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 protease K, a lysis buffer solution, magnetic beads and a binding buffer solution, by limiting the composition of the lysis buffer solution and the binding buffer solution, not only can the extraction efficiency of cfDNA be effectively improved, but also the purity of the cfDNA can be remarkably improved, the cfDNA of 100-200 bp can be effectively enriched, and the kit is convenient in component acquisition, low in cost and suitable for industrial production. Meanwhile, the extraction operation is simple and suitable for high-throughput treatment.

Description

Technical Field

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

[0002] Cell-free DNA (cfDNA) refers to DNA fragments that are free outside cells, with a length between 100 and 200 base pairs. It is mainly released into the extracellular environment of the human body through pathways such as apoptosis and necrosis, and is often present in physiological extracellular environments such as blood, lymph, milk, urine, and amniotic fluid.

[0003] Currently, cfDNA detection is a common form of liquid biopsy in the market and has been widely used in multiple aspects such as tumor detection, guiding evaluation, and prognostic evaluation. For example, by detecting cfDNA of tumor origin, tumor-related mutations, loss of heterozygosity, gene amplification, oncovirus DNA, hypermethylation of the promoter region of tumor suppressor genes, etc. can be found, thereby enabling the study of tumor DNA under non-invasive conditions. However, cfDNA has a low content and small fragments, making extraction difficult, and the extraction process is prone to deletion, resulting in low detection sensitivity, which to a certain extent limits its application in clinical diagnosis. Therefore, there is an urgent need 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, which can improve the extraction efficiency of cfDNA, increase the purity of the obtained cfDNA, and effectively enrich cfDNA of 100 - 200 bp.

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

[0006] The present invention provides a cfDNA extraction kit, which includes proteinase K, lysis buffer, magnetic beads, and binding buffer;

[0007] The lysis buffer includes 0.1 - 0.5 M proline, 1 - 6 mM dithiothreitol, 0.1 - 0.8 M thiourea, pH 7.5 - 8.5, 50 - 100 mM tris(hydroxymethyl)aminomethane hydrochloride, 1 - 5 mM ethylenediaminetetraacetic acid, and 0.1 - 1.0% (v / v) Triton X-100;

[0008] The binding buffer includes 2 - 10 M guanidine hydrochloride, 4 - 16% (w / v) polyethylene glycol 8000, pH 6 - 6.5, 25 - 35 mM tris(hydroxymethyl)aminomethane hydrochloride, 1.0 - 2.0 mM ethylenediaminetetraacetic acid, and 0.01 - 0.1 wt% Triton X-100.

[0009] The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises a first washing solution;

[0010] The first washing solution includes 0.5-3 M guanidine hydrochloride, pH 7.0-8.0, 0.4-0.6% (w / v) tris hydrochloride, and 70-80% (v / v) ethanol.

[0011] The cfDNA extraction kit as described above, wherein the cfDNA extraction kit further comprises a second washing solution;

[0012] The second wash solution includes 0.05-0.5 M sodium chloride, 0.05-0.15 mM EDTA, and 70-80% (v / v) ethanol.

[0013] The cfDNA extraction kit as described above, wherein the concentration of proteinase K is 0.5-1.0 mg / mL.

[0014] In the cfDNA extraction kit as described above, the magnetic microbeads are of a core-shell structure, comprising a ferroferric oxide core and a silica shell coating at least a portion of the surface of the core, wherein the silica shell is modified with hydroxyl groups.

[0015] The cfDNA extraction kit as described above, wherein the modification density of the hydroxyl groups is 2-8 hydroxyl groups / nm 2 .

[0016] In the cfDNA extraction kit as described above, the particle size of the magnetic microbeads is 0.4-0.8 μm.

[0017] 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:

[0018] The body fluid sample is incubated with proteinase K and lysis buffer to obtain a lysis mixture;

[0019] mixing the lysis mixture, magnetic microbeads and binding buffer to obtain a binding mixture;

[0020] washing the binding mixture with a first washing solution and a second washing solution in sequence to obtain a washing product;

[0021] The washed product is eluted with an elution buffer to obtain cfDNA.

[0022] The cfDNA extraction method as described above, wherein the volume ratio of the body fluid sample, proteinase K, lysis buffer, magnetic beads and binding buffer is (95-105):(4-6):(4-6):(1-4):(105-115).

[0023] The cfDNA extraction method as described above, wherein the body fluid sample is selected from at least one of blood, lymph fluid, milk, urine, amniotic fluid

[0024] The present invention provides a cfDNA extraction kit, comprising proteinase K, lysis buffer, magnetic beads and binding buffer. By restricting the composition of the lysis buffer and the binding buffer, it can not only effectively improve the extraction efficiency of cfDNA, but also significantly improve the purity of cfDNA, and effectively enrich cfDNA of 100-200 bp. Moreover, the components of the kit are convenient to obtain and low in cost, and at the same time, the extraction operation is simple and suitable for high-throughput processing. Detailed implementation manners

[0025] To enable those skilled in the art to better understand the solution of the present invention, the present invention will be further described in detail below. The specific implementation manners listed below only describe the principles and features of the present invention, and the examples given are only used to explain the present invention, not to limit the scope of the present invention. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] Currently, the extraction methods of cell-free DNA (cfDNA) can be mainly divided into traditional phenol / chloroform 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. It can directly extract cfDNA from crude samples such as plasma, reducing the pretreatment steps, and its shearing force on DNA is small, which helps to maintain the integrity of cfDNA.

[0027] However, the concentration of cfDNA in body fluids is low and the content is small, and the extraction efficiency of existing magnetic bead extraction kits is often not high, which may lead to insufficient sensitivity in downstream detection. At the same time, cfDNA is easily contaminated by genomic DNA, especially when cell lysis is incomplete or plasma is not processed in time, which will significantly affect the detection of low-abundance cfDNA such as circulating tumor DNA (ctDNA). In addition, some application scenarios require enrichment of cfDNA fragments within a specific size range, while existing magnetic bead extraction kits lack flexibility in this regard. The cost of some commercial magnetic bead extraction kits is also relatively high, which also limits their application in resource-limited environments.

[0028] To solve the above problems, a first aspect of the present invention provides a cfDNA extraction kit, comprising proteinase K, lysis buffer, magnetic beads, and binding buffer; the lysis buffer comprises 0.1-0.5 M proline, 1-6 mM dithiothreitol, 0.1-0.8 M thiourea, pH 7.5-8.5, 50-100 mM tris(hydroxymethyl)aminomethane hydrochloride, 1-5 mM ethylenediaminetetraacetic acid, and 0.1-1.0% (v / v) polyethylene glycol octylphenyl ether; the binding buffer comprises 2-10 M guanidine hydrochloride, 4-16% (w / v) polyethylene glycol 8000, pH 6-6.5, 25-35 mM tris(hydroxymethyl)aminomethane hydrochloride, 1.0-2.0 mM ethylenediaminetetraacetic acid, and 0.01-0.1 wt% polyethylene glycol octylphenyl ether.

[0029] First, in cfDNA extraction, proteinase K is a broad-spectrum serine protease that can efficiently degrade histones, nucleoproteins, and other proteins (such as hemoglobin and albumin) bound to DNA, thereby releasing cfDNA. In addition, proteinase K can also avoid the degradation of cfDNA during extraction by degrading nucleases (such as DNAase), thus ensuring the integrity of cfDNA.

[0030] Second, the composition of the lysis buffer is crucial for the successful release and purification of high-quality cfDNA. Through long-term experiments, it has been found that the lysis buffer prepared by compounding proline, dithiothreitol, thiourea, tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, and polyethylene glycol octylphenyl ether can effectively improve the cfDNA extraction efficiency and enhance the purity of the obtained cfDNA.

[0031] Specifically, proline can disrupt the hydrophobic cavity of proteins, enhance the denaturation effect of thiourea on histones, thereby promoting the improvement of cfDNA release efficiency; it can act as an osmoprotectant to maintain the stability of the cfDNA phosphate backbone in the lysis system and reduce base damage during high-temperature incubation; it can also bind to heme in blood, reducing its inhibitory effect on proteinase K, and is thus particularly suitable for the extraction of blood samples.

[0032] On the one hand, dithiothreitol, as a strong reducing agent, can disrupt the disulfide bonds in proteins, causing protein denaturation and removal, thus contributing to the release of cfDNA; on the other hand, it can maintain a reducing environment to protect cfDNA from oxidative damage.

[0033] Thiourea is a denaturant that can interfere with the hydrogen bonds and hydrophobic interactions of proteins, thereby promoting protein denaturation and dissolution, and can also chelate metal ions to protect the integrity of cfDNA.

[0034] Tris hydrochloride is a commonly used pH buffer that can effectively maintain the stability of the pH value of the solution, thereby protecting the stability of cfDNA.

[0035] Ethylenediaminetetraacetic acid can chelate metal ions (such as calcium ions, magnesium ions, etc.), thereby inhibiting the activity of nucleases and avoiding the degradation of cfDNA.

[0036] Triton X-100 is a non-ionic detergent that can reduce the surface tension of the solution, promote the uniform mixing of various reagents, ensure that all components can evenly contact cfDNA, and improve the extraction efficiency; it can also improve the purity of cfDNA by disrupting cell membranes and dissolving membrane proteins to reduce non-specific binding of proteins during extraction.

[0037] In the present invention, Proteinase K directly degrades proteins, dithiothreitol enhances protein denaturation by reducing disulfide bonds, and thiourea further disrupts the protein structure. The three work together to efficiently release cfDNA from protein complexes. Triton X-100 assists cell lysis through membrane disruption, accelerates protein exposure, and forms a synergy of "chemical degradation + physical membrane disruption" with the protease system, significantly enhancing the release efficiency.

[0038] Ethylenediaminetetraacetic acid and thiourea can also inhibit nuclease activity by chelating metal ions, dithiothreitol maintains a reducing environment to prevent oxidative damage, and Tris hydrochloride stabilizes the pH to avoid acid-base damage. Thus, a cfDNA protection network can be constructed multi-dimensionally to reduce degradation losses during extraction.

[0039] Triton X-100 reduces non-specific binding of proteins to cfDNA, Proteinase K degrades free proteins, and thiourea and ethylenediaminetetraacetic acid remove impurities through denaturation / chelating effects, jointly reducing protein contamination; the synergistic effect of each component avoids the massive release of genomic DNA (gDNA) (because the lysis buffer is mild and specifically acts on the cfDNA-protein complex), further improving the purity of cfDNA.

[0040] The combination of the pH buffering ability of Tris hydrochloride and the metal ion chelating effects of ethylenediaminetetraacetic acid and thiourea can also maintain the stability of the system chemical environment, ensure the activity of reagents such as Proteinase K, and avoid a decrease in extraction efficiency caused by environmental fluctuations.

[0041] The component design of the above lysis buffer follows the multiple logics of "efficient release - precise protection - impurity removal - system stability". Each component synergistically complements through chemical actions (such as enzymatic hydrolysis, denaturation, chelation) and physical actions (such as membrane disruption, detergency), not only achieving the efficient release of cfDNA, but also maintaining its integrity through a multi-dimensional protection mechanism, while significantly reducing protein and genomic DNA contamination, and ultimately improving the extraction efficiency and purity. This compounding strategy is designed based on the core pain points of "difficult release, easy degradation, and easy contamination" in the cfDNA extraction process, and the synergistic effect of each component is the key to achieving the technical effect.

[0042] Meanwhile, through a large number of experiments, the present invention has found that when the contents of proline, dithiothreitol, thiourea, tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, and Triton X-100 in the lysis buffer meet the above ranges, the effect of the lysis buffer can be enhanced to ensure the effective lysis of cells, the sufficient removal of proteins, and the integrity and purity of cfDNA.

[0043] In one embodiment of the present invention, the lysis buffer is prepared from 0.3 M proline, pH 7.5, 50 mM tris(hydroxymethyl)aminomethane hydrochloride, 2 mM ethylenediaminetetraacetic acid, 0.5% (v / v) Triton X-100, 3 mM dithiothreitol, and 0.4 M thiourea.

[0044] Next, in cfDNA extraction, magnetic beads provide an efficient and convenient method for separating and purifying cfDNA. The magnetic beads can capture cfDNA, and under the action of an external magnetic field, the magnetic beads captured with cfDNA can be easily separated from the solution, thereby effectively removing impurities in the lysis buffer and only retaining the cfDNA bound to the magnetic beads.

[0045] Finally, in cfDNA extraction, the composition of the binding buffer is crucial for promoting the successful binding of cfDNA to magnetic beads, thereby separating and purifying high-quality cfDNA. Through long-term experiments, it has been found that compounding guanidine hydrochloride, polyethylene glycol 8000, tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, and Triton X-100 to obtain the binding buffer can effectively improve the cfDNA extraction efficiency and enhance the purity of the obtained cfDNA.

[0046] Specifically, in the binding buffer, guanidine hydrochloride is a potent denaturant that can disrupt the tertiary and secondary structures of proteins, helping to remove protein impurities and making cfDNA more likely to bind to magnetic beads; guanidine hydrochloride can neutralize the negative charge of cfDNA by providing a high-salt environment, reducing the electrostatic repulsion between cfDNA molecules and facilitating the interaction between cfDNA and the binding groups on the surface of magnetic beads; guanidine hydrochloride can inhibit the activity of nucleases, preventing the degradation of cfDNA during extraction and helping to maintain the integrity and quality of cfDNA; guanidine hydrochloride helps to dissolve cell debris and other organic impurities, thus improving the purity of the extracted cfDNA.

[0047] Polyethylene glycol 8000 is a high-molecular-weight polymer that can reduce the solvation water layer around cfDNA molecules. It can not only reduce the probability of cfDNA hydrolysis reaction but also promote the aggregation and precipitation of cfDNA, thereby facilitating the interaction between cfDNA and magnetic beads.

[0048] In the present invention, the high-salt environment provided by guanidine hydrochloride neutralizes the negative charge of cfDNA, and polyethylene glycol 8000 promotes the aggregation of cfDNA through the polymer effect. The two work together to make cfDNA more likely to adsorb onto the hydroxyl groups on the surface of magnetic beads, forming a dual promotion mechanism of "chemical neutralization + physical aggregation".

[0049] Tris hydrochloride maintains an appropriate pH environment. Under this condition, the binding efficiency between the silanol groups (Si-OH) on the surface of magnetic beads and the phosphate backbone of cfDNA through hydrogen bonds and salt bridges is the highest, forming an environmental adaptation synergy with guanidine hydrochloride and PEG 8000.

[0050] Guanidine hydrochloride can also inhibit the activity of nucleases, and ethylenediaminetetraacetic acid chelates metal ions to achieve dual blocking of nuclease catalysis and prevent the degradation of cfDNA; Triton X-100 removes proteins and membrane impurities. The three work together to reduce the degradation and contamination of cfDNA, forming a protection network of "enzyme inhibition - metal chelation - impurity removal".

[0051] The denaturing effect of guanidine hydrochloride combined with the detergency of Triton X-100 can more thoroughly remove protein impurities, avoid non-specific binding of impurities to magnetic beads, and thus improve the purity of cfDNA.

[0052] The pH buffering capacity of Tris hydrochloride combined with the metal ion chelating effect of ethylenediaminetetraacetic acid maintains the chemical environment stability of the system, ensures that the denaturing efficiency of guanidine hydrochloride and the precipitation-promoting effect of polyethylene glycol 8000 are not disturbed, and avoids the decrease in binding efficiency caused by environmental fluctuations.

[0053] The component design of the above-mentioned binding buffer aims at the core goals of "promoting binding - protecting cfDNA molecules - removing impurities - stabilizing the system". Each component synergistically complements through chemical actions (such as denaturation, chelation) and physical actions (such as aggregation, detergency): guanidine hydrochloride and polyethylene glycol 8000 jointly drive the binding of cfDNA to magnetic beads; tris(hydroxymethyl)aminomethane hydrochloride and ethylenediaminetetraacetic acid maintain environmental stability and protect cfDNA; octylphenoxypolyethoxyethanol reduces non-specific adsorption. This compounding strategy enables the binding buffer to efficiently capture cfDNA under high-salt conditions, simultaneously achieve impurity removal and molecular protection, and ultimately significantly improve the extraction efficiency (such as the increase in the total DNA content in the test example) and the purity of cfDNA (the proportion of 100 - 200 bp fragments increases), reflecting the key value of the synergistic effect of each component.

[0054] Meanwhile, through a large number of experiments, the present invention has found that when the contents of guanidine hydrochloride, polyethylene glycol 8000, tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid and octylphenoxypolyethoxyethanol in the binding buffer meet the above ranges, the effect of the binding buffer can be enhanced to ensure the effective binding of cfDNA to magnetic microspheres.

[0055] In one embodiment of the present invention, the binding buffer is prepared from 5 M guanidine hydrochloride, 8% (w / v) polyethylene glycol 8000, pH 6.2, 30 mM tris(hydroxymethyl)aminomethane hydrochloride, 1.5 mM ethylenediaminetetraacetic acid and 0.05 wt% octylphenoxypolyethoxyethanol.

[0056] Therefore, the cfDNA extraction kit provided by the present invention can not only effectively improve the extraction efficiency of cfDNA, but also significantly improve the purity of cfDNA, effectively enrich cfDNA of 100 - 200 bp, and the components of the kit are convenient to obtain and low in cost. At the same time, the extraction operation is simple and suitable for high-throughput processing.

[0057] In the above technical solution, the cfDNA extraction kit further includes a first washing solution; the first washing solution includes 0.5 - 3 M guanidine hydrochloride, pH 7.0 - 8.0, 0.4 - 0.6% (w / v) tris(hydroxymethyl)aminomethane hydrochloride, 70 - 80% (v / v) ethanol.

[0058] Adding ethanol can play a role in reducing the solubility of cfDNA, promoting the precipitation of cfDNA, and making cfDNA easier to bind to magnetic beads. Ethanol can also effectively remove water, salts, proteins and other organic impurities in the solution, helping to enhance the washing effect.

[0059] The first washing solution of the present invention is a high-salt buffer solution. By increasing the ionic strength of the solution, the solubility of proteins and other impurities can be reduced, thereby effectively removing the proteins and other organic impurities bound to cfDNA. At the same time, the high-salt condition helps to enhance the binding force between cfDNA and magnetic beads, ensuring that cfDNA will not be eluted during the washing process.

[0060] Through a large number of experiments, the present invention has found that when the contents of guanidine hydrochloride, tris(hydroxymethyl)aminomethane hydrochloride, and ethanol in the first washing solution meet the above ranges, the effect of the first washing solution can be enhanced, effectively removing water, salts, proteins, and other organic impurities in the solution, while ensuring the effective binding of cfDNA and magnetic beads during the washing process.

[0061] In an embodiment of the present invention, the first washing solution is prepared from 1 M guanidine hydrochloride, pH 7.5, 0.5% (w / v) tris(hydroxymethyl)aminomethane hydrochloride, and 70% (v / v) ethanol.

[0062] In the above technical solution, the cfDNA extraction kit further includes a second washing solution; the second washing solution includes 0.05 - 0.5 M sodium chloride, 0.05 - 0.15 mM ethylenediaminetetraacetic acid, and 70 - 80% (v / v) ethanol.

[0063] Due to the presence of ethylenediaminetetraacetic acid and ethanol, the second washing solution can also remove salts, proteins, and other organic impurities, and strengthen the binding of cfDNA and magnetic beads. In addition, the sodium chloride in the second washing solution provides a low-salt condition, which can remove the residual salts during the high-salt washing process, help to reduce the ionic strength, and reduce the inhibitory effect on downstream enzyme reactions (such as PCR). Further washing with the second washing solution can also remove non-specifically bound impurities and improve the purity of cfDNA. At the same time, the low-salt condition helps to prepare for the elution step of cfDNA, promoting the efficient recovery of cfDNA in the subsequent elution step.

[0064] Through a large number of experiments, the present invention has found that when the contents of sodium chloride, ethylenediaminetetraacetic acid, and ethanol in the second washing solution meet the above ranges, the effect of the second washing solution can be enhanced, effectively removing salts, proteins, and other organic impurities in the solution, and preparing for the subsequent elution of cfDNA.

[0065] In an embodiment of the present invention, the second washing solution is prepared from 0.1 M sodium chloride (NaCl), 0.1 mM EDTA, and 70% (v / v) ethanol.

[0066] Furthermore, the cfDNA extraction kit of the present invention may further include an elution solution, and the elution solution may be pure water.

[0067] In the above technical solution, the concentration of Proteinase K is 0.5 - 1.0 mg / mL, which can enhance the proteolytic effect of Proteinase K.

[0068] In the above technical solution, in order to capture more cfDNA and enhance the binding of magnetic beads to cfDNA, while reducing the non-specific binding of magnetic beads to genomic DNA, the present invention can use magnetic beads with a core-shell structure, and the magnetic beads include a magnetite core and a silica shell layer coated on at least part of the surface of the core, and the silica shell layer is modified with hydroxyl groups.

[0069] Among them, the magnetite core provides magnetism, and by cooperating with an external magnetic field, the separation of cfDNA from the solution can be simply and conveniently achieved. Under high-salt and low-pH conditions, the silanol groups (Si-OH) on the surface of the magnetic beads can bind to the phosphate backbone of nucleic acids through hydrogen bonding and salt bridge effects, thereby realizing the adsorption of cfDNA.

[0070] Furthermore, by precisely regulating the modification density of hydroxyl groups, the non-specific binding to large-fragment genomic DNA can be reduced, while promoting the magnetic beads to more effectively capture small-fragment cfDNA, especially improving the selectivity for cfDNA fragments of a specific size of 100 - 200 bp. Specifically, the modification density of hydroxyl groups is 2 - 8 hydroxyl groups / nm 2 。Through such a high-density hydroxyl modification, more DNA binding sites can be provided, and the mutual interference of adjacent groups will cause steric hindrance, which can hinder the stretching and binding of long-fragment DNA, and finally selectively enrich cfDNA of 100 - 200 bp

[0071] In an alternative embodiment, the particle size of the magnetic beads is 0.4 - 0.8 μm. The smaller magnetic beads within this particle size range have a larger specific surface area, which can provide more cfDNA binding sites, thereby improving the binding efficiency of cfDNA and being more easily maintained in uniform suspension, helping to ensure that cfDNA uniformly contacts the surface of the magnetic beads.

[0072] In addition, the magnetic beads can be suspended in a solvent during use, and the concentration can be 5 - 20 mg / mL; in one embodiment, the magnetic beads are suspended in a 20% ethanol aqueous solution.

[0073] The second aspect of the present invention provides a method for extracting cfDNA, which is carried out using the cfDNA extraction kit provided in the first aspect of the present invention, and includes the following steps:

[0074] Incubate the body fluid sample with Proteinase K and lysis buffer to obtain a lysis mixture;

[0075] Mix the lysis mixture, magnetic beads, and binding buffer to obtain a binding mixture;

[0076] Wash the binding mixture successively with a first wash solution and a second wash solution to obtain a washed product;

[0077] Elute the washed product with an elution solution to obtain cfDNA.

[0078] In a specific embodiment, a body fluid sample, proteinase K, and lysis buffer can be first added to a 15 mL centrifuge tube. After vortexing and mixing evenly, incubate at 55 °C for 30 min, and then place at room temperature (25 °C) for 5 - 10 min to restore to room temperature (25 °C) to obtain a lysis mixture.

[0079] Then, add binding buffer and magnetic beads to the lysis mixture, invert and mix evenly at room temperature (25 °C) for 5 min to obtain a binding mixture. Transfer the centrifuge tube containing the binding mixture to a magnetic stand, magnetize for 5 min, and after clarification, discard the supernatant.

[0080] Next, remove the centrifuge tube from the magnetic stand, add the first wash solution to the tube, vortex and mix evenly for 5 - 10 s, and transfer it to a new 1.5 mL centrifuge tube; place the 1.5 mL centrifuge tube on the magnetic stand, magnetize for 5 min, and after clarification, aspirate the supernatant back into the original 15 mL centrifuge tube, rinse the tube wall and tube cap, and transfer it to a 1.5 mL centrifuge tube. Place the 1.5 mL centrifuge tube on the magnetic stand, magnetize for 5 min, and after clarification, discard the supernatant.

[0081] Subsequently, add the second wash solution to the 1.5 mL centrifuge tube, vortex and mix evenly for 5 - 10 s, centrifuge instantaneously, place the 1.5 mL centrifuge tube on the magnetic stand, magnetize for 2 min, and after clarification, discard the supernatant. Repeat this step. After discarding the supernatant, centrifuge instantaneously, and use a small - volume pipette to carefully aspirate the remaining supernatant. Open the 1.5 mL centrifuge tube and air - dry the magnetic beads at room temperature for 5 minutes.

[0082] Finally, add an elution solution (which can be pure water) to the 1.5 mL centrifuge tube, vortex and mix evenly for 5 - 10 s, let it stand at 60 °C for 5 min after mixing evenly. Centrifuge instantaneously, place the centrifuge tube on the magnetic stand, magnetize for 3 - 5 min, and after clarification, aspirate the DNA solution and transfer it to a new centrifuge tube to obtain a cfDNA sample.

[0083] In the above technical solution, the volume ratio of the body fluid sample, proteinase K, lysis buffer, magnetic beads and binding buffer is (95 - 105):(4 - 6):(4 - 6):(1 - 4):(105 - 115). Within this range, each reagent in the cfDNA extraction kit can exert the best effect. For example, the body fluid sample can be 4 mL, proteinase K can be 200 μL, lysis buffer can be 200 μL, magnetic beads can be 80 μL, and binding buffer can be 4.5 mL.

[0084] In the above technical solution, the body fluid sample is selected from at least one of blood, lymph fluid, milk, urine, and amniotic fluid.

[0085] Hereinafter, the technical solution of the present application will be further explained and illustrated in conjunction with specific embodiments. For the experimental methods without specific conditions noted in the following embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The reagents used, unless otherwise specified, are commercially available or can be obtained through public channels.

[0086] Example 1:

[0087] This example provides a cfDNA extraction kit, including proteinase K, lysis buffer, magnetic beads, binding buffer, first washing solution, second washing solution and elution solution.

[0088] Among them, the concentration of proteinase K is 0.8 mg / mL.

[0089] The lysis buffer is prepared from 0.3 M proline, pH 7.5, 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5% (v / v) octylphenoxypolyethoxyethanol (Triton X-100), 3 mM dithiothreitol (DTT) and 0.4 M thiourea.

[0090] The magnetic beads are PuriMag Si-COOH carboxyl-modified magnetic beads produced by PuriMag with the product number PuriMag Si-COOH, and the particle size is 0.4 - 0.8 μm. The magnetic beads have a core-shell structure, including a magnetite (Fe3O4) core and a silica (SiO2) shell layer coated on the surface of the core, and hydroxyl groups are modified on the SiO2 shell layer, and the modification density of the hydroxyl groups is 5 hydroxyl groups / nm 2 . The magnetic beads are suspended in a 20% ethanol aqueous solution, and the volume used in Test Example 1 is 80 μL.

[0091] The binding buffer is prepared from 5 M guanidine hydrochloride (GuHCl), 8% (w / v) polyethylene glycol 8000 (PEG 8000), pH 6.2, 30 mM Tris-HCl, 1.5 mM EDTA, and 0.05 wt% Triton X-100.

[0092] The first washing solution is prepared from 1 M GuHCl, pH 7.5, 0.5% (w / v) Tris-HCl, and 70% (v / v) ethanol.

[0093] The second washing solution is prepared from 0.1 M sodium chloride (NaCl), 0.1 mM EDTA, and 70% (v / v) ethanol.

[0094] The elution solution is pure water.

[0095] Example 2

[0096] This example provides a cfDNA extraction kit, including proteinase K, lysis buffer, magnetic beads, binding buffer, the first washing solution, the second washing solution, and elution solution. The specific components and contents can refer to Example 1. The difference is only that in this example, the lysis buffer is prepared from 0.3 M proline, pH 7.5, 50 mM Tris-HCl, 2 mM EDTA, 0.5% (v / v) Triton X-100, 1 mM DTT, and 0.1 M thiourea.

[0097] Example 3

[0098] This example provides a cfDNA extraction kit, including proteinase K, lysis buffer, magnetic beads, binding buffer, the first washing solution, the second washing solution, and elution solution. The specific components and contents can refer to Example 1. The difference is only that in this example, the lysis buffer is prepared from 0.3 M proline, pH 7.5, 50 mM Tris-HCl, 2 mM EDTA, 0.5% (v / v) Triton X-100, 6 mM DTT, and 0.8 M thiourea.

[0099] Example 4

[0100] This example provides a cfDNA extraction kit, including proteinase K, lysis buffer, magnetic beads, binding buffer, the first washing solution, the second washing solution, and elution solution. The specific components and contents can refer to Example 1. The difference is only that in this example, the usage volume of magnetic beads in Test Example 1 is 40 μL.

[0101] Example 5

[0102] This embodiment provides a cfDNA extraction kit, which includes Proteinase K, lysis buffer, magnetic beads, binding buffer, first wash solution, second wash solution and elution solution. The specific components and contents can be referred to Example 1, with the only difference being that in this embodiment, the usage volume of the magnetic beads in Test Example 1 is 160 μL.

[0103] Example 6

[0104] This embodiment provides a cfDNA extraction kit, which includes Proteinase K, lysis buffer, magnetic beads, binding buffer, first wash solution, second wash solution and elution solution. The specific components and contents can be referred to Example 1, with the only difference being that in this embodiment, the binding buffer is prepared from 2 M GuHCl, 4% (w / v) PEG 8000, pH 6.2, 30 mM Tris-HCl, 1.5 mM EDTA and 0.05 wt% Triton X-100.

[0105] Example 7

[0106] This embodiment provides a cfDNA extraction kit, which includes Proteinase K, lysis buffer, magnetic beads, binding buffer, first wash solution, second wash solution and elution solution. The specific components and contents can be referred to Example 1, with the only difference being that in this embodiment, the binding buffer is prepared from 10 M GuHCl, 16% (w / v) PEG 8000, pH 6.2, 30 mM Tris-HCl, 1.5 mM EDTA and 0.05 wt% Triton X-100.

[0107] Example 8

[0108] This embodiment provides a cfDNA extraction kit, which includes Proteinase K, lysis buffer, magnetic beads, binding buffer, first wash solution, second wash solution and elution solution. The specific components and contents can be referred to Example 1, with the only difference being that in this embodiment, the first wash solution is prepared from 0.5 M GuHCl, pH 7.5, 0.5% (w / v) Tris-HCl, 70% (v / v) ethanol.

[0109] Example 9

[0110] This embodiment provides a cfDNA extraction kit, which includes Proteinase K, lysis buffer, magnetic beads, binding buffer, first wash solution, second wash solution and elution solution. The specific components and contents can be referred to Example 1, with the only difference being that in this embodiment, the first wash solution is prepared from 3 M GuHCl, pH 7.5, 0.5% (w / v) Tris-HCl, 70% (v / v) ethanol.

[0111] Example 10

[0112] This example provides a cfDNA extraction kit, which includes proteinase K, lysis buffer, magnetic beads, binding buffer, the first washing solution, the second washing solution and eluent. For the specific components and their contents, reference can be made to Example 1. The only difference is that in this example, the second washing solution is prepared from 0.05 M NaCl, 0.1 mM EDTA, and 70% (v / v) ethanol.

[0113] Example 11

[0114] This example provides a cfDNA extraction kit, which includes proteinase K, lysis buffer, magnetic beads, binding buffer, the first washing solution, the second washing solution and eluent. For the specific components and their contents, reference can be made to Example 1. The only difference is that in this example, the second washing solution is prepared from 0.2 M NaCl, 0.1 mM EDTA, and 70% (v / v) ethanol.

[0115] Comparative Example 1

[0116] This comparative example provides a cfDNA extraction kit, which includes proteinase K, lysis buffer, magnetic beads, binding buffer, the first washing solution, the second washing solution and eluent. For the specific components and their contents, reference can be made to Example 1. The only difference is that in this example, the lysis buffer does not contain proline.

[0117] Test Example

[0118] This test example provides a cfDNA extraction method. The kits provided in Examples 1 - 11 and Comparative Example 1 are respectively used for cfDNA extraction, including the following steps:

[0119] (1) Lysis: In a 15 mL centrifuge tube, add 4 mL of human plasma sample, 200 μL of proteinase K, and 200 μL of lysis buffer. After vortexing and mixing evenly, incubate at 55°C for 30 min, and then place it at room temperature (25°C) for 5 - 10 min to restore to room temperature (25°C) to obtain a lysis mixture.

[0120] (2) Binding: Add 4.5 mL of binding solution and the magnetic beads with the specified volume in the above examples to the lysis mixture, and invert and mix evenly at room temperature (25°C) for 5 min to obtain a binding mixture.

[0121] (3) Magnetic absorption: Move the centrifuge tube containing the binding mixture to a magnetic rack, perform magnetic absorption for 5 min. After clarification, discard the supernatant.

[0122] (4) First washing: Remove the above-mentioned centrifuge tube from the magnetic rack, add 1 mL of the first washing solution into the tube, vortex and mix for 5 - 10 s, and transfer it to a new 1.5 mL centrifuge tube; place the 1.5 mL centrifuge tube on the magnetic rack, magnetically attract for 5 min, after clarification, aspirate the supernatant back into the original 15 mL centrifuge tube, rinse the tube wall and tube cap, and transfer it to a 1.5 mL centrifuge tube.

[0123] (5) Magnetic attraction: Place the 1.5 mL centrifuge tube on the magnetic rack, magnetically attract for 5 min, after clarification, discard the supernatant.

[0124] (6) Second washing: Add 1 mL of the second washing solution into the 1.5 mL centrifuge tube, vortex and mix for 5 - 10 s, centrifuge instantaneously, place the 1.5 mL centrifuge tube on the magnetic rack, magnetically attract for 2 min, after clarification, discard the supernatant. Repeat this step.

[0125] (7) Drying: Centrifuge instantaneously, use a small - volume pipette to carefully aspirate the remaining supernatant. Open the 1.5 mL centrifuge tube and dry the magnetic beads at room temperature for 5 minutes.

[0126] (8) Elution: Add 50 μL of elution solution into the 1.5 mL centrifuge tube, vortex and mix for 5 - 10 s, after mixing, let it stand at 60 °C for 5 min. Centrifuge instantaneously, place the centrifuge tube on the magnetic rack, magnetically attract for 3 - 5 min, after clarification, aspirate the DNA solution and transfer it to a new centrifuge tube to obtain cfDNA samples 1 - 12 (corresponding to Examples 1 - 11 and Comparative Example 1 respectively).

[0127] Test example

[0128] Use the RC1101 - Plasma Free DNA Extraction Kit produced by Company A (Kaishuo Biotechnology (Xiamen) Co., Ltd.) and the DP720 - Enhanced Magnetic Bead Method Large - Volume Free Nucleic Acid Extraction Kit produced by Company B (Tiangen Biochemical Technology (Beijing) Co., Ltd.) sold on the market to extract cfDNA from 4 mL of human plasma samples according to the instructions respectively to obtain cfDNA (A) samples and cfDNA (B) samples.

[0129] Use capillary electrophoresis (using the Qsep100 instrument of Guangding Biotechnology with S'more Cartridge) to detect the proportion of cfDNA in cfDNA samples 1 - 12, the above - mentioned cfDNA (A) samples and the above - mentioned cfDNA (B) samples in the test example. The calculation formula is as follows: W / Y×100%, where W is the DNA content with a fragment size of 100 - 200 bp, and Y is the total DNA content in the cfDNA sample. The results can be seen in Table 1.

[0130] The total DNA content in the cfDNA samples 1-12, the above-mentioned cfDNA (A) sample, and the above-mentioned cfDNA (B) sample in the test case was detected using a Qubit fluorescence quantitative instrument. The results can be seen in Table 2.

[0131] Table 1

[0132]

[0133] Table 2

[0134]

[0135] According to the results in Table 1, it can be found that the cfDNA extraction kit and cfDNA extraction method provided by the present invention can effectively enrich cfDNA and significantly improve the purity of cfDNA. The proportion of cfDNA in the cfDNA samples extracted is significantly higher than that of the kits developed by Company A and Company B.

[0136] According to the results in Table 2, it can be found that the cfDNA extraction kit and cfDNA extraction method provided by the present invention can effectively improve the extraction efficiency of cfDNA, and the total DNA content in the cfDNA samples extracted is significantly higher than that of the kits developed by Company A and Company B.

[0137] In summary, the present invention provides a cfDNA extraction kit and extraction method, which can not only effectively improve the extraction efficiency of cfDNA, but also effectively enrich 100-200 bp cfDNA and significantly improve the purity of cfDNA. The kit components are easy to obtain and low-cost, and the extraction operation is simple and suitable for high-throughput processing.

[0138] 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, It includes Proteinase K, lysis buffer, magnetic beads, and binding buffer; The lysis buffer includes 0.1 - 0.5 M proline, 1 - 6 mM dithiothreitol, 0.1 - 0.8 M thiourea, pH 7.5 - 8.5, 50 - 100 mM tris(hydroxymethyl)aminomethane hydrochloride, 1 - 5 mM ethylenediaminetetraacetic acid, and 0.1 - 1.0% (v / v) Triton X-100; The binding buffer includes 2 - 10 M guanidine hydrochloride, 4 - 16% (w / v) polyethylene glycol 8000, pH 6 - 6.5, 25 - 35 mM tris(hydroxymethyl)aminomethane hydrochloride, 1.0 - 2.0 mM ethylenediaminetetraacetic acid, and 0.01 - 0.1 wt% Triton X-100.

2. The cfDNA extraction kit according to claim 1, wherein The cfDNA extraction kit further includes a first washing solution; The first washing solution includes 0.5 - 3 M guanidine hydrochloride, pH 7.0 - 8.0, 0.4 - 0.6% (w / v) tris(hydroxymethyl)aminomethane hydrochloride, and 70 - 80% (v / v) ethanol.

3. The cfDNA extraction kit according to claim 1 or 2, characterized in that, The cfDNA extraction kit further includes a second washing solution; The second washing solution includes 0.05 - 0.5 M sodium chloride, 0.05 - 0.15 mM ethylenediaminetetraacetic acid, and 70 - 80% (v / v) ethanol.

4. The cfDNA extraction kit according to claim 1 or 2, characterized in that, The concentration of the Proteinase K is 0.5 - 1.0 mg / mL.

5. The cfDNA extraction kit according to claim 1 or 2, characterized in that, The magnetic beads have a core-shell structure, including a magnetite core and a silica shell layer coated on at least part of the core surface, and the silica shell layer is modified with hydroxyl groups.

6. The cfDNA extraction kit according to claim 5, wherein, The modification density of the hydroxyl groups is 2 - 8 hydroxyl groups / nm 2 .

7. The cfDNA extraction kit according to claim 5, wherein The particle size of the magnetic beads is 0.4 - 0.8 μm.

8. A method for extracting cfDNA, characterized in that, It is carried out using the cfDNA extraction kit according to any one of claims 1 - 7, and includes the following steps: Incubate the body fluid sample with the Proteinase K and the lysis buffer to obtain a lysis mixture; Mix the lysis mixture, the magnetic beads, and the binding buffer to obtain a binding mixture; Wash the binding mixture successively with the first washing solution and the second washing solution to obtain a washed product; Elute the washed product with an eluent to obtain the cfDNA.

9. The cfDNA extraction method according to claim 8, wherein, The volume ratio of the body fluid sample, the Proteinase K, the lysis buffer, the magnetic beads, and the binding buffer is (95 - 105):(4 - 6):(4 - 6):(1 - 4):(105 - 115).

10. The cfDNA extraction method according to claim 8, wherein, The body fluid sample is selected from at least one of blood, lymph fluid, milk, urine, and amniotic fluid.

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