Reagent combination, kit and method for extracting free DNA in plasma

Abstract

A reagent combination for extracting free DNA in plasma, a kit comprising the reagent combination, and a method for extracting free DNA in plasma by using the reagent combination or the kit. The reagent combination comprises a binding solution and a first washing solution, wherein the binding solution comprises a first chaotropic agent and a first alcohol, and the first washing solution comprises a second chaotropic agent and a second alcohol. The present invention is suitable for large-scale clinical detection, and has a relatively high DNA yield and DNA product purity.

Description

Reagent combination, kit and method for extracting free DNA from plasma

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202311611763.5 filed on November 28, 2023, the entire contents of which are expressly incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of liquid biopsy, and in particular to a reagent combination for extracting free DNA in plasma, a kit comprising the reagent combination, and a method for extracting free DNA in plasma using the reagent combination or the kit. Background Art

[0004] Liquid biopsy refers to a non-invasive testing method that obtains tissue-derived biomarkers from body fluids and analyzes them to obtain information about the tissue of origin. It has been used for prenatal diagnosis and the diagnosis, screening, and monitoring of diseases such as cancer. The most commonly used biomarker in liquid biopsy is cell-free DNA (cfDNA), the genetic material that exists outside cells in plasma. This DNA may contain genetic information from embryonic or cancerous tissue.

[0005] The content of free DNA in plasma is usually very low (the average free DNA concentration in healthy human plasma is about 4 ng / mL) and is usually small fragments (enriched in 170 bp). Therefore, in order to obtain sufficient free DNA from a limited sample volume for downstream testing, the nucleic acid extraction method needs to have the highest possible DNA extraction yield. At the same time, in order to improve the sensitivity of downstream testing, the DNA product extracted by the nucleic acid extraction method also needs to have the highest possible purity. In addition, in order to facilitate large-scale clinical testing applications, extraction methods that are easy to implement high-throughput automated operations are also desired by those skilled in the art.

[0006] Currently, nucleic acid extraction methods are mainly divided into organic solvent extraction, centrifugal column extraction, and magnetic bead extraction. Organic solvent extraction uses organic solvents such as phenol and chloroform to denature and remove impurities such as proteins, followed by precipitation and separation of nucleic acids using alcohols. However, these methods require highly toxic and carcinogenic volatile organic solvents, are complex procedures, and require high operator skill, making them difficult to automate and unsuitable for large-scale clinical testing. Centrifugal column extraction utilizes the principle of nucleic acid adsorption to a solid phase material (such as a silica gel membrane) under specific aqueous solution conditions and separation of nucleic acid from the solid phase under another specific aqueous solution condition to separate and purify nucleic acids from a sample. However, these methods are costly and rely on specialized filtration and high-speed centrifugation equipment, making automation difficult and unsuitable for large-scale clinical testing. Magnetic bead extraction is a variant of the centrifugal column method, employing superparamagnetic nanoparticles (magnetic beads) as the solid phase material. It utilizes the principle of nucleic acid adsorption to the magnetic beads under specific solution conditions and the separation of the adsorbed magnetic beads from the solution by an external magnetic field to separate and purify nucleic acids from a sample. This type of method only requires the action of a magnetic field to quickly separate nucleic acids from the liquid phase to the solid phase. It is easy to implement high-throughput automated operations and can be used for large-scale clinical testing. However, the nucleic acid extraction yield of the traditional magnetic bead method is not as good as that of the centrifugal column method. In addition, the traditional magnetic bead method usually requires a pH environment that deviates from the neutrality during the nucleic acid extraction process (for example, sample lysis and DNA binding stages), and a pH environment that deviates from the neutrality is not conducive to the stability of DNA, which results in poor quality of DNA products and low purity of DNA products, which is not conducive to downstream testing. Moreover, due to the low content of free DNA in plasma, the magnetic bead method is usually unable to extract free DNA in plasma.

[0007] Therefore, in order to apply the magnetic bead method that is easy to implement high-throughput automated operation when extracting free DNA from plasma in large-scale clinical testing, technicians in this field have been seeking to solve how to improve the DNA yield and DNA product purity of the magnetic bead method.

[0008] Summary of the Invention

[0009] The present invention provides a reagent combination for extracting free DNA in plasma, a kit comprising the reagent combination, and a method for extracting free DNA in plasma using the reagent combination or the kit. The reagent combination, the kit, and the method are suitable for large-scale clinical testing and have a high DNA yield and DNA product purity.

[0010] In one aspect, the present invention provides a reagent combination for extracting free DNA from plasma, comprising a binding solution and a first wash solution, wherein the binding solution comprises a first chaotrope and a first alcohol, and the first wash solution comprises a second chaotrope and a second alcohol. In one embodiment, the first chaotrope and the second chaotrope are the same chaotrope.

[0011] In one embodiment, the first chaotropic agent and the second chaotropic agent are different chaotropic agents.

[0012] In one embodiment, the first alcohol and the second alcohol are the same alcohol.

[0013] In one embodiment, the first alcohol and the second alcohol are different alcohols.

[0014] In one embodiment, the reagent combination comprises magnetic beads capable of binding to DNA, which bind to DNA via functional groups on their surfaces, such as carboxyl groups, hydroxyl groups, or a combination thereof. In a preferred embodiment, the magnetic beads capable of binding to DNA are nanomagnetic beads with surface modified silanol groups.

[0015] In one embodiment, for 1 mL of plasma sample, about 15 μL to 30 μL of magnetic beads (25 mg / mL) are required.

[0016] In one embodiment, the binding fluid contains a surfactant.

[0017] In one embodiment, the binding solution contains a buffer. In a preferred embodiment, the buffer comprises Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

[0018] In one embodiment, the binding solution comprises an EDTA solution.

[0019] In one embodiment, the first washing solution contains a surfactant.

[0020] In one embodiment, the first washing solution contains a buffer. In a preferred embodiment, the buffer comprises Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

[0021] In one embodiment, the first washing solution comprises an EDTA solution.

[0022] In one embodiment, the reagent combination comprises a lysis buffer combination.

[0023] The lysis solution combination can be any reagent combination that can separate DNA from other impurities.

[0024] In one embodiment, the lysis solution combination comprises a digestive enzyme solution. In a preferred embodiment, the lysis solution combination comprises a protease solution. In a preferred embodiment, the lysis solution combination comprises a serine protease solution. In a preferred embodiment, the digestive enzyme solution comprises a proteinase K solution.

[0025] In one embodiment, the lysis solution combination comprises a digestion solution.

[0026] The digestion solution can be any reagent that can separate DNA from other impurities, any reagent that can prevent DNA degradation, or a combination thereof.

[0027] In one embodiment, the digestive fluid comprises a protein denaturant.

[0028] In one embodiment, the digestive fluid comprises a surfactant.

[0029] In one embodiment, the digestion solution comprises an EDTA solution.

[0030] In one embodiment, the digestion solution comprises a buffer. In a preferred embodiment, the buffer comprises Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

[0031] In one embodiment, the surfactants contained in the digestion solution, the combined solution, and the first washing solution may be different or the same.

[0032] In one embodiment, the buffers contained in the digestion solution, binding solution, first washing solution, second washing solution, and elution solution may be different or the same.

[0033] In one embodiment, the ratio of the volume of the digestive enzyme solution to the volume of the plasma sample is 0.01-0.08, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or a sub-range consisting of any value within these ranges.

[0034] In one embodiment, the ratio of the volume of the digestive fluid to the volume of the plasma sample is 0.05-0.20, for example, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, or a sub-range consisting of any value within these ranges.

[0035] In one embodiment, the ratio of the volume of the binding fluid to the volume of the plasma sample is 1.00-1.75, for example, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35 , 1.36, 1.37, 1.38, 1.39, 1.40, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, or a sub-range consisting of any value within these ranges.

[0036] In one embodiment, the reagent combination comprises a second wash solution.

[0037] In one embodiment, the second washing liquid contains a third alcohol.

[0038] In one embodiment, the third alcohol is the same as the first alcohol or the second alcohol.

[0039] In one embodiment, the third alcohol is different from both the first and second alcohols.

[0040] In one embodiment, the second washing solution contains a buffer. In a preferred embodiment, the buffer comprises Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

[0041] In one embodiment, the second washing solution contains EDTA solution.

[0042] In one embodiment, the reagent combination comprises an eluent.

[0043] The elution solution can be any reagent that can elute DNA from the solid phase material (eg, nanomagnetic beads with silanol groups modified on the surface).

[0044] In one embodiment, the eluent comprises a buffer. In a preferred embodiment, the eluent comprises a Tris-HCl buffer, a Bis-Tris buffer, a glycine buffer or a combination thereof.

[0045] Chaotropic agents can be any substance that destroys the hydrogen bond network between water molecules in aqueous solution (i.e., exerts chaotropic activity). This affects the natural stability of other molecules (mainly macromolecules (proteins, nucleic acids)) in solution by weakening the hydrophobic effect. For example, chaotropic agents can reduce the amount of protein structural order formed by water molecules, either in the bulk or in the hydration shell around hydrophobic amino acids, causing them to denature.

[0046] In one embodiment, the first chaotropic agent or the second chaotropic agent comprises perchlorate, hexafluorophosphate, tetrafluoroborate, an amide, an ammonium salt, a potassium salt, a sodium salt, a lithium salt or a combination thereof, for example, sodium perchlorate, potassium perchlorate, guanidine hydrochloride, guanidine isothiocyanate, urea or a combination thereof.

[0047] In one embodiment, the first alcohol, the second alcohol or the third alcohol comprises a monohydric alcohol, a polyhydric alcohol or a combination thereof, for example, methanol, polyethylene glycol, ethanol, isopropanol, glycerol, isoamyl alcohol, butanol or a combination thereof.

[0048] In one embodiment, the surfactant includes an ionic surfactant, a nonionic surfactant, an amphoteric surfactant, or a combination thereof.

[0049] In one embodiment, the ionic surfactant includes an anionic surfactant, a cationic surfactant, or a combination thereof.

[0050] In one embodiment, the anionic surfactant includes carboxylates (e.g., potassium, sodium, ammonium and triethanolammonium salts of fatty acids), sulfonates (e.g., sodium alkylbenzene sulfonate (LAS or ABS), α-olefin sulfonate (AOS), alkyl sulfonates (AS and SAS) and α-sulfomonocarboxylic acid and its derivatives (MES)), sulfates / esters (e.g., fatty alcohol sulfates, secondary alkyl sulfates, fatty alcohol polyoxyethylene ether sulfates, sulfates of fatty acid derivatives and sulfates of unsaturated alcohols), phosphates / esters (e.g., alkyl / aryl phosphates) or a combination thereof.

[0051] In one embodiment, the cationic surfactant comprises an amine (eg, alkylamines, alkoxylated amines, amine oxides, and alkanolamides), an alkylammonium salt (eg, alkyltrimethylammonium salt), or a combination thereof.

[0052] In one embodiment, the nonionic surfactant includes a polyoxyethylene nonionic surfactant, a polyol nonionic surfactant, or a combination thereof.

[0053] In one embodiment, the polyoxyethylene nonionic surfactant includes polyoxyethylene fatty alcohol ether, polyoxyethylene fatty acid ester or polyoxyethylene sugar alcohol or a combination thereof. For example, the general formula is RO(CH2CH2O) n H fatty alcohol polyoxyethylene ether, R can be saturated or unsaturated, linear or branched (for example, C 10 ~C 18 , preferably C 12 -C 16 ) hydrocarbon group; n is the number of ethylene oxide additions, n ≥ 1, for example, n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or a greater natural number, or a combination thereof, specific examples of which include polyethylene glycol cetyl ether, cetearyl alcohol polyoxyethylene ether-10, or a combination thereof. Another example is polyoxyethylene sorbitol (e.g., Tween, such as Tween 20, 40, 60, 80, or 85), polyoxyethylene sorbitan (e.g., Span, such as Span 20, 40, 60, 80, or 85), or a combination thereof.

[0054] In one embodiment, the polyol-type nonionic surfactant includes fatty acid glycerides.

[0055] In one embodiment, the amphoteric surfactant includes an amino acid type (eg, laurylaminopropionic acid), a betaine type (eg, alkyl dimethyl betaine, alkyl dimethyl sulfoethyl betaine, and alkyl dimethyl hydroxypropyl phosphobetaine), or a combination thereof.

[0056] In one embodiment, specific examples of surfactants include sodium dodecyl sulfate (SDS), sodium deoxycholate (NaDOC), Triton X-100, NP-40, Tween 20, polyethylene glycol cetyl ether, ceteareth-10, or a combination thereof.

[0057] In one embodiment, the protein denaturant comprises an ionic surfactant, an amino compound, or a combination thereof.

[0058] In one embodiment, the ionic surfactant includes an anionic surfactant, a cationic surfactant, or a combination thereof.

[0059] In one embodiment, the anionic surfactant includes carboxylates (e.g., potassium, sodium, ammonium and triethanolammonium salts of higher fatty acids), sulfonates (e.g., sodium alkylbenzene sulfonate (LAS or ABS), α-olefin sulfonate (AOS), alkyl sulfonates (AS and SAS) and α-sulfomonocarboxylic acid and its derivatives (MES)), sulfates / esters (e.g., fatty alcohol sulfates, secondary alkyl sulfates, fatty alcohol polyoxyethylene ether sulfates, sulfates of fatty acid derivatives and sulfates of unsaturated alcohols), phosphates / esters (e.g., alkyl / aryl phosphates) or a combination thereof.

[0060] In one embodiment, the cationic surfactant comprises an amine (eg, alkylamines, alkoxylated amines, amine oxides, and alkanolamides), an alkylammonium salt (eg, alkyltrimethylammonium salt), or a combination thereof.

[0061] In one embodiment, the amino compound includes urea, a guanidine salt (eg, guanidine hydrochloride, guanidine isothiocyanate), or a combination thereof.

[0062] In one embodiment, the digestive enzyme solution has a concentration of 10-30 mg / mL, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mg / mL, or a sub-range consisting of any value within these ranges.

[0063] In one embodiment, the concentration of the protein denaturant is 4.0-6.0 M, e.g., 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 M, or a sub-range consisting of any value within these ranges.

[0064] %, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0 wt %, or a subrange consisting of any value within these ranges.

[0065] In one embodiment, the mass percentage of the surfactant in the combining fluid is 6.0-10.0 wt %, for example, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, or a sub-range consisting of any value within these ranges.

[0066] , 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 wt %, or a sub-range consisting of any value within these ranges.

[0067] In one embodiment, the concentration of the buffer in the digestion solution is 20-30 mM, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mM, or a sub-range consisting of any value within these ranges.

[0068] In one embodiment, the pH of the buffer in the digestion solution is 6.5-8.5, e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or a sub-range consisting of any value within these ranges.

[0069] In one embodiment, the concentration of the buffer in the binding solution is 15-25 mM, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mM, or a sub-range consisting of any value within these ranges.

[0070] In one embodiment, the pH of the buffer in the binding solution is 6.5-8.5, e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or a sub-range consisting of any value within these ranges.

[0071] In one embodiment, the concentration of the buffer in the first wash solution is 15-25 mM, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mM, or a sub-range consisting of any value within these ranges.

[0072] In one embodiment, the pH of the buffer in the first wash solution is 6.5-8.5, e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or a sub-range consisting of any value within these ranges.

[0073] In one embodiment, the concentration of the buffer in the second wash solution is 45-55 mM, e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 mM, or a sub-range consisting of any value within these ranges.

[0074] In one embodiment, the pH of the buffer in the second wash solution is 6.5-8.5, e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or a sub-range consisting of any value within these ranges.

[0075] In one embodiment, the concentration of the buffer in the eluent is 5-15 mM, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM, or a sub-range consisting of any value within these ranges.

[0076] In one embodiment, the pH of the buffer in the eluent is 6.5-8.5, e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or a sub-range consisting of any value within these ranges.

[0077] In one embodiment, the concentration of the EDTA solution in the digestive fluid is 0.8-1.8 mM, e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 mM, or a sub-range consisting of any value within these ranges.

[0078] In one embodiment, the concentration of the EDTA solution in the binding solution is 1.00-1.10 mM, for example, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10 mM, or a sub-range consisting of any value within these ranges.

[0079] In one embodiment, the concentration of the EDTA solution in the first wash solution is 1.5-2.5 mM, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 mM, or a sub-range consisting of any value within these ranges.

[0080] In one embodiment, the concentration of the EDTA solution in the second wash solution is 0.3-0.8 mM, for example, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mM, or a sub-range consisting of any value within these ranges.

[0081] In one embodiment, the concentration of the first chaotropic agent in the binding solution is 4.00-5.00 M, for example, 4.00, 4.01, 4.02, 4.03, 4.04, 4.05, 4.06, 4.07, 4.08, 4.09, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, 4.19, 4.20 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.30, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.40, 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48 , 4.49, 4.50, 4.51, 4.52, 4.53, 4.54, 4.55, 4.56, 4.57, 4.58, 4.59, 4.60, 4.61, 4.62, 4.63, 4.64, 4.65, 4.66, 4.67, 4.68, 4.69, 4.70, 4.71, 4.72, 4.73, 4.74, 4.75, 4.76 , 4.77, 4.78, 4.79, 4.80, 4.81, 4.82, 4.83, 4.84, 4.85, 4.86, 4.87, 4.88, 4.89, 4.90, 4.91, 4.92, 4.93, 4.94, 4.95, 4.96, 4.97, 4.98, 4.99, 5.00M, or a sub-range consisting of any value within these ranges.

[0082] In one embodiment, the concentration of the second chaotropic agent in the first wash solution is 2.5-3.5 M, e.g., 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 M, or a sub-range consisting of any value within these ranges.

[0083] In one embodiment, the volume percentage of the first alcohol contained in the combination solution is 15.0-60.0%, for example, 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24.0, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25.0, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26.0, 26.1, 26.2, 26.3, 26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27.0, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28.0, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 29.0, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30.0, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31.0, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32.0, 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, 33.0, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34.0, 34.1、34.2、34.3、34.4、34.5、34.6、34.7、34.8、34.9、35.0、35.1、35.2、35.3、35.4、35.5、35.6、35.7、35.8、35.9、36.0、36.1、36.2、36.3、36.4、36.5、36.6、36.7、36.8、36.9、37.0、37.1、37.2、37.3、37.4、37.5、37.6、37.7、37.8、37.9、38.0、38.1、38.2、38.3、38.4、38.5、38.6、38.7、38.8、38.9、39.0、39.1、39.2、39.3、39.4、39.5、39.6、39.7、39.8、39.9、40.0、40.1、40.2、40.3、40.4、40.5、40.6、40.7、40.8、40.9、41.0、41.1、41.2、41.3、41.4、41.5、41.6、41.7、41.8、41.9、42.0、42.1、42.2、42.3、42.4、42.5、42.6、42.7、42.8、42.9、43.0、43.1、43.2、43.3、43.4、43.5、43.6、43.7、43.8、43.9、44.0、44.1、44.2、44.3、44.4、44.5、44.6、44.7、44.8、44.9、45.0、45.1、45.2、45.3、45.4、45.5、45.6、45.7、45.8、45.9、46.0、46.1、46.2、46.3、46.4、46.5、46.6、46.7、46.8、46.9、47.0、47.1、47.2、47.3、47.4、47.5、47.6、47.7、47.8、47.9、48.0、48.1、48.2、48.3、48.4、48.5、48.6、48.7、48.8、48.9、49.0、49.1、49.2、49.3、49.4、49.5、49.6、49.7、49.8、49.9、50.0、50.1、50.2、50.3、50.4、50.5、50.6、50.7、50.8、50.9、51.0、51.1、51.2、51.3、51.4、51.5、51.6、51.7、51.8、51.9、52.0、52.1、52.2、52.3、52.4、52.5、52.6、52.7、52.8、52.9、53.0、53.1、53.2、53.3、53.4、53.5、53.6、53.7、53.8、53.9、54.0、54.1, 54.2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55.0, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56.0, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9, 57.0, 57.1, 57.2, 5 7.3, 57.4, 57.5, 57.6, 57.7, 57.8, 57.9, 58.0, 58.1, 58.2, 58.3, 58.4, 58.5, 58.6, 58.7, 58.8, 58.9, 59.0, 59.1, 59.2, 59.3, 59.4, 59.5, 59.6, 59.7, 59.8, 59.9, 60.0%, or a subrange consisting of any value within these ranges.

[0084] In one embodiment, the volume percentage of the second alcohol contained in the first wash liquid is 10-35%, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35%, or a sub-range consisting of any value within these ranges.

[0085] In one embodiment, the volume percentage of the third alcohol contained in the second washing liquid is 75-85%, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85%, or a sub-range consisting of any value in these ranges.

[0086] In one embodiment, in a mixture of a lysate combination and plasma, the concentration is 0.32-1.2 M (e.g., 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 0.91 69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20 M, or a subrange consisting of any value in these ranges) of protein denaturants and / or a mass fraction of 0.64-2.4% (e.g., 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 2.91, 2.92, 2.93, 2.94, 2.95, 2.96, 2.97, 2.98, 2.99, 3.91, 3.99, 4.91, 4.92, 4.93, 4.94, 4.95, 4.96, 4.97, 4.98, 4.99, 5.91, 5.91, 5.99, .75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.00, 1.01, 1. 02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1 .29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1 .81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.30, 2.31, 2.32, 2.33, 2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.40%, or any subranges consisting of values ​​within these ranges) can fully separate free DNA from impurities such as proteins while inhibiting the activity of DNase and avoiding the inactivation of digestive enzymes.

[0087] In one embodiment, in the system, a chaotropic agent at a concentration of 2.1-3.5 M (e.g., 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 M, or a sub-range consisting of any value in these ranges) and / or an alcohol at a volume percentage of 10-35% (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35%, or a sub-range consisting of any value in these ranges) can effectively bind DNA to magnetic beads. The system can be a stock system (e.g., a stock solution), for example, for the first wash solution. The system may also be a working system (eg, a mixture comprising a chaotropic agent and / or alcohol and other substances (eg, plasma, lysate, etc.)), for example, for a binding solution.

[0088] In a preferred embodiment, in the working system comprising the binding liquid, the concentration of the first chaotropic agent is 2.1-3.0 M (for example, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 M, or a sub-range consisting of any values ​​in these ranges) and / or the volume percentage of the first alcohol is 10-25% (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25%, or a sub-range consisting of any values ​​in these ranges).

[0089] In a preferred embodiment, the concentration of the second chaotropic agent in the first wash solution is 2.1-3.5 M (e.g., 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 M, or a sub-range consisting of any values ​​in these ranges) and / or the volume percentage of the second alcohol is 10-35% (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35%, or a sub-range consisting of any values ​​in these ranges).

[0090] In one embodiment, the presence of a surfactant can effectively prevent non-specific binding of impurities to the magnetic beads, for example, a polyoxyethylene nonionic surfactant (eg, polyethylene glycol hexadecyl ether).

[0091] In a preferred embodiment, the reagent combination for extracting free DNA from plasma includes a binding solution comprising a 4.00-5.00 M first chaotropic agent and 15.0-60.0% by volume of a first alcohol. Preferably, the binding solution may also include a surfactant. Further preferably, the surfactant accounts for 6.0-10.0% by weight. More preferably, the surfactant may include a polyoxyethylene nonionic surfactant (e.g., polyethylene glycol cetyl ether).

[0092] In a preferred embodiment, the reagent combination for extracting free DNA from plasma includes a first wash solution; the first wash solution includes 2.5-3.5M of a second chaotropic agent and 10.0-35.0% by volume of a second alcohol. Preferably, the first wash solution may also include a surfactant. Further preferably, the surfactant accounts for 3.0-7.0% by weight. More preferably, the surfactant may include a polyoxyethylene nonionic surfactant (e.g., polyethylene glycol cetyl ether).

[0093] In a preferred embodiment, the reagent combination for extracting free DNA in plasma includes a binding solution and a first washing solution; the binding solution includes a first chaotropic agent at 4.00-5.00M and a first alcohol at a volume percentage of 15.0-60.0%, and the first washing solution includes a second chaotropic agent at 2.5-3.5M and a second alcohol at a volume percentage of 10.0-35.0%. Preferably, the binding solution and / or the first washing solution may also include a surfactant. Further preferably, the mass percentage of the surfactant in the binding solution is 6.0-10.0% and / or the mass percentage of the surfactant in the first washing solution is 3.0-7.0%. More preferably, the surfactant in the binding solution and / or the first washing solution may include a polyoxyethylene nonionic surfactant (e.g., polyethylene glycol hexadecyl ether).

[0094] In another aspect, the present invention provides a kit for extracting cell-free DNA in plasma, comprising the reagent combination for extracting cell-free DNA in plasma according to the aforementioned aspect.

[0095] In yet another aspect, the present invention provides a method for extracting free DNA in plasma using the reagent combination or kit of the aforementioned aspects, comprising the following steps:

[0096] a) Digest the sample; b) Allow free DNA to bind to magnetic beads; c) Wash the magnetic beads; d) Elute the DNA.

[0097] In one embodiment, in step a), the sample is digested using a lysis solution combination comprising a digestion enzyme solution and a digestion solution.

[0098] In one embodiment, in step a), the ratio of the volume of the digestive enzyme solution to the volume of the plasma sample is 0.01-0.08, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or a sub-range consisting of any value within these ranges.

[0099] In one embodiment, in step a), the ratio of the volume of the digestive fluid to the volume of the plasma sample is 0.05-0.20, for example, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, or a sub-range consisting of any value within these ranges.

[0100] In one embodiment, step a) includes an incubation process.

[0101] In one embodiment, in step a), the incubation temperature is 55-65°C, for example, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65°C, or a sub-range consisting of any value in these ranges.

[0102] In one embodiment, in step a), the incubation time is at least 1 minute, e.g., at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 16 minutes, at least 17 minutes, at least 18 minutes, at least 19 minutes, at least 20 minutes, at least 21 minutes, at least 22 minutes, at least 23 minutes, at least 24 minutes, at least 25 minutes, or a sub-range consisting of any value within these ranges.

[0103] In one embodiment, in step b), magnetic beads and binding solution are added.

[0104] In one embodiment, in step b), the ratio of the volume of the binding solution added to the volume of the plasma sample is 1.00-1.75, for example, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 70, 1.71, 1.72, 1.73, 1.74, 1.75, or a sub-range consisting of any value within these ranges.

[0105] In one embodiment, step b) includes an incubation process.

[0106] In one embodiment, in step b), the incubation temperature is room temperature, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35°C, or a sub-range consisting of any value in these ranges.

[0107] In one embodiment, in step b), the incubation time is at least 1 minute, e.g., at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, or a sub-range consisting of any value within these ranges.

[0108] In one embodiment, in step b), the sample is thoroughly mixed during the incubation to prevent sedimentation of the magnetic beads.

[0109] In one embodiment, in step b), the magnetic beads are separated by, for example, adsorption using an external magnetic field.

[0110] In one embodiment, step c) includes washing the magnetic beads with a first washing solution.

[0111] In one embodiment, in step c), after washing the magnetic beads with the first washing solution, the magnetic beads are washed with the first washing solution at least once, for example, at least once, at least twice, at least three times, or a sub-range consisting of any value within these ranges.

[0112] In one embodiment, step c) includes washing the magnetic beads with a second washing solution.

[0113] In one embodiment, in step c), after washing the magnetic beads with the second washing solution, the magnetic beads are washed with the second washing solution at least once, for example, at least once, at least twice, at least three times, or a sub-range consisting of any value within these ranges.

[0114] In one embodiment, in step c), the magnetic beads are separated by, for example, adsorption using an external magnetic field.

[0115] In one embodiment, in step d), the DNA on the magnetic beads is eluted with an elution buffer.

[0116] In one embodiment, step d) includes an incubation process.

[0117] In one embodiment, in step d), the incubation temperature is room temperature, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35°C, or a sub-range consisting of any value in these ranges.

[0118] In one embodiment, in step d), the incubation time is at least 1 minute, e.g., at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or a sub-range consisting of any value within these ranges.

[0119] In one embodiment, in step d), the sample is thoroughly mixed during the incubation to prevent sedimentation of the magnetic beads.

[0120] In one embodiment, in step d), the magnetic beads are separated by, for example, adsorption using an external magnetic field.

[0121] In the present invention, the concentration of the chaotropic agent in the mixed system comprising the binding solution and / or the first washing solution and / or the volume percentage of the alcohol can enable efficient binding of free DNA to the nanomagnetic beads. At the same time, the presence of the surfactant can prevent non-specific binding of impurities to the magnetic beads, thereby making the magnetic bead method suitable for extracting high-yield and high-purity DNA from plasma. In the mixed system of the lysis solution combination and plasma, the concentrations of the protein denaturant and the surfactant can fully separate free DNA from impurities such as proteins, while also inhibiting the activity of DNA enzymes and avoiding the inactivation of digestive enzymes, thereby facilitating simultaneous improvement in the yield and purity of DNA extraction. In addition, the concentrations of the components in the reagent combination of the present invention enable DNA extraction under neutral pH conditions, overcoming the disadvantage of the traditional magnetic bead method that relies on a pH environment that deviates from neutrality to extract DNA. This advantageously ensures the quality of the extracted DNA and facilitates downstream detection.

[0122] The "room temperature" mentioned in the present invention refers to a temperature of 10-35°C.

[0123] The "neutral pH value" described in the present invention refers to a pH value of 6.5-8.5.

[0124] Unless otherwise specified, the percentages, ratios, and concentrations described herein for alcohols are by volume; and the percentages and ratios described herein for other substances are by weight.

[0125] Unless otherwise indicated, when describing the percentage, ratio, or concentration of a specific component in a system, the percentage, ratio, or concentration refers to the percentage, ratio, or concentration of the specific component in the current system. The system can be a reserve system (e.g., a reserve solution (e.g., a digestive enzyme solution, a digestive fluid, a binding solution, a first wash solution, a second wash solution, or an eluent)). The system can also be a working system (e.g., a mixture comprising a chaotropic agent and / or alcohol and other substances (e.g., plasma, a lysis solution, etc.)). In some cases, the reserve system can also be a working system, for example, for the first or second wash solution or the eluent.

[0126] The "working concentration" mentioned in the present invention refers to the percentage, ratio, or concentration of a specific component (e.g., chaotropic agent, alcohol) in a working system (e.g., a mixture containing chaotropic agent and / or alcohol and other substances (e.g., plasma, lysate, etc.)). BRIEF DESCRIPTION OF THE DRAWINGS

[0127] FIG1 is a comparative diagram of the yields of free DNA extracted from 1 mL of plasma sample (the average of two repeated sets of values ​​is taken and one decimal place is retained) in Example 1, Example 2, Comparative Example 1 and Comparative Example 2.

[0128] Figure 2 is a comparative schematic diagram of the fluorescence quantitative PCR (qPCR) detection results (taking the average of two groups of replicates and retaining one decimal place) of the internal reference gene (ALU element) of Test Example 1, Test Example 2, Test Comparison Example 1 and Test Comparison Example 2. DETAILED DESCRIPTION

[0129] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples. The experimental procedures described in the following examples are all conventional procedures unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0130] Human whole blood samples stored in Streck BCT blood collection tubes were centrifuged at 1600g and 4°C for 10 minutes. The supernatant was then centrifuged at 16,000g and 4°C for 10 minutes to remove impurities such as cell debris. Plasma samples were obtained by removing 1 mL of this plasma from each of Example 1, Example 2, Comparative Example 1, and Comparative Example 1. The same conditions were repeated for Example 1, Example 2, Comparative Example 1, and Comparative Example 1, as well as for Test Example 1, Test Example 2, Comparative Example 1, and Comparative Example 2.

[0131] Example 1

[0132] a) To 1 mL of plasma sample, add 40 μL of 20 mg / mL protease solution and 0.1 mL of digestion solution (5.5 M guanidine thiocyanate, 1.3 mM EDTA, 10.4 wt% polyethylene glycol hexadecyl ether, 26 mM Tris-HCl pH 7.5). The working concentrations of each component are: 0.5 M guanidine thiocyanate, 0.1 mM EDTA, 0.9 wt% polyethylene glycol hexadecyl ether, 2.3 mM Tris-HCl pH 7.5.

[0133] b) Add 15 μL of 25 mg / mL silanol-modified magnetic beads and 1.25 mL of binding solution (4.44 M guanidine isothiocyanate, 1.05 mM EDTA, 8.0 wt% polyethylene glycol hexadecyl ether, 19.3% (v / v) isopropanol, 21 mM Tris-HCl pH 7.5) to the digested sample. The working concentrations are: 2.6 M guanidine isothiocyanate, 0.6 mM EDTA, 5% polyethylene glycol hexadecyl ether, 10.6% (v / v) isopropanol, 12.3 mM Tris-HCl pH 7.5. Mix thoroughly and incubate at room temperature for 10 minutes, mixing the sample thoroughly to prevent beads from settling. After incubation, apply a magnetic field to separate the beads and discard the solution.

[0134] c) resuspending the magnetic beads in 1 mL of a first wash solution (3 M guanidine isothiocyanate, 4.9 wt % polyethylene glycol hexadecyl ether, 2 mM EDTA, 30% (v / v) isopropanol, 20 mM Tris-HCl, pH 7.5); mixing thoroughly, applying an external magnetic field to separate the magnetic beads, and discarding the solution; repeating step c) once;

[0135] d) resuspending the magnetic beads in 1 mL of a second washing solution (80% (v / v) ethanol, 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA); after thorough mixing, separating the magnetic beads; and repeating step d) once.

[0136] e) Resuspend the magnetic beads in 30 μL of elution buffer (10 mM Tris-HCl pH 8.0); incubate at room temperature for 5 minutes, mixing the sample thoroughly to prevent sedimentation of the magnetic beads; the resulting solution is the free DNA in the extracted plasma.

[0137] Example 2

[0138] a) To 1 mL of plasma sample, add 40 μL of 20 mg / mL protease solution and 0.2 mL of digestion buffer (5.52 M guanidine thiocyanate, 8 wt% Triton X-100, 26 mM Tris-HCl pH 7.5). Working concentrations of the following components are: 0.9 M guanidine thiocyanate, 1.3% Triton X-100, 4.3 mM Tris-HCl pH 7.5.

[0139] b) Add 15 μL of 25 mg / mL silanol-modified magnetic beads and 1.25 mL of binding solution (4.44 M guanidine isothiocyanate, 6 wt% Triton X-100, 25% (v / v) isopropanol, 20 mM Tris-HCl pH 7.5) to the digested sample. Working concentrations of the following components are: 2.7 M guanidine isothiocyanate, 3.7% Triton X-100, 12.7% (v / v) isopropanol, 12.3 mM Tris-HCl pH 7.5. Mix thoroughly and incubate at room temperature for 10 minutes, mixing the sample thoroughly to prevent beads from settling. After incubation, apply a magnetic field to separate the beads and discard the solution.

[0140] c) resuspending the magnetic beads in 1 mL of a first washing solution (3 M guanidine isothiocyanate, 5 wt% Tween 20, 30% (v / v) isopropanol, 20 mM Tris-HCl, pH 7.5); after thorough mixing, applying an external magnetic field to separate the magnetic beads, and discarding the solution; repeating step c) once;

[0141] d) resuspending the magnetic beads in 1 mL of a second washing solution (80% (v / v) ethanol, 50 mM Tris-HCl, pH 8.0); mixing thoroughly, separating the magnetic beads; and repeating step d) once.

[0142] e) Resuspend the magnetic beads in 30 μL of elution buffer (10 mM Tris-HCl pH 8.0); incubate at room temperature for 5 minutes, mixing the sample thoroughly to prevent sedimentation of the magnetic beads; the resulting solution is the free DNA in the extracted plasma.

[0143] Comparative Example 1

[0144] As described above, 1 mL of plasma sample was taken. Plasma DNA was extracted from the plasma sample using a commercially available magnetic bead extraction kit (manufacturer: ApostleBio; model: MiniMax cfDNA Isolation kit) according to the manufacturer's instructions, and finally eluted with 30 μL of eluent to obtain a plasma DNA product solution of the same volume as in Examples 1 and 2. TM Qubit TMThe dsDNA quantification kit was used to detect the DNA concentration in the product to calculate the yield of free DNA in the extracted plasma (see Figure 1).

[0145] Comparative Example 2

[0146] As described above, a 1 mL plasma sample was taken. The plasma DNA extraction procedure was performed on the plasma sample as follows:

[0147] a) To 1 mL of plasma sample, add 40 μL of 20 mg / mL protease solution and 0.65 mL of digestion solution (5 M guanidine thiocyanate, 25 mM EDTA, 25 wt% Tween 20, 40 mM Bis-Tris pH 6.0). The working concentrations of the components are: 2 M guanidine thiocyanate, 10 mM EDTA, 10 wt% Tween 20, 15 mM Bis-Tris pH 6.0.

[0148] b) Add 15 μL of 25 mg / mL silanol-modified magnetic beads and 3.3 mL of binding solution (3.5 M guanidine thiocyanate, 1 mM EDTA, 2.5 wt% Tween 20, 45% (v / v) isopropanol, 40 mM Bis-Tris pH 6.0) to the digested sample. The working concentrations of the components are: 3 M guanidine thiocyanate, 4 mM EDTA, 5 wt% Tween 20, 30% (v / v) isopropanol, 31 mM Bis-Tris pH 6.0. Mix thoroughly and incubate at room temperature for 10 minutes, mixing the sample thoroughly to prevent beads from settling. After incubation, apply a magnetic field to separate the beads and discard the solution.

[0149] c) resuspending the magnetic beads in 1 mL of a first wash solution (4 M guanidine isothiocyanate, 10 wt% Tween 20, 40% (v / v) isopropanol, 40 mM Bis-Tris pH 6.0); after thorough mixing, applying an external magnetic field to separate the magnetic beads, and discarding the solution; repeating step c) once;

[0150] d) resuspending the magnetic beads in 1 mL of a second washing solution (80% (v / v) ethanol, 50 mM Tris-HCl, pH 8.6); after thorough mixing, separating the magnetic beads; and repeating step d) once.

[0151] e) Resuspend the magnetic beads in 30 μL of elution buffer (10 mM Tris-HCl pH 8.6); incubate at room temperature for 5 minutes, mixing the sample thoroughly to prevent the magnetic beads from settling; the resulting solution is the free DNA in the extracted plasma. TM Qubit TMThe dsDNA quantification kit was used to detect the DNA concentration in the product to calculate the yield of free DNA in the extracted plasma (see Table 1 and Figure 1).

[0152] Referring to Table 1 and Figure 1, comparing the above results, it was found that compared with Comparative Example 1 and Comparative Example 1, the free DNA yields obtained in Example 1 and Example 2 were higher, that is, the DNA yields in Example 1 and Example 2 were higher.

[0153] Table 1 Free DNA yields of Examples 1-2 and Comparative Examples 1-2

[0154] Test Example 1

[0155] 2 μL of the free DNA solution obtained in Example 1 was collected and subjected to fluorescent quantitative PCR using a commercially available fluorescent quantitative PCR kit (Vazyme, Model: Q112) according to the manufacturer's instructions to measure the levels of the human genomic reference gene (ALU element). The resulting Ct values ​​are shown in Table 2 and Figure 2.

[0156] Test Example 2

[0157] 2 μL of the free DNA solution obtained in Example 2 was collected and subjected to fluorescent quantitative PCR using a commercially available fluorescent quantitative PCR kit (Vazyme, Model: Q112) according to the manufacturer's instructions to measure the levels of the human genomic reference gene (ALU element). The resulting Ct values ​​are shown in Table 2 and Figure 2.

[0158] Comparative Test Example 1

[0159] 2 μL of the free DNA solution obtained in Comparative Example 1 was collected and subjected to fluorescent quantitative PCR (qPCR) using a commercially available Vazyme kit (Model: Q112) according to the manufacturer's instructions to measure the level of the human genomic reference gene (ALU element). The resulting Ct values ​​are shown in Table 2 and Figure 2.

[0160] Comparative Test Example 2

[0161] 2 μL of the free DNA solution obtained in Comparative Example 2 was collected and subjected to fluorescent quantitative PCR (qPCR) using a commercially available Vazyme kit (Model: Q112) according to the manufacturer's instructions to measure the level of the human genomic reference gene (ALU element). The resulting Ct values ​​are shown in Table 2 and Figure 2.

[0162] Referring to Figure 2, comparing the above results, it was found that compared with Test Example 1 and Test Example 1, Test Example 1 and Test Example 2 detected higher internal reference gene levels (lower Ct values), which shows that the purity of the DNA products of Example 1 and Example 2 is suitable for the qPCR detection process and can improve the sensitivity of downstream detection.

[0163] Table 2 Ct value results of test examples 1-2 and comparative test examples 1-2 for detecting the level of human genome reference genes (ALU elements)

Claims

1. A reagent combination for extracting free DNA in plasma, characterized in that: The method comprises a binding solution and a first washing solution, wherein the binding solution comprises a first chaotropic agent and a first alcohol, and the first washing solution comprises a second chaotropic agent and a second alcohol.

2. The reagent combination for extracting free DNA in plasma according to claim 1, wherein: The binding solution further contains a surfactant, a buffer, an EDTA solution or a combination thereof; preferably, the surfactant includes an anionic surfactant, a cationic surfactant, a nonionic surfactant, an amphoteric surfactant or a combination thereof; preferably, the surfactant includes sodium dodecyl sulfate (SDS), sodium deoxycholate (NaDOC), Triton X-100, NP-40, Tween 20, a polyoxyethylene nonionic surfactant or a combination thereof; preferably, the surfactant includes a polyoxyethylene nonionic surfactant; preferably, the buffer includes Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

3. A reagent combination for extracting free DNA in plasma according to any one of the preceding claims, wherein: The first washing solution also contains a surfactant, a buffer, an EDTA solution or a combination thereof; preferably, the surfactant includes an anionic surfactant, a cationic surfactant, a nonionic surfactant, an amphoteric surfactant or a combination thereof; preferably, the surfactant includes sodium dodecyl sulfate (SDS), sodium deoxycholate (NaDOC), Triton X-100, NP-40, Tween 20, a polyoxyethylene nonionic surfactant or a combination thereof; preferably, the surfactant includes a polyoxyethylene nonionic surfactant; preferably, the buffer includes Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

4. A reagent combination for extracting free DNA in plasma according to any one of the preceding claims, wherein: The reagent combination comprises a lysis solution combination; preferably, the lysis solution combination comprises a digestive enzyme solution, a digestive solution or a combination thereof; more preferably, the digestive enzyme solution comprises a proteinase K solution; more preferably, the digestive solution comprises a protein denaturant, a surfactant, an EDTA solution, a buffer or a combination thereof; more preferably, the protein denaturant comprises an anionic surfactant, a cationic surfactant, an amino compound or a combination thereof; more preferably, the protein denaturant comprises urea, a guanidine salt; more preferably, the protein denaturant comprises guanidine isothiocyanate; more preferably, the surfactant comprises sodium dodecyl sulfate (SDS), sodium deoxycholate (NaDOC), Triton X-100, NP-40, Tween 20, a polyoxyethylene nonionic surfactant or a combination thereof; more preferably, the surfactant comprises a polyoxyethylene nonionic surfactant; more preferably, the buffer comprises Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

5. The reagent combination for extracting free DNA in plasma according to any one of the preceding claims, wherein: The reagent combination comprises a second washing solution; preferably, the second washing solution comprises alcohol, buffer, EDTA solution or a combination thereof; preferably, the alcohol comprises a monohydric alcohol, a polyhydric alcohol or a combination thereof; preferably, the alcohol comprises methanol, butanol, polyethylene glycol, ethanol, isopropanol, isoamyl alcohol, glycerol or a combination thereof; preferably, the alcohol comprises ethanol; preferably, the buffer comprises Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

6. The reagent combination for extracting free DNA in plasma according to any one of the preceding claims, wherein: The reagent combination comprises an eluent; preferably, the eluent comprises a buffer; more preferably, the eluent comprises Tris-HCl buffer, Bis-Tris buffer, glycine buffer or a combination thereof.

7. A reagent combination for extracting free DNA in plasma according to any one of the preceding claims, wherein: The first or second chaotropic agent comprises perchlorate, hexafluorophosphate, tetrafluoroborate, amide, ammonium salt, potassium salt, sodium salt, lithium salt or a combination thereof; preferably, the first or second chaotropic agent comprises sodium perchlorate, potassium perchlorate, guanidine hydrochloride, guanidine isothiocyanate, urea or a combination thereof.

8. The reagent combination for extracting free DNA in plasma according to any one of the preceding claims, wherein: The alcohol in the combined liquid and the first washing liquid each includes a monohydric alcohol, a polyhydric alcohol or a combination thereof; preferably, the alcohol in the combined liquid and the first washing liquid each includes methanol, butanol, polyethylene glycol, ethanol, isopropanol, isoamyl alcohol, glycerol or a combination thereof; more preferably, the alcohol in the combined liquid and the first washing liquid each includes isopropanol.

9. The reagent combination for extracting free DNA in plasma according to any one of claims 4 to 8, wherein: The concentration of the digestive enzyme solution is 10-30 mg / mL; preferably, the concentration of the digestive enzyme solution is 15-25 mg / mL.

10. The reagent combination for extracting free DNA in plasma according to any one of claims 4 to 8, wherein: The concentration of the protein denaturant is 4.0-6.0M; preferably, the concentration of the protein denaturant is 4.3-5.8M.

11. The reagent combination for extracting free DNA in plasma according to any one of claims 4 to 8, wherein: The mass percentage of the surfactant in the digestive liquid is 8.0-12.0wt%; preferably, the mass percentage of the surfactant in the digestive liquid is 9.0-11.0wt%.

12. The reagent combination for extracting free DNA in plasma according to any one of claims 2 to 8, wherein: The mass percentage of the surfactant in the combined liquid is 6.0-10.0wt%; preferably, the mass percentage in the combined liquid is 7.0-9.0wt%.

13. The reagent combination for extracting free DNA in plasma according to any one of claims 3 to 8, wherein: The mass percentage of the surfactant in the first washing liquid is 3.0-7.0 wt %; preferably, the mass percentage of the surfactant in the first washing liquid is 4.0-6.0 wt %.

14. The reagent combination for extracting free DNA in plasma according to any one of claims 4 to 8, wherein: The concentration of the buffer in the digestion solution is 20-30 mM, and the pH value thereof is 6.5-8.5; preferably, the concentration of the buffer in the digestion solution is 23-28 mM, and the pH value thereof is 7.3-7.

8.

15. The reagent combination for extracting free DNA in plasma according to any one of claims 2 to 8, wherein: The concentration of the buffer in the binding solution is 15-25 mM, and the pH value thereof is 6.5-8.5; preferably, the concentration of the buffer in the binding solution is 18-23 mM, and the pH value thereof is 7.3-7.

8.

16. The reagent combination for extracting free DNA in plasma according to any one of claims 3 to 8, wherein: The concentration of the buffer in the first washing solution is 15-25 mM, and the pH value thereof is 6.5-8.5; preferably, the concentration of the buffer in the first washing solution is 18-23 mM, and the pH value thereof is 7.3-7.

8.

17. The reagent combination for extracting free DNA in plasma according to any one of claims 5 to 8, wherein: The concentration of the buffer in the second washing solution is 45-55 mM, and the pH value thereof is 6.5-8.5; preferably, the concentration of the buffer in the second washing solution is 48-53 mM, and the pH value thereof is 7.7-8.

0.

18. The reagent combination for extracting free DNA in plasma according to any one of claims 6 to 8, wherein: The concentration of the buffer in the eluent is 5-15 mM, and the pH value thereof is 6.5-8.5; preferably, the concentration of the buffer in the eluent is 8-13 mM, and the pH value thereof is 7.7-8.

0.

19. The reagent combination for extracting free DNA in plasma according to any one of claims 4 to 8, wherein: The concentration of the EDTA solution in the digestive solution is 0.8-1.8 mM; preferably, the concentration of the EDTA solution in the digestive solution is 1.1-1.6 mM.

20. The reagent combination for extracting free DNA in plasma according to any one of claims 2 to 8, wherein: The concentration of the EDTA solution in the binding solution is 1.00-1.10 mM; preferably, the concentration of the EDTA solution in the binding solution is 1.03-1.08 mM.

21. The reagent combination for extracting free DNA in plasma according to any one of claims 3 to 8, wherein: The concentration of the EDTA solution in the first washing solution is 1.5-2.5 mM; preferably, the concentration of the EDTA solution in the first washing solution is 1.8-2.3 mM.

22. The reagent combination for extracting free DNA in plasma according to any one of claims 5 to 8, wherein: The concentration of the EDTA solution in the second washing solution is 0.3-0.8 mM; preferably, the concentration of the EDTA solution in the second washing solution is 0.4-0.6 mM.

23. The reagent combination for extracting free DNA in plasma according to any one of claims 1 to 8, wherein: The concentration of the first chaotropic agent in the binding solution is 4.00-5.00M; preferably, the concentration of the first chaotropic agent in the binding solution is 4.20-4.80M.

24. The reagent combination for extracting free DNA in plasma according to any one of claims 1 to 8, wherein: The concentration of the second chaotropic agent in the first washing solution is 2.5-3.5M; preferably, the concentration of the second chaotropic agent in the first washing solution is 2.8-3.3M.

25. The reagent combination for extracting free DNA in plasma according to any one of claims 1 to 8, wherein: The volume percentage of the first alcohol contained in the combination liquid is 15.0-60.0%; preferably, the volume percentage of the first alcohol contained in the combination liquid is 20.0-50.0%.

26. The reagent combination for extracting free DNA in plasma according to any one of claims 1 to 8, wherein: The volume percentage of the second alcohol contained in the first washing liquid is 10-35%; preferably, the volume percentage of the second alcohol contained in the first washing liquid is 15-33%.

27. The reagent combination for extracting free DNA in plasma according to any one of claims 5 to 8, wherein: The volume percentage of the third alcohol contained in the second washing liquid is 75-85%; preferably, the volume percentage of the third alcohol contained in the second washing liquid is 78-83%.

28. A kit for extracting free DNA in plasma, comprising the reagent combination for extracting free DNA in plasma according to any one of claims 1 to 27.

29. A method for extracting free DNA in plasma using the reagent combination according to any one of claims 1 to 27 or the kit according to claim 28, comprising the following steps: a) digest the sample; b) allow free DNA to bind to magnetic beads; c) wash the magnetic beads; d) elute the DNA.

30. The method of claim 29, wherein: In step a), the sample is digested with a lysis solution combination comprising a digestive enzyme solution and a digestive solution; preferably, the ratio of the volume of the digestive enzyme solution to the volume of the plasma sample is 0.01-0.08, and the ratio of the volume of the digestive solution to the volume of the plasma sample is 0.05-0.20; more preferably, the ratio of the volume of the digestive enzyme solution to the volume of the plasma sample is 0.04-0.08, and the ratio of the volume of the digestive solution to the volume of the plasma sample is 0.08-0.

20.

31. The method according to claim 29 or 30, wherein: In step b), magnetic beads and binding solution are added; preferably, the ratio of the volume of the added binding solution to the volume of the plasma sample is 1.00-1.75; more preferably, the ratio of the volume of the added binding solution to the volume of the plasma sample is 1.20-1.

50.

32. The method according to claim 29 or 30, wherein: Step c) includes washing the magnetic beads with a first washing solution; preferably, in step c), after washing the magnetic beads with the first washing solution, the magnetic beads are washed with the first washing solution at least once.

33. The method according to claim 29 or 30, wherein: Step c) includes washing the magnetic beads with a second washing solution; preferably, after washing the magnetic beads with the second washing solution, the magnetic beads are washed with the second washing solution at least once.

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