected. Meanwhile, the magnetic microsphere also has the problem of nonspecific adsorption of long fragment genome DNA, and the difficulty in cfDNA extraction by a magnetic bead method is further increased.
To solve the above problems, the first aspect of the present invention provides a cfDNA extraction kit, which includes a first binding solution, a second binding solution, and magnetic microspheres.
In the present invention, the magnetic microspheres include a first magnetic microsphere and a second magnetic microsphere, which are magnetic microspheres having the same structure but different uses. The first magnetic microsphere and the second magnetic microsphere are of a core-shell structure, and comprise a ferroferric oxide inner core and a silicon dioxide shell layer coated on at least part of the inner core surface, wherein carboxyl groups are modified on the silicon dioxide shell layer.
In the cfDNA extraction process, the magnetic microspheres can be matched with the first binding solution or the second binding solution to provide two different purposes, namely (1) the first magnetic microspheres can capture impurities under the matching of the first binding solution and then can be easily separated from the solution under the action of an external magnetic field, and (2) the cfDNA can be captured by the second magnetic microspheres under the matching of the second binding solution and then can be easily separated from the solution under the action of the external magnetic field. The object captured by the magnetic microsphere can be regulated and controlled through the arrangement of the first binding liquid and the second binding liquid, so that the staged separation of impurities and cfDNA is realized.
In the present invention, the first binding liquid can drive impurities such as proteins and lipids to adsorb on the surface of the first magnetic microsphere. The pH of the first binding solution is 4.0-5.0, including 0.5-1.5M guanidine hydrochloride and 5-15% (w/v) polyethylene glycol 8000.
First, under the acidic (pH 4-5) condition provided by the first binding solution, the carboxyl (-COOH) modified on the surface of the first magnetic microsphere can be protonated, so that the negative charge (-COO-) on the surface of the first magnetic microsphere is reduced, and at this time, the net negative charge density of the surface of the first magnetic microsphere is reduced, but the whole surface of the first magnetic microsphere is still mainly negative. Under acidic conditions, amino (-NH 2) carried by protein, lipid and other impurities can be protonated to form positively charged ammonium (-NH 3 +), so that the impurities are positively charged as a whole. cfDNA consists of nucleotides whose phosphate backbone is negatively charged (-PO 4 3-), so that even under acidic conditions its net negative charge density is slightly reduced, but the overall negative charge is still dominant. Thus, positively charged impurities will preferentially electrostatically attract the remaining negative charge of the first magnetic microsphere surface, while negatively charged cfDNA will electrostatically repel the negative charge of the first magnetic bead surface.
Next, the first binding solution contains guanidine hydrochloride at a low concentration (0.5-1.5M). The low-concentration guanidine hydrochloride can reduce the ionic strength in a system, so that competitive adsorption of salt ions to impurity molecules on the surface of the first magnetic microsphere is reduced, and at the moment, the interaction of the impurity molecules (such as proteins) and the surface of the first magnetic microsphere is dominant in hydrophobic interaction, hydrogen bonding and the like, and compared with cfDNA, the impurity molecules are more easily adsorbed on the surface of the first magnetic microsphere. Meanwhile, the guanidine hydrochloride with low concentration can effectively avoid the damage of impurity structure, so that the guanidine hydrochloride with low concentration keeps natural overall conformation, and is easier to combine with the first magnetic microsphere through the surface functional group, for example, the hydrophobic region of the protein is easier to expose under the guanidine hydrochloride with low concentration, and the hydrophobic effect of the hydrophobic region and the surface of the magnetic bead is enhanced, so that the hydrophobic region is preferentially adsorbed.
Finally, the first binding solution contains polyethylene glycol 8000. Polyethylene glycol 8000 is a water-soluble macromolecular polymer, and can abstract water molecules on the surfaces of impurities such as proteins and lipids through osmotic pressure effect, compress hydration layers of the impurities, so that the impurity molecules are easier to be close to the surfaces of the magnetic microspheres, thereby strengthening the hydrophobic effect and electrostatic attraction between the impurities and the first magnetic microspheres and promoting the impurities to be preferentially adsorbed on the first magnetic microspheres. The macromolecular structure of polyethylene glycol 8000 can also form steric hindrance in the solution, so that nonspecific contact of negatively charged short cfDNA segments with residual negative charges on the surface of the first magnetic microsphere is reduced, cfDNA is prevented from being wrapped and precipitated by impurities, cfDNA is ensured to be stably reserved in supernatant, and conditions are created for capturing cfDNA specifically by a subsequent second binding solution. Therefore, polyethylene glycol 8000 and guanidine hydrochloride in the first binding solution cooperate, and the staged separation effect of preferential adsorption of impurities and efficient retention of cfDNA is realized by regulating and controlling the interface behavior of the impurities and cfDNA.
In short, the acidic environment enhances the electrostatic attraction of the impurities to the first magnetic microsphere by adjusting the charge state, while the low chaotropic salt concentration in combination with polyethylene glycol 8000 facilitates the adsorption of the impurities by reducing the ionic interference, enhancing the hydrophobic effect, etc. Under the synergistic effect of the two, impurities (such as proteins and lipids) are preferentially bound to the surface of the first magnetic microsphere, and cfDNA is relatively high in solubility under the conditions of charge repulsion and low salt, and remains in the supernatant, so that a foundation is laid for the specific capture of cfDNA in the subsequent second step.
In the invention, the second binding solution can effectively promote cfDNA to be adsorbed on the surface of the second magnetic microsphere. The pH of the second binding solution is 6.5-7.5, and comprises 2-4M guanidine hydrochloride, 10-30% (v/v) isopropanol and 5-15 g/L beta-cyclodextrin.
In one aspect, the second binding solution provides a neutral (pH 6.5-7.5) environment in which the surface-modified carboxyl groups (-COOH) of the second magnetic microspheres undergo deprotonation to become negatively charged carboxyl ions (-COO-), causing an increase in the negative charge density on the surfaces of the second magnetic microspheres. At this time, while the phosphate backbone of cfDNA is still negatively charged (-PO 4 3-), isopropanol in the second binding solution can neutralize some of the negative charge of cfDNA, reducing its solubility, while β -cyclodextrin reduces competition of lipid impurities for the hydrophobic sites of the second magnetic microsphere. Therefore, the improvement of the surface negative charge density of the second magnetic microsphere in the neutral environment is cooperated with the charge neutralization state of the cfDNA under the action of isopropanol, so that the cfDNA is more easily combined with the second magnetic microsphere in a hydrophobic action, an ion bridge and other modes, the cfDNA is effectively captured, the cfDNA and the first binding solution preferentially adsorb impurities in the acidic environment to form staged regulation and control, and the extraction efficiency and purity of the cfDNA are finally improved.
On the other hand, the high concentration (2-4M) guanidine hydrochloride in the second binding solution can destroy the binding of cfDNA to residual impurities, enhance the interaction of cfDNA with the second magnetic microsphere, and inhibit nuclease activity. Isopropyl alcohol (Isopropyl alcohol, IPA) can neutralize the negative charge of cfDNA, reduce the solubility of cfDNA, promote cfDNA precipitation and the combination of cfDNA with the hydrophobic area of the surface of the second magnetic microsphere, and dissolve impurities such as salt and small molecular substances, so that the purity of cfDNA is improved. Beta-cyclodextrin forms inclusion compound with lipid impurities preferentially, so that competitive adsorption of the lipid impurities to the hydrophobic region on the surface of the second magnetic microsphere is reduced. The isopropanol and the beta-cyclodextrin exert a synergistic effect through double hydrophobic effects, and jointly drive cfDNA to be adsorbed on the surface of the second magnetic microsphere in a more ordered conformation, so that the adsorption order of the cfDNA is enhanced, and the capture specificity is improved. The interaction of the guanidine hydrochloride enhanced cfDNA and the second magnetic microsphere has a synergistic effect with the charge neutralization effect of isopropanol and the lipid competition reducing effect of beta-cyclodextrin, so that the high-efficiency and specific capturing of the cfDNA are realized together, and the extraction efficiency and purity are improved.
Therefore, the cfDNA extraction kit provided by the invention can promote the good matching effect with the magnetic microspheres by limiting the components and the contents of the first binding liquid and the second binding liquid, and further breaks through the bottleneck of the traditional single-step magnetic bead method by regulating the magnetic microsphere binding environment in stages, thereby effectively improving the extraction efficiency and the extraction purity of cfDNA, and having the advantages of simplicity and high repeatability in operation. The invention provides reliable technical support for liquid biopsy, and is suitable for application scenes such as liquid biopsy of tumors, noninvasive prenatal diagnosis and the like.
In the technical scheme, the cfDNA extraction kit further comprises proteinase K, wherein the concentration of proteinase K is 0.1-2 mg/mL.
Proteinase K is a broad-spectrum serine protease that can efficiently degrade histones, nucleoproteins and other proteins (e.g., hemoglobin, albumin) that bind cfDNA, thereby releasing cfDNA. In addition, proteinase K can also protect cfDNA from degradation during extraction by degrading nucleases (e.g., DNAase), thereby ensuring the integrity of cfDNA.
Experiments show that when the concentration of proteinase K in the cfDNA extraction kit is 0.1-2 mg/mL, the cfDNA extraction kit is suitable for most body fluid samples, can exert better protein degradation effect, further improves the cfDNA extraction efficiency, and can avoid excessive enzyme activity and cost increase caused by overhigh concentration. Illustratively, the concentration of proteinase K may 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 and any value between any two of the ranges described above.
In the technical scheme, the cfDNA extraction kit also comprises a lysate, wherein the lysate comprises 5-7M guanidine hydrochloride and 0.5-1.5% (v/v) triton X-100.
The lysate containing the components can efficiently destroy cell structures possibly existing in a body fluid sample, release cfDNA, and inhibit nuclease activity to avoid cfDNA degradation. Wherein, 5-7M guanidine hydrochloride can be used as strong chaotropic agent to destroy the hydrogen bond and hydrophobic effect of protein, denatured histone, nucleoprotein and nuclease in body fluid sample to release cfDNA, and 0.5-1.5% (v/v) Triton X-100 (Triton X-100) can be used as nonionic detergent to destroy phospholipid bilayer of cell membrane, and together with guanidine hydrochloride to promote cell lysis and dissolve lipid impurity.
In the technical scheme, the cfDNA extraction kit also comprises a washing liquid, wherein the washing liquid comprises 70-90% (v/v) ethanol and sodium acetate with the pH of 5.0-6.0 and 0.1-0.5M.
In one aspect, 70-90% (v/v) ethanol can reduce cfDNA solubility, promote cfDNA precipitation, thereby promoting cfDNA binding to the second magnetic microsphere, and can also solubilize moisture, salt, protein and other organic impurities in the solution, helping to enhance the washing effect. Sodium acetate with pH of 5.0-6.0 and 0.1-0.5M is dissociated into acetate ions and sodium ions in the system, and the sodium ions weaken electrostatic repulsive force among cfDNA molecules by neutralizing negative charges on a cfDNA phosphate skeleton, so that the cfDNA molecules are more easily combined with functional groups (such as carboxyl) on the surface of the second magnetic microsphere, thereby reducing the loss of cfDNA in the washing process and improving the content of the cfDNA.
On the other hand, ethanol has a synergistic effect with sodium acetate. The ethanol can reduce the dielectric constant of the solution, promote the cfDNA to be dehydrated and precipitated, and the existence of sodium acetate can further reduce the solubility of the cfDNA, and meanwhile, the residual salt ions (such as guanidine salt) are combined through ionic bonds, so that the residual salt ions are removed along with the washing step, thereby reducing the impurity residues, avoiding the inhibition effect of the salt ions on downstream detection (such as PCR and sequencing), and improving the experimental reliability.
In the technical scheme, the cfDNA extraction kit further comprises an eluent, wherein the eluent comprises TE buffer with the pH of 7.9-8.1.
The TE buffer is a buffer consisting of Tris (hydroxymethyl) aminomethane (Tris (hydroxymethyl) aminomethane, tris) and ethylenediamine tetraacetic acid (ETHYLENE DIAMINE TETRAACETIC ACID, EDTA). The weak alkalinity (pH 7.9-8.1) of the TE buffer solution can break the ionic bond between cfDNA and the surface of the second magnetic microsphere, so that cfDNA is promoted to fall off from the surface of the second magnetic microsphere, and EDTA can also chelate metal ions to prevent nuclease from degrading cfDNA.
In the technical scheme, the carboxyl density of the first magnetic microsphere and the second magnetic microsphere is 1-5 mu mol/m < 2 >.
In the present invention, impurities are adsorbed to the hydrophobic region of the silica shell of the first magnetic microsphere mainly by hydrophobic interaction in the first binding phase, and cfDNA is also adsorbed to the hydrophobic region of the silica shell of the second magnetic microsphere mainly by hydrophobic interaction in the second binding phase.
Controlling the carboxyl density of the first magnetic microsphere and the second magnetic microsphere may optimize the degree of exposure of the hydrophobic region. The carboxyl groups are modified on the surface of the silicon dioxide shell layer, so that the density of the carboxyl groups determines the hydrophilic-hydrophobic balance of the surface of the shell layer, when the density of the carboxyl groups is too low (< 1 mu mol/m < 2 >) the hydrophobic region of the shell layer is excessively exposed, so that nonspecific adsorption (such as long fragment DNA and RNA) is increased, when the density of the carboxyl groups is too high (> 5 mu mol/m < 2 >), the carboxyl groups cover the hydrophobic region of the shell layer, hydrophobic binding of cfDNA and the surface of the second magnetic microsphere is weakened, when the density of the carboxyl groups of the first magnetic microsphere and the second magnetic microsphere is 1-5 mu mol/m < 2 >, the carboxyl groups are properly exposed on the hydrophobic region of the shell layer, only short fragment cfDNA (concentrated hydrophobic bases) is allowed to bind, and long fragment cfDNA (dispersed hydrophobic region is repelled and difficult to anchor).
The carboxyl density of the first magnetic microsphere and the second magnetic microsphere can be controlled to regulate and control the bonding strength of an ion bridge. The carboxyl density determines the number of binding sites of the ion bridge, if the carboxyl density is too low, the cfDNA is easy to run off in washing, if the ion bridge is too high, the ion bridge is required to be eluted at higher pH or temperature, and the cfDNA integrity is easy to be destroyed.
Controlling the carboxyl density of the first magnetic microsphere and the second magnetic microsphere may also indirectly affect impurity competition. The carboxyl groups are negatively charged under neutral conditions, which reduces hydrophobic competition of negatively charged impurities (e.g., RNA) by electrostatic repulsion.
Experiments show that when the carboxyl density of the first magnetic microsphere and the second magnetic microsphere is 1-5 mu mol/m < 2 >, the first magnetic microsphere can well play a role of binding impurities under the condition of a first binding solution, and the second magnetic microsphere can well play a role of binding 100-200 bp cfDNA under the condition of a second binding solution.
In the above technical solution, the particle diameters of the first magnetic microsphere and the second magnetic microsphere are 0.5-1.5 μm.
The larger the particle size is, the larger the specific surface area of the magnetic microsphere is, the more binding sites can be exposed, and the higher the adsorption efficiency of cfDNA is, namely, the larger the particle size of the magnetic microsphere is, the smaller the specific surface area of the magnetic microsphere is, the adsorption capacity is lower, but the surface binding sites are sparser in distribution, the non-specific adsorption can be reduced, and the magnetic microsphere is suitable for the scene with higher purity requirements.
The separation of the first magnetic microsphere and the second magnetic microsphere depends on an external magnetic field, the particle size directly influences the response speed of the magnetic microsphere in the magnetic field, the magnetic response of the small-particle-size magnetic microsphere is slower and can be absorbed by the magnetic field completely, the magnetic substance content of the large-particle-size magnetic microsphere is higher, the sedimentation in the magnetic field is faster, the separation time is short, and the magnetic microsphere is suitable for high-flux automatic operation.
In addition, the small-particle-size magnetic microspheres have better dispersibility, can be more fully contacted with a sample, reduce adsorption deviation caused by local concentration non-uniformity, and have poorer dispersibility, are easy to agglomerate due to gravity sedimentation, and possibly cause insufficient local binding sites.
In consideration of the above factors, experiments show that the particle sizes of the first magnetic microspheres and the second magnetic microspheres are 0.5-1.5 mu m, the particle sizes are proper, the specific surface area is moderate, the extraction efficiency and the purity requirement can be considered, and the operation is convenient and quick.
In the technical scheme, the first magnetic microspheres and the second magnetic microspheres are suspended in a phosphate buffer solution or a tris hydrochloride buffer solution with the pH of 7.0-8.0, and the concentration of the first magnetic microspheres and the second magnetic microspheres is 5-15 mg/mL.
The first magnetic microsphere and the second magnetic microsphere are suspended in the buffer solution, so that the dispersion stability of the magnetic microsphere can be maintained, and the agglomeration of the magnetic microsphere is avoided. The Phosphate Buffer Solution (PBS) contains sodium chloride, potassium chloride, phosphate and the like, can provide physiological ionic strength and prevent the magnetic microspheres from aggregating due to electrostatic action, and the Tris (Tris-HCl) buffer solution can stabilize the surface charge of the magnetic microspheres by adjusting the pH value and ensure that the magnetic microspheres keep activity in a suspension state.
When the concentration of the first magnetic microsphere and the second magnetic microsphere is 5-15 mg/mL, the adsorption efficiency and the operation convenience can be both considered, if the concentration of the magnetic microsphere is too low, cfDNA capture can be incomplete, and if the concentration of the magnetic microsphere is too high, the aggregation risk of the magnetic microsphere can be increased, and the separation efficiency is affected.
The second aspect of the present invention provides a cfDNA extraction method, which is performed using the cfDNA extraction kit, and comprises the following steps:
The body fluid sample is cracked to obtain a lysate, and the lysate, the first binding solution and the first magnetic microspheres are mixed to obtain first magnetic microspheres carrying impurities;
Mixing the supernatant, the second binding solution and the second magnetic microspheres to obtain the second magnetic microspheres carrying cfDNA, and washing and eluting to obtain cfDNA.
Specifically, the plasma sample can be mixed with proteinase K and lysate, and incubated at 55-65deg.C for 20-40 min to obtain lysate. Adding the first binding solution and the first magnetic microsphere suspension into the lysate, carrying out vortex oscillation and mixing uniformly, standing at room temperature for 3-10 min so as to promote the impurities to be adsorbed on the surfaces of the first magnetic microspheres and obtain the first magnetic microspheres carrying the impurities, removing the first magnetic microspheres carrying the impurities through magnetic separation, and keeping the supernatant. And then adding a second binding solution and a second magnetic microsphere suspension into the supernatant, uniformly mixing by vortex oscillation, standing at room temperature for 3-10 min so as to promote cfDNA to be adsorbed on the surfaces of the second magnetic microspheres to obtain second magnetic microspheres carrying cfDNA, and obtaining the second magnetic microspheres carrying cfDNA by magnetic separation. Adding a washing solution into the second magnetic microsphere carrying cfDNA, carrying out vortex oscillation for 5-20 s, standing at room temperature for 0.5-5 min, removing supernatant by magnetic separation, washing repeatedly for one time, and airing the second magnetic microsphere at room temperature. And finally, adding an eluent into the dried second magnetic microspheres, lightly blowing and uniformly mixing, incubating for 5-20 min at 50-70 ℃ to promote the dissociation of cfDNA and the second magnetic microspheres, removing the second magnetic microspheres through magnetic separation, and transferring the supernatant to a new centrifuge tube to obtain a cfDNA sample.
Further, the volume ratio of the first binding liquid 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.
Further, the volume ratio of the second binding liquid 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. Mu.L.
Further, the volume ratio of the first binding liquid to the second binding liquid is (1-2): 1-2. For example, the first binding fluid may be 2 mL and the second binding fluid may be 2 mL.
In an embodiment of the present invention, the plasma sample may be 2 mL, proteinase K may be 50 μl, the lysate may be 2 mL, the first conjugate may be 2 mL, the second conjugate may be 2 mL, the first magnetic microsphere suspension may be 50 μl, the second magnetic microsphere suspension may be 50 μl, the wash solution may be 500 μl, and the eluent may be 50 μl, which helps to improve cfDNA extraction efficiency and extraction purity.
Further, the body fluid sample is at least one selected from blood, lymph, emulsion, urine, amniotic fluid.
The technical scheme of the application will be further explained below with reference to specific examples. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The reagents used, unless otherwise specified, are commercially available or publicly available.
Example 1:
the embodiment provides a cfDNA extraction kit, which comprises proteinase K, lysate, first binding solution, second binding solution, first magnetic microsphere suspension, second magnetic microsphere suspension, washing solution and eluent;
wherein the concentration of proteinase K is 0.5 mg/mL;
the lysate is prepared from 6M guanidine hydrochloride and 1% (v/v) Triton X-100 (Triton X-100);
the pH of the first binding solution was 4.5, which was formulated from 1M guanidine hydrochloride and 10% (w/v) polyethylene glycol 8000;
The pH of the second binding solution was 7.0, which was formulated from 3M guanidine hydrochloride, 20% (v/v) isopropanol, and 10 g/L beta-cyclodextrin;
The magnetic microsphere is carboxylated magnetic microsphere with the particle size of 0.5-1.5 mu m, has a core-shell structure, comprises a ferroferric oxide (Fe 3O4) core and a silicon dioxide (SiO 2) shell layer coated on the surface of the core, wherein carboxyl is modified on the silicon dioxide shell layer, and the carboxyl density is 3 mu mol/m < 2 >, and is suspended in Phosphate Buffer Saline (PBS) with the pH of 7.4 to prepare magnetic microsphere suspension with the concentration of 10 mg/mL (the magnetic microsphere suspension is divided into a first magnetic microsphere suspension and a second magnetic microsphere suspension);
The washing liquid is prepared from 80% (v/v) ethanol and sodium acetate with pH of 5.5 and 0.3M;
the eluent was TE (Tris-EDTA) buffer (10 mM Tris-HCl, 1mM EDTA,pH 8.0.+ -. 0.1).
The embodiment also provides a method for extracting cfDNA by using the kit, which comprises the following steps:
(1) Lysis, namely mixing a2 mL plasma sample with 50 mu L of proteinase K and 2 mL lysate, and incubating at 60 ℃ for 30min to obtain a lysate;
(2) Adding 2 mL first binding solution and 50 mu L first magnetic microsphere suspension into the lysate, standing at room temperature (25 ℃) for 5 min after vortex oscillation and mixing to promote the impurities to be adsorbed on the surfaces of the first magnetic microspheres so as to obtain first magnetic microspheres carrying the impurities;
(3) Capturing, namely adding 2 mL parts of a second binding solution and 50 mu L of a second magnetic microsphere suspension into the supernatant, standing at room temperature (25 ℃) for 5 min parts after vortex oscillation and uniform mixing so as to promote cfDNA to be adsorbed on the surfaces of the second magnetic microspheres and obtain second magnetic microspheres carrying cfDNA;
(4) Washing, namely adding 500 mu L of washing liquid into the second magnetic microsphere carrying cfDNA, carrying out vortex oscillation for 10 s, standing for 1 to min at room temperature (25 ℃), removing supernatant by magnetic separation, repeatedly washing once, and airing the second magnetic microsphere at room temperature (25 ℃);
(5) And (3) eluting, namely adding 50 mu L of eluent into the dried second magnetic microspheres, lightly blowing and uniformly mixing, incubating at 60 ℃ for 10min to promote dissociation of cfDNA and the second magnetic microspheres, removing the second magnetic microspheres through magnetic separation, and transferring the supernatant into a new centrifuge tube to obtain a cfDNA sample.
Comparative example 1:
the present comparative example uses a large amount of free DNA extraction kit of magnetic bead method manufactured by guangzhou meiji biotechnology limited company under the product number IVD5435 for cfDNA extraction, the extraction kit comprises proteinase K, buffer SDS, binding solution MLK, magnetic bead solution MPF, washing solution MAW1, washing solution MAW2 and eluent EB, and the extraction process comprises the following steps:
(1) Lysing, namely transferring 100 mu L of proteinase K to a centrifuge tube of 15 mL, transferring 2 mL plasma samples to the centrifuge tube, adding 100 mu LBuffer SDS to the centrifuge tube, mixing the mixture for a plurality of times in a reversed manner, incubating the mixture at 55 ℃ for 30 min, reversing the mixture for a plurality of times, and standing the mixture at room temperature (25 ℃) for 5min to restore the lysate to room temperature;
(2) Adding 3.8 mL binding solution MLK and 150 mu L magnetic bead MPF into the lysate, mixing uniformly at room temperature (25 ℃) upside down for 10min, transferring to a magnetic rack, standing for 5min to enable cfDNA to adsorb the magnetic beads, sucking and discarding the solution, and sucking and discarding residual liquid after short centrifugation;
(3) Washing, namely adding 1.0 mL of washing solution MAW1 into the magnetic beads, swirling 5 s, reversing for 10-15 times, transferring to a magnetic rack to adsorb 1 min, absorbing and discarding the solution, repeatedly washing once by using the washing solution MAW1, adding 1.0 mL of washing solution MW2, swirling 5 s, reversing for 10-15 times, transferring to the magnetic rack to adsorb 1 min, absorbing and discarding the solution, repeatedly washing once by using the washing solution MAW2, absorbing and discarding residual liquid after short centrifugation, and drying the magnetic beads by using 37 ℃ metal bath for 10-15min to completely dry the magnetic beads;
(4) And (3) eluting, namely adding 50 mu L of eluent EB into the dried magnetic beads, carrying out shaking incubation at 37 ℃ for 5 min ℃ to enable cfDNA to be fully dissolved, transferring to a magnetic rack for adsorbing 1 min, transferring supernatant containing cfDNA to a new centrifuge tube, carrying out short centrifugation on the original centrifuge tube, and collecting residual liquid into the new centrifuge tube to obtain a cfDNA sample.
Comparative example 2:
This comparative example provides a cfDNA extraction kit comprising proteinase K, lysate, first binding solution, second binding solution, first magnetic microsphere suspension, second magnetic microsphere suspension, washing solution, and eluent, wherein the components and contents thereof can be referred to example 1, except that the second binding solution does not contain beta-cyclodextrin.
The comparative example also provides a method for cfDNA extraction using the above kit, and specific steps can be referred to example 1 to obtain cfDNA samples.
Comparative example 3:
The present comparative example provides a cfDNA extraction kit comprising proteinase K, lysate, first binding solution, second binding solution, first magnetic microsphere suspension, second magnetic microsphere suspension, washing solution, and eluent, wherein the components and contents thereof can be referred to example 1, except that the second binding solution does not contain isopropanol.
The comparative example also provides a method for cfDNA extraction using the above kit, and specific steps can be referred to example 1 to obtain cfDNA samples.
Test case
(1) The absorbance at 260 nm (a 260) and the absorbance at 280 nm (a 280) of the cfDNA sample described above were tested using an 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.
(2) The cfDNA content (in ng) of 100-200 bp in the cfDNA samples described above was tested according to capillary electrophoresis using a fully automated nucleic acid protein analysis system Qsep, the results of which are shown in table 2.
TABLE 1
TABLE 2
According to the results of table 1 and table 2, it can be found that the cfDNA purity (a260/a280=1.75-1.85) extracted by using the cfDNA extraction kit and the cfDNA extraction method provided by the invention is obviously better than that of the single-step magnetic bead method (a260/a280=1.65-1.67) of comparative example 1, and the cfDNA content (16.9-18.8 ng) of 100-200 bp is obviously improved compared with that of the single-step magnetic bead method (10.9-12.6) of comparative example 1, so that the invention can break through the bottleneck of the traditional single-step magnetic bead method by regulating the magnetic microsphere combination environment in stages, has the advantages of simplicity of operation and high repeatability (CV is less than or equal to 5%), effectively improves the cfDNA extraction efficiency and purity, provides reliable technical support for liquid biopsy, and is suitable for application scenes such as tumor liquid biopsy and non-invasive prenatal diagnosis.
Furthermore, it can be found from the results of tables 1 and 2 that guanidine hydrochloride, isopropyl alcohol and beta-cyclodextrin in the second binding solution of the present invention have a synergistic effect, and that only when the three are used together, a significant improvement in cfDNA extraction efficiency and purity can be achieved.
Finally, it should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not deviate the essence of the corresponding technical solution from the scope of the technical solution of the embodiments of the present invention.
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