A cfDNA extraction kit and extraction method

Abstract

The present invention provides a cfDNA extraction kit and method. The cfDNA extraction kit provided by the present invention includes proteinase K, a lysis buffer, magnetic microbeads, and a binding buffer. By limiting the composition of the lysis buffer and the binding buffer, it can not only effectively improve the extraction efficiency of cfDNA, but also significantly increase the purity of cfDNA and effectively enrich cfDNA of 100-200 bp. In addition, the kit components are easily available and low-cost, and the extraction operation is simple and suitable for high-throughput processing.

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 outside cells, with a length between 100 and 200 base pairs. It is mainly released into the extracellular environment of the human body through cell apoptosis, necrosis and other pathways, and is often present in physiological extracellular environments such as blood, lymph, milk, urine and amniotic fluid.

[0003] Currently, cfDNA testing is a common form of liquid biopsy on the market and has been widely used in various applications, including tumor detection, guidance evaluation, and prognostic assessment. For example, testing tumor-derived cfDNA can reveal tumor-related mutations, loss of heterozygosity, gene amplification, oncoviral DNA, and hypermethylation of tumor suppressor gene promoter regions, enabling non-invasive study of tumor DNA. However, cfDNA is low in content and small in fragment size, making extraction difficult. Losses are prone to occur during the extraction process, resulting in low detection sensitivity, which to some 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 cfDNA purity. Summary of the Invention

[0004] The present invention provides a cfDNA extraction kit that 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 that is simple to operate and suitable for high-throughput processing.

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

[0007] The lysis buffer consisted of 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 hydrochloride, 1-5 mM ethylenediaminetetraacetic acid, and 0.1-1.0% (v / v) polyethylene glycol octylphenyl ether;

[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 hydrochloride, 1.0-2.0 mM ethylenediaminetetraacetic acid, and 0.01-0.1 wt% polyethylene glycol octylphenyl ether.

[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] In the cfDNA extraction method as described above, the volume ratio of the body fluid sample, proteinase K, lysis buffer, magnetic microbeads 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, milk, urine, and amniotic fluid.

[0024] The present invention provides a cfDNA extraction kit, comprising proteinase K, a lysis buffer, magnetic microbeads, and a binding buffer. By limiting the composition of the lysis buffer and the binding buffer, not only can the extraction efficiency of cfDNA be effectively improved, but also the purity of cfDNA can be significantly improved, and 100-200 bp of cfDNA can be effectively enriched. In addition, the kit components are easy to obtain and low in cost, and the extraction operation is simple and suitable for high-throughput processing. DETAILED DESCRIPTION

[0025] 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.

[0026] Currently, cell-free DNA (cfDNA) extraction methods can be primarily categorized into traditional phenol / chloroform extraction, silica gel column extraction, and magnetic bead extraction. Compared to the first two extraction methods, magnetic bead extraction offers advantages such as ease of use, reduced time consumption, ease of automation, and high-throughput processing. It can directly extract cfDNA from crude samples such as plasma, reducing pretreatment steps. Furthermore, it exerts minimal shear stress on DNA, helping to maintain cfDNA integrity.

[0027] However, cfDNA has low concentrations and low amounts in bodily fluids, and existing magnetic bead extraction kits often have low extraction efficiencies, which can lead to insufficient sensitivity in downstream testing. Furthermore, cfDNA is susceptible to contamination with genomic DNA, particularly in cases of incomplete cell lysis or delayed plasma processing. This can significantly impact the detection of low-abundance cfDNA, such as circulating tumor DNA (ctDNA). Furthermore, some applications require enrichment of cfDNA fragments within a specific size range, a requirement for which existing magnetic bead extraction kits lack flexibility. Some commercial magnetic bead extraction kits are also costly, limiting their application in resource-limited settings.

[0028] To solve the above problems, the first aspect of the present invention provides a cfDNA extraction kit, comprising proteinase K, a lysis buffer, magnetic microbeads, and a 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 efficiently degrades histones, nucleoproteins, and other proteins (such as hemoglobin and albumin) bound to DNA, thereby releasing cfDNA. Furthermore, proteinase K can prevent cfDNA degradation during the extraction process by degrading nucleases (such as DNAase), thereby ensuring the integrity of the cfDNA.

[0030] Secondly, the composition of the lysis buffer is crucial for the successful release and purification of high-quality cfDNA. Long-term experiments have shown that a lysis buffer formulated with proline, dithiothreitol, thiourea, tris(hydroxymethylaminomethane) hydrochloride, ethylenediaminetetraacetic acid, and polyethylene glycol octylphenyl ether can effectively improve the efficiency of cfDNA extraction and enhance the purity of the resulting cfDNA.

[0031] Specifically, proline can destroy the hydrophobic cavity of proteins and enhance the denaturing effect of thiourea on histones, thereby promoting the improvement of cfDNA release efficiency; it can act as an osmotic protectant 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 hemoglobin in the blood, reducing its inhibitory effect on proteinase K, and is therefore particularly suitable for blood sample extraction.

[0032] On the one hand, dithiothreitol, as a strong reducing agent, can destroy the disulfide bonds in proteins, causing protein denaturation and removal, thereby facilitating 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 solubilization. It can also chelate metal ions to protect the integrity of cfDNA.

[0034] Tris(hydroxymethyl)aminomethane hydrochloride is a commonly used pH buffer that can effectively maintain the pH stability 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 preventing the degradation of cfDNA.

[0036] Polyethylene glycol octylphenyl ether is a non-ionic detergent that can reduce the surface tension of the solution, promote uniform mixing of various reagents, ensure that all components can evenly contact cfDNA, and improve extraction efficiency; it can also improve the purity of cfDNA by destroying cell membranes and dissolving membrane proteins, reducing nonspecific binding of proteins during the extraction process.

[0037] In this study, proteinase K directly degrades proteins, dithiothreitol enhances protein denaturation by reducing disulfide bonds, and thiourea further disrupts protein structure. Together, these three enable efficient release of cfDNA from protein complexes. Poly(ethylene glycol) octylphenyl ether aids cell lysis by disrupting the membrane, accelerating protein exposure and forming a synergistic "chemical degradation + physical membrane disruption" approach with the protease system, significantly improving 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(hydroxymethylaminomethane) hydrochloride stabilizes the pH to avoid acid-base damage. This can construct a cfDNA protection network in multiple dimensions and reduce degradation losses during the extraction process.

[0039] Polyethylene glycol octylphenyl ether reduces the nonspecific binding of proteins to cfDNA, proteinase K degrades free proteins, and thiourea and ethylenediaminetetraacetic acid remove impurities through denaturation / chelation, jointly reducing protein contamination; the synergistic effect of each component avoids the large-scale release of genomic DNA (gDNA) (because the lysis buffer is mild and acts specifically on the cfDNA-protein complex), further improving the purity of cfDNA.

[0040] The pH buffering capacity of tris(hydroxymethyl)aminomethane hydrochloride combined with the metal ion chelation effect of ethylenediaminetetraacetic acid and thiourea can also maintain the chemical environment stability of the system, ensure the activity of reagents such as proteinase K, and avoid the decrease in extraction efficiency due to environmental fluctuations.

[0041] The components of the aforementioned lysis buffer are designed following a multi-faceted principle of "efficient release - precise protection - impurity removal - system stability." These components complement each other through chemical reactions (such as enzymatic hydrolysis, denaturation, and chelation) and physical actions (such as membrane disruption and decontamination). This not only enables efficient cfDNA release but also maintains its integrity through multi-dimensional protection mechanisms, significantly reducing protein and genomic DNA contamination, ultimately improving extraction efficiency and purity. This complex strategy addresses the core pain points of cfDNA extraction: difficult release, easy degradation, and contamination. The synergistic effect of the components is key to achieving this technical success.

[0042] At the same time, the present invention has found through a large number of experiments that when the contents of proline, dithiothreitol, thiourea, tris(hydroxymethylaminomethane) hydrochloride, ethylenediaminetetraacetic acid and polyethylene glycol octylphenyl ether in the lysis buffer meet the above ranges, the effect of the lysis buffer can be enhanced to ensure effective cell lysis, 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) polyethylene glycol octylphenyl ether, 3 mM dithiothreitol and 0.4 M thiourea.

[0044] Next, during cfDNA extraction, magnetic microbeads provide an efficient and simple method for isolating and purifying cfDNA. Magnetic microbeads can capture cfDNA. Under the influence of an external magnetic field, the captured cfDNA can be easily separated from the solution, effectively removing impurities in the lysis buffer and retaining only the cfDNA bound to the magnetic microbeads.

[0045] Finally, in cfDNA extraction, the composition of the binding buffer is crucial for promoting successful binding of cfDNA to magnetic microbeads, thereby isolating and purifying high-quality cfDNA. Long-term experiments have shown that a binding buffer formulated with guanidine hydrochloride, polyethylene glycol 8000, tris(hydroxymethylaminomethane) hydrochloride, ethylenediaminetetraacetic acid, and polyethylene glycol octylphenyl ether can effectively improve cfDNA extraction efficiency and enhance the purity of the resulting cfDNA.

[0046] Specifically, in the binding buffer, guanidine hydrochloride is a powerful denaturant that can destroy the tertiary and secondary structures of proteins, help remove protein impurities, and make cfDNA easier to bind to magnetic microbeads; guanidine hydrochloride can neutralize the negative charge of cfDNA by providing a high-salt environment, reduce the electrostatic repulsion between cfDNA molecules, and help enhance the interaction between cfDNA and the binding groups on the surface of magnetic microbeads; guanidine hydrochloride can inhibit the activity of nucleases, prevent cfDNA from being degraded during the extraction process, and help maintain the integrity and quality of cfDNA; guanidine hydrochloride helps dissolve cell debris and other organic impurities, thereby improving the purity of the extracted cfDNA.

[0047] Polyethylene glycol 8000 is a high molecular weight polymer that can reduce the solvated water layer around cfDNA molecules, which not only reduces the probability of cfDNA hydrolysis reaction, but also promotes cfDNA aggregation and precipitation, thereby promoting the interaction between cfDNA and magnetic microbeads.

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

[0049] Tris(hydroxymethyl)aminomethane hydrochloride maintains a suitable pH environment. Under this condition, the silanol (Si-OH) groups on the surface of the magnetic microbeads bind most efficiently to the phosphate backbone of cfDNA through hydrogen bonds and salt bridges, forming an environmental adaptation synergy with guanidine hydrochloride and PEG 8000.

[0050] Guanidine hydrochloride can also inhibit nuclease activity, and ethylenediaminetetraacetic acid chelates metal ions to achieve double blocking of nuclease catalysis and prevent cfDNA degradation; polyethylene glycol octylphenyl ether removes protein and membrane impurities. The three work together to reduce the degradation and contamination of cfDNA, forming a protective network of "enzyme inhibition-metal chelation-impurity removal".

[0051] The denaturing effect of guanidine hydrochloride combined with the decontamination effect of polyethylene glycol octylphenyl ether can also more thoroughly remove protein impurities, avoid nonspecific binding of impurities to magnetic beads, and thus improve the purity of cfDNA.

[0052] The pH buffering capacity of tris(hydroxymethyl)aminomethane hydrochloride) combined with the metal ion chelation effect of EDTA maintains the stability of the system's chemical environment, ensuring that the denaturation efficiency of guanidine hydrochloride and the precipitation-promoting effect of polyethylene glycol 8000 are not interfered with, avoiding the decrease in binding efficiency caused by environmental fluctuations.

[0053] The components of the binding buffer are designed with the core goal of "promoting binding, protecting cfDNA molecules, removing impurities, and stabilizing the system." Each component works synergistically and complementary through chemical interactions (such as denaturation and chelation) and physical interactions (such as aggregation and decontamination): Guanidine hydrochloride and polyethylene glycol 8000 jointly promote cfDNA binding to the magnetic beads; tris (hydroxymethyl)aminomethane hydrochloride) and ethylenediaminetetraacetic acid maintain a stable environment and protect cfDNA; and polyethylene glycol octylphenyl ether reduces nonspecific adsorption. This complex strategy enables the binding buffer to efficiently capture cfDNA while simultaneously removing impurities and protecting molecules under high-salt conditions. Ultimately, this significantly improves extraction efficiency (e.g., increased total DNA content in the test case) and cfDNA purity (increased proportion of 100-200 bp fragments), demonstrating the critical value of the synergistic effect of each component.

[0054] At the same time, the present invention has found through a large number of experiments that when the contents of guanidine hydrochloride, polyethylene glycol 8000, trishydroxymethylaminomethane hydrochloride, ethylenediaminetetraacetic acid and polyethylene glycol octylphenyl ether in the binding buffer meet the above ranges, the binding buffer effect can be enhanced to ensure that cfDNA is effectively bound to the magnetic microbeads.

[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 % polyethylene glycol octylphenyl ether.

[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 and effectively enrich 100-200 bp of cfDNA. The components of the kit are easy 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 also 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.

[0058] Adding ethanol can reduce the solubility of cfDNA, promote its precipitation, and make it easier for cfDNA to bind to magnetic microbeads. Ethanol can also effectively remove water, salt, protein, and other organic impurities from the solution, helping to enhance the washing effect.

[0059] The first wash solution of the present invention is a high-salt buffer. By increasing the ionic strength of the solution, it can reduce the solubility of proteins and other impurities, thereby effectively removing 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 microbeads, ensuring that cfDNA is not eluted during the washing process.

[0060] After a large number of experiments, the present invention found that when the contents of guanidine hydrochloride, tris(hydroxymethylaminomethane) hydrochloride and ethanol in the first washing solution meet the above-mentioned ranges, the effect of the first washing solution can be enhanced, effectively removing water, salt, protein and other organic impurities in the solution, while ensuring the effective binding of cfDNA and magnetic microbeads during the washing process.

[0061] In one 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 also 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] The second wash solution, containing ethylenediaminetetraacetic acid and ethanol, also removes salt, protein, and other organic impurities, and strengthens the binding of cfDNA to the magnetic beads. Furthermore, the sodium chloride in the second wash solution provides low-salt conditions, removing residual salt from the high-salt wash process. This helps lower the ionic strength and minimizes inhibition of downstream enzyme reactions (such as PCR). Further washing with the second wash solution also removes non-specifically bound impurities, improving the purity of the cfDNA. Furthermore, the low-salt conditions help prepare for the cfDNA elution step, facilitating efficient recovery of the cfDNA in the subsequent elution step.

[0064] After a large number of experiments, the present invention 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 salt, protein and other organic impurities in the solution, and preparing for subsequent cfDNA elution.

[0065] In one 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 also include an eluent, which 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 proteolysis effect of proteinase K.

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

[0069] The ferroferric oxide core provides magnetic properties, enabling simple and convenient separation of cfDNA from the solution by combining it with an external magnetic field. Under high-salt, low-pH conditions, the silanol (Si-OH) groups on the surface of the magnetic microbeads can bind to the phosphate backbone of the nucleic acid through hydrogen bonds and salt bridges, thereby adsorbing the cfDNA.

[0070] Furthermore, by precisely controlling the modification density of hydroxyl groups, nonspecific binding to large genomic DNA fragments can be reduced, while enabling magnetic microbeads to more effectively capture small cfDNA fragments, 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 Such high-density hydroxyl modification can provide more DNA binding sites, and the interference between adjacent groups will cause steric hindrance, thereby hindering the stretching and binding of long DNA fragments, and ultimately selectively enriching 100-200 bp cfDNA.

[0071] In an optional embodiment, the magnetic microbeads have a particle size of 0.4-0.8 μm. Smaller magnetic microbeads within this particle size range have a larger specific surface area, can provide more cfDNA binding sites, thereby improving the binding efficiency of cfDNA, and are easier to maintain uniform suspension, helping to ensure that cfDNA evenly contacts the surface of the magnetic microbeads.

[0072] Furthermore, the magnetic microbeads may be suspended in a solvent at a concentration of 5-20 mg / mL during use. In one embodiment, the magnetic microbeads are suspended in a 20% ethanol aqueous solution.

[0073] The second aspect of the present invention provides a cfDNA extraction method, which is performed using the cfDNA extraction kit provided by the first aspect of the present invention, comprising the following steps:

[0074] The body fluid sample is incubated with proteinase K and lysis buffer to obtain a lysis mixture;

[0075] mixing the lysis mixture, magnetic microbeads and binding buffer to obtain a binding mixture;

[0076] washing the binding mixture with a first washing solution and a second washing solution in sequence to obtain a washing product;

[0077] The washed product is eluted with an elution buffer to obtain cfDNA.

[0078] In a specific embodiment, the body fluid sample, proteinase K and lysis buffer can be first added to a 15 mL centrifuge tube, vortexed to mix, incubated at 55°C for 30 minutes, and then placed at room temperature (25°C) for 5-10 minutes to return to room temperature (25°C) to obtain a lysis mixture.

[0079] Add binding buffer and magnetic beads to the lysate mixture and mix by inversion at room temperature (25°C) for 5 minutes to obtain a binding mixture. Transfer the centrifuge tube containing the binding mixture to a magnetic stand and magnetize for 5 minutes. After clarification, discard the supernatant.

[0080] Next, remove the centrifuge tube from the magnetic rack, add the first wash solution to the tube, vortex and mix for 5-10 seconds, and transfer it to a new 1.5 mL centrifuge tube. Place the 1.5 mL centrifuge tube on the magnetic rack and magnetize for 5 minutes. After clarification, aspirate the supernatant back to the original 15 mL centrifuge tube, rinse the tube walls and tube cap, and transfer it to the 1.5 mL centrifuge tube. Place the 1.5 mL centrifuge tube on the magnetic rack and magnetize for 5 minutes. After clarification, discard the supernatant.

[0081] Next, add the second wash buffer to the 1.5 mL centrifuge tube, vortex to mix for 5-10 seconds, and centrifuge briefly. Place the 1.5 mL centrifuge tube on a magnetic rack and magnetize for 2 minutes. After clarification, discard the supernatant. Repeat this step. After discarding the supernatant, centrifuge briefly and carefully aspirate the remaining supernatant using a small-range pipette. Open the 1.5 mL centrifuge tube and allow the magnetic beads to dry at room temperature for 5 minutes.

[0082] Finally, add the eluent (which can be pure water) to a 1.5 mL centrifuge tube, vortex for 5-10 seconds, mix thoroughly, and incubate at 60°C for 5 minutes. Centrifuge briefly, place the tube on a magnetic rack, and magnetize for 3-5 minutes. After clarification, transfer the DNA solution to a new centrifuge tube to obtain the cfDNA sample.

[0083] In the above technical solution, the volume ratios of the body fluid sample, proteinase K, lysis buffer, magnetic microbeads, and binding buffer are (95-105):(4-6):(4-6):(1-4):(105-115). Within this range, the individual reagents in the cfDNA extraction kit can achieve optimal results. For example, the body fluid sample can be 4 mL, the proteinase K can be 200 μL, the lysis buffer can be 200 μL, the magnetic microbeads can be 80 μL, and the 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, milk, urine, and amniotic fluid.

[0085] 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.

[0086] Example 1:

[0087] This embodiment provides a cfDNA extraction kit, including proteinase K, a lysis buffer, magnetic microbeads, a binding buffer, a first washing solution, a second washing solution, and an eluent.

[0088] The concentration of proteinase K was 0.8 mg / mL.

[0089] The lysis buffer was prepared with 0.3 M proline, pH 7.5, 50 mM tris-HCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5% (v / v) polyethylene glycol octylphenyl ether (Triton X-100), 3 mM dithiothreitol (DTT), and 0.4 M thiourea.

[0090] The magnetic microbeads are PuriMag Si-COOH carboxyl-modified magnetic beads, manufactured by PuriMag and marketed as PuriMag Si-COOH. They have a particle size of 0.4-0.8 μm. These magnetic microbeads have a core-shell structure, consisting of a core of ferroferric oxide (Fe3O4) and a silicon dioxide (SiO2) shell coating the core. The SiO2 shell is modified with hydroxyl groups at a density of 5 hydroxyl groups / nm. 2 The magnetic microbeads were suspended in a 20% ethanol aqueous solution, and the volume used in Experimental Example 1 was 80 μL.

[0091] The binding buffer was 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 was prepared from 1 M GuHCl, pH 7.5, 0.5% (w / v) Tris-HCl, and 70% (v / v) ethanol.

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

[0094] The eluent was pure water.

[0095] Example 2

[0096] This example provides a cfDNA extraction kit, including proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the lysis buffer is prepared with 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, comprising proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the lysis buffer is prepared with 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 microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the magnetic microbeads are used in a volume of 40 μL.

[0101] Example 5

[0102] This example provides a cfDNA extraction kit, including proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the magnetic microbeads are used in a volume of 160 μL in Experiment 1.

[0103] Example 6

[0104] This example provides a cfDNA extraction kit, comprising proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the binding buffer is prepared with 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 example provides a cfDNA extraction kit, comprising proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the binding buffer comprises 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 example provides a cfDNA extraction kit, comprising proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the first wash solution is prepared from 0.5 M GuHCl, pH 7.5, 0.5% (w / v) Tris-HCl, and 70% (v / v) ethanol.

[0109] Example 9

[0110] This example provides a cfDNA extraction kit, comprising proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the first wash solution is prepared with 3 M GuHCl, pH 7.5, 0.5% (w / v) Tris-HCl, and 70% (v / v) ethanol.

[0111] Example 10

[0112] This example provides a cfDNA extraction kit, comprising proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, with the only difference being that in this example, the second wash solution is prepared with 0.05 M NaCl, 0.1 mM EDTA, and 70% (v / v) ethanol.

[0113] Example 11

[0114] This example provides a cfDNA extraction kit, comprising proteinase K, lysis buffer, magnetic microbeads, binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts are similar to those in Example 1, with the only difference being that in this example, the second wash solution is prepared with 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, including proteinase K, a lysis buffer, magnetic microbeads, a binding buffer, a first wash solution, a second wash solution, and an eluent. The specific components and amounts can be found in Example 1, except that in this example, the lysis buffer does not contain proline.

[0117] Test example

[0118] This test example provides a cfDNA extraction method, which uses the kits provided in Examples 1-11 and Comparative Example 1 to extract cfDNA, 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. Vortex to mix, incubate at 55°C for 30 min, and then place at room temperature (25°C) for 5-10 min to return to room temperature (25°C) to obtain a lysis mixture.

[0120] (2) Binding: Add 4.5 mL of binding solution and the volume of magnetic microbeads specified in the above example to the lysis mixture, mix by inversion 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 the magnetic rack, absorb it for 5 minutes, and discard the supernatant after clarification.

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

[0123] (5) Magnetic absorption: Place a 1.5 mL centrifuge tube into a magnetic rack and magnetize for 5 min. After clarification, discard the supernatant.

[0124] (6) Second wash: Add 1 mL of the second wash solution to a 1.5 mL centrifuge tube, vortex and mix for 5-10 seconds, centrifuge briefly, place the 1.5 mL centrifuge tube on a magnetic rack, magnetize for 2 minutes, clarify, and discard the supernatant. Repeat this step.

[0125] (7) Drying: Centrifuge briefly and carefully remove the remaining supernatant using a small-range pipette. 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 buffer to a 1.5 mL centrifuge tube, vortex for 5-10 seconds, mix thoroughly, and then let stand at 60°C for 5 minutes. Centrifuge briefly, place the centrifuge tube on a magnetic rack, and magnetize for 3-5 minutes. After clarification, transfer the DNA solution to a new centrifuge tube to obtain cfDNA samples 1-12 (corresponding to Examples 1-11 and Comparative Example 1, respectively).

[0127] Test Case

[0128] cfDNA was extracted from 4 mL of human plasma using the RC1101 plasma cell-free DNA extraction kit produced by Company A (Kaishuo Biotechnology (Xiamen) Co., Ltd.) and the DP720 enhanced magnetic bead-based large-volume cell-free nucleic acid extraction kit produced by Company B (Tiangen Biochemical Technology (Beijing) Co., Ltd.) according to the manufacturer's instructions, respectively. cfDNA was obtained as cfDNA (A) and cfDNA (B).

[0129] Capillary electrophoresis (using a Qsep100 instrument equipped with an S3 cartridge from Guangding Bio) was used to detect the cfDNA content in cfDNA samples 1-12, the aforementioned cfDNA (A) sample, and the aforementioned cfDNA (B) sample. The cfDNA content was calculated using the following formula: 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: Includes 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 includes 2-10 M guanidine hydrochloride, 4-16% (w / v) polyethylene glycol 8000, pH 6-6.5, 25-35 mM tris hydrochloride, 1.0-2.0 mM ethylenediaminetetraacetic acid and 0.01-0.1 wt % polyethylene glycol octylphenyl ether.

2. The cfDNA extraction kit according to claim 1, characterized in that 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 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 microbeads are of a core-shell structure, comprising a ferroferric oxide core and a silicon dioxide shell layer covering at least a portion of the surface of the core, wherein the silicon dioxide shell layer is modified with hydroxyl groups.

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

7. The cfDNA extraction kit according to claim 5, characterized in that The particle size of the magnetic microbeads is 0.4-0.8 μm.

8. A cfDNA extraction method, characterized in that: The method is performed using the cfDNA extraction kit according to any one of claims 1 to 7, comprising the following steps: incubating the body fluid sample with the proteinase K and the lysis buffer to obtain a lysis mixture; mixing the lysis mixture, the magnetic microbeads and the binding buffer to obtain a binding mixture; washing the binding mixture with a first washing solution and a second washing solution in sequence to obtain a washing product; The washed material is eluted using an elution solution to obtain the cfDNA.

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

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

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