Kit and method for extracting free DNA

Abstract

The invention relates to the technical field of free DNA extraction, and provides a kit and a method for extracting free DNA, the kit comprises a lysis binding solution A, first magnetic beads, a lysis binding solution B, a washing solution A, a washing solution B and an eluent, and the lysis binding solution B comprises second magnetic beads; wherein the first magnetic beads tend to adsorb large-fragment nucleic acid in the environment of the splitting combination solution A, the second magnetic beads in the splitting combination solution B fully adsorb small-fragment nucleic acid, and a magnetic bar module of an instrument or an external magnetic field in other forms is used in a matched manner to complete the purification and elution process of free DNA; the purposes of improving the distribution ratio of free DNA in a nucleic acid separation product and reducing background pollution of large-fragment nucleic acid and loss of small-fragment nucleic acid are achieved; and free DNA meeting downstream application requirements can be separated under the condition of different sample qualities.

Description

Technical Field

[0001] The invention relates to the technical field of free DNA extraction, in particular to a kit and a method for extracting free DNA. Background Art

[0002] Free DNA (also known as cfDNA) is a type of DNA that is free outside cells in blood or other body fluids (urine, tissue fluid, saliva, follicular fluid, etc.). Since Mandel and Metais first reported the existence of free DNA in human plasma in 1948, it has played an increasingly important role in the diagnosis and screening of genetics and diseases as an ideal biomarker. It is widely used in tumor screening and diagnosis, prenatal screening, cardiovascular disease detection, transplantation medical monitoring, autoimmune disease detection, infection detection and other scenarios, providing new methods for the diagnosis of clinically related diseases, auxiliary decision-making and prognosis of diseases.

[0003] Free DNA has the characteristics of small fragments and low concentration. Sufficient yield and low background nucleic acid contamination are the key to ensure the accuracy and reliability of free DNA detection results. According to current research progress, different free DNA release pathways may be the reason for the different lengths of its fragments, but overall free DNA is mainly distributed at 166bp.

[0004] At present, commercial free DNA extraction reagents are mainly based on the silicon-based membrane centrifugal column method and the magnetic bead adsorption method in terms of methodology. The nucleic acid extracted by the silicon-based membrane centrifugal column method has high purity and good yield, but it requires the use of high-speed centrifugation equipment multiple times, making it difficult to achieve high-throughput and fully automated nucleic acid extraction. The multiple tube transfer operations also introduce the risk of contamination. The nucleic acid yield of the magnetic bead adsorption method is not as good as that of the centrifugal column method, but its operation process with magnetic separation as the core can be perfectly implemented on automated equipment, which is conducive to improving experimental efficiency, reducing manual errors and ensuring the consistency of results. It can also achieve cost savings in terms of manpower, instruments, consumables, etc. However, no matter which method is used to prepare nucleic acid extraction reagents, it is impossible to solve the problems of low free DNA yield, large-fragment nucleic acid background contamination and small-fragment nucleic acid loss simply from the choice of methodology.

[0005] Therefore, how to increase the distribution ratio of free DNA in nucleic acid separation products while ensuring the free DNA extraction yield is a research focus in this field. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a kit and method for increasing the distribution ratio of free DNA in a nucleic acid separation product, so as to solve the problems in the prior art.

[0007] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0008] One of the purposes of the present invention is to protect a kit; the kit includes a lysis and binding solution A; the lysis and binding solution A includes: a hydrophobic agent, a first surfactant, isopropanol, lauryl magnesium sulfate and magnesium chloride.

[0009] In certain embodiments, the concentration of the hydrophobic agent in the lysis and binding solution A is 10% to 30 v / v%.

[0010] In certain embodiments, the hydrophobic agent includes one or more of cinnamon oil and mineral oil.

[0011] In some embodiments, the hydrophobic agent is cinnamon oil.

[0012] In certain embodiments, the concentration of the first surfactant in the lysis and binding solution A is 5-10 w / v%.

[0013] In certain embodiments, the first surfactant in the lysis and binding solution A is an anionic surfactant.

[0014] In certain embodiments, the anionic surfactant includes one or more of sodium dodecyl sulfate (SDS), sodium lauroyl sarcosinate, sodium alkylbenzene sulfonate, and sodium dodecyl sulfonate.

[0015] In some specific embodiments, the anionic surfactant is preferably SDS, and may also be sodium lauroyl sarcosinate.

[0016] In certain embodiments, the concentration of isopropanol in the lysis and binding solution A is 20-30 v / v%.

[0017] In certain embodiments, the concentration of magnesium lauryl sulfate in the lysis and binding solution A is 1-5 w / v%.

[0018] In certain embodiments, the concentration of magnesium chloride in the lysis and binding solution A is 2-20 w / v%.

[0019] In certain embodiments, the lysis and binding solution A comprises 10-30 v / v% of a hydrophobic agent, 5-10 w / v% of a first surfactant, 20-30 v / v% of isopropanol, 1-5 w / v% of magnesium lauryl sulfate, and 2-20 w / v% of magnesium chloride.

[0020] In certain embodiments, the kit further comprises a lysis and binding solution B, wherein the lysis and binding solution B comprises a guanidine salt, a second surfactant, tris(hydroxymethyl)aminomethane, sodium chloride, polyethylene glycol (PEG) and acetone.

[0021] In certain embodiments, the concentration of guanidine salt in the lysis and binding solution B is 20-30 w / v%.

[0022] In certain embodiments, the guanidine salt includes at least one of guanidine isothiocyanate and guanidine hydrochloride.

[0023] In certain embodiments, the guanidine salt is guanidine isothiocyanate.

[0024] In certain embodiments, the concentration of the second surfactant in the lysis and binding solution B is 0.1-2 v / v%.

[0025] In certain embodiments, the second surfactant in the lysis and binding solution B is a non-ionic surfactant.

[0026] In certain embodiments, the nonionic surfactant is one or more of Triton, polysorbate, and ethylphenyl polyethylene glycol (NP40).

[0027] In certain embodiments, the Triton is Triton X-100.

[0028] In certain embodiments, the polysorbate includes polysorbate-20 (Tween-20) and polysorbate-80 (Tween-80).

[0029] In certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the lysis and binding solution B is 0.05-3w / w%.

[0030] In certain embodiments, the concentration of sodium chloride in the lysis and binding solution B is 10-20 w / w%.

[0031] In certain embodiments, the concentration of polyethylene glycol in the lysis and binding solution B is 15-30 w / v%.

[0032] In certain embodiments, the molecular weight of the polyethylene glycol (PEG) is 4000-8000; it can also be PEG4000, PEG6000, PEG8000, etc.

[0033] In certain embodiments, the concentration of acetone in the lysis and binding solution B is 20-40 v / v%.

[0034] In certain embodiments, the kit comprises first magnetic beads and / or second magnetic beads.

[0035] In certain embodiments, the first magnetic beads are carboxylated magnetic beads.

[0036] In certain embodiments, the working concentration of the first magnetic bead solution is 0.03 w / w% to 0.06 w / w%.

[0037] In certain embodiments, the particle size of the first magnetic beads is 500 nm to 1 μm.

[0038] In certain embodiments, the working concentration of the second magnetic beads is 0.5 w / w% to 0.8 w / w%.

[0039] In certain embodiments, the second magnetic beads are modified differently from the first magnetic beads.

[0040] In some embodiments, the second magnetic beads are amino-modified magnetic beads.

[0041] In certain embodiments, the particle size of the second magnetic beads is 50 nm to 200 nm.

[0042] In certain embodiments, the lysis and binding solution B comprises: 20-30w / v% guanidine salt, 0.1-2v / v% second surfactant, 0.05-3w / w% tris(hydroxymethyl)aminomethane, 10-20w / w% sodium chloride, 15-30w / v% polyethylene glycol, and 20-40v / v% acetone. The lysis and binding solution B preferably also comprises second magnetic beads at a working concentration of 0.5w / w% to 0.8w / w%.

[0043] In certain embodiments, the kit further comprises one or more of proteinase K, washing solution A, washing solution B, and elution solution.

[0044] In certain embodiments, the working concentration of proteinase K is 0.5-4 mg / mL.

[0045] In some embodiments, the washing solution A comprises guanidine salt, tris(hydroxymethyl)aminomethane, a third surfactant and alcohol. In some embodiments, the concentration of guanidine salt in the washing solution A is 2M to 3M.

[0046] In certain embodiments, the guanidine salt includes at least one of guanidine isothiocyanate and guanidine hydrochloride.

[0047] In certain embodiments, the guanidine salt is guanidine isothiocyanate.

[0048] In certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the washing solution A is 5 mM to 10 mM.

[0049] In certain embodiments, the concentration of the third surfactant in the washing solution A is 1-5 v / v%.

[0050] In certain embodiments, the third surfactant in the washing solution A is a nonionic surfactant.

[0051] In certain embodiments, the nonionic surfactant is one or more of Triton, polysorbate, and ethylphenyl polyethylene glycol (NP40).

[0052] In certain embodiments, the Triton is Triton X-100.

[0053] In certain embodiments, the polysorbate includes Tween-20 and Tween-80.

[0054] In certain embodiments, the concentration of alcohol in the washing solution A is 40-60 v / v%.

[0055] In certain embodiments, the alcohol in the washing solution A includes ethanol.

[0056] In some embodiments, the ethanol is anhydrous ethanol.

[0057] In certain embodiments, the washing solution B comprises tris(hydroxymethyl)aminomethane, potassium chloride and alcohol.

[0058] In certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the washing solution B is 5 mM to 10 mM.

[0059] In certain embodiments, the concentration of potassium chloride in the washing solution B is 1-10 w / v %.

[0060] In certain embodiments, the concentration of alcohol in the washing solution B is 70-80 v / v%.

[0061] In certain embodiments, the alcohol in the washing solution B includes ethanol.

[0062] In some embodiments, the ethanol is anhydrous ethanol.

[0063] In certain embodiments, the eluent comprises tris(hydroxymethylaminomethane).

[0064] In certain embodiments, the concentration of the tris(hydroxymethyl)aminomethane is 5 mM to 50 mM.

[0065] The second object of the present invention is to protect the use of the kit as described above in extracting free DNA from a sample or preparing a product for extracting free DNA from a sample.

[0066] In certain embodiments, the sample comprises: at least one of whole blood, serum, plasma, saliva, urine, pleural effusion, ascites, sputum, tissue fluid, and follicular fluid samples.

[0067] In certain embodiments, the product is a reagent, a kit, or a system.

[0068] In certain embodiments, the system is a nucleic acid extraction and / or detection system.

[0069] The third object of the present invention is to protect a product, which comprises: the kit as described above.

[0070] In certain embodiments, the product further comprises a nucleic acid extractor.

[0071] In some specific embodiments, the nucleic acid extractor is a fully automatic nucleic acid extractor or a manual nucleic acid extraction device.

[0072] In certain embodiments, the product further comprises other means capable of providing an external magnetic field.

[0073] The fourth object of the present invention is to protect a method for extracting free DNA, which comprises the step of extracting free DNA using the product as described above;

[0074] The method is for non-disease diagnosis and treatment purposes.

[0075] In certain embodiments, the method comprises the steps of:

[0076] S1: Mix proteinase K, cleavage binding solution A and the sample to be tested, lyse, add the first magnetic beads for the first incubation, magnetic adsorption, and then discard the first magnetic beads to obtain a mixed solution 1;

[0077] S2: adding lysis binding solution B and / or the second magnetic beads to the mixed solution 1, performing a second incubation and magnetic adsorption, and then washing with washing solution A and washing solution B, eluting with elution solution to obtain a free DNA extract.

[0078] In some embodiments, the volume ratios of the test sample, proteinase K, cleavage binding solution A, first magnetic beads, cleavage binding solution B, washing solution A, washing solution B and eluent are: 1, 0.08-0.12, 0.2-0.6, 0.8-1.2, 0.4-0.8, 1.2-1.8, 1.4-2, 0.08-0.2.

[0079] In certain embodiments, the lysis condition is shaking for 4 to 8 minutes.

[0080] In certain embodiments, the first incubation time is 2 to 5 minutes.

[0081] In certain embodiments, the second incubation time is 5 to 20 minutes.

[0082] Compared with the prior art, the present invention has the following beneficial effects:

[0083] In order to separate free DNA that meets the requirements of downstream applications under the conditions of different sample qualities, the present invention studies the formula and use process of a free DNA extraction reagent, and obtains a kit, which includes proteinase K, a first magnetic bead, a second magnetic bead, a lysis binding solution A, a lysis binding solution B, a washing solution A, a washing solution B and an elution solution, wherein the first magnetic beads in the lysis binding solution A environment tend to adsorb large-fragment nucleic acids, and the second magnetic beads in the lysis binding solution B environment fully adsorb small-fragment nucleic acids, and then the magnetic bar module of the instrument or other forms of external magnetic fields are used in combination to complete the purification and elution process of the free DNA; the purpose of increasing the distribution ratio of free DNA in the nucleic acid separation product and reducing the background contamination of large-fragment nucleic acids and the loss of small-fragment nucleic acids is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is the detection result of capillary electrophoresis of sample A in Example 6.

[0085] Figure 2 This is the detection result of capillary electrophoresis of sample B in Example 6.

[0086] Figure 3 The test results of capillary electrophoresis of samples 1 to 8 in test example 1 are shown in FIG.

[0087] Figure 4 The capillary electrophoresis test results of samples 1 to 8 using the Tiangen magnetic bead extraction kit in Test Example 1 are shown. DETAILED DESCRIPTION

[0088] One of the purposes of the present invention is to protect a kit for extracting free DNA; the kit comprises a lysis and binding solution A; the lysis and binding solution A comprises: a hydrophobic agent, a first surfactant, isopropanol, lauryl magnesium sulfate and magnesium chloride.

[0089] In certain embodiments, the concentration of the hydrophobic agent in the lysis and binding solution A is 10-30 v / v%, 10-20 v / v%, 20-30 v / v%, 15-25 v / v%, or 20 v / v%.

[0090] In certain embodiments, the hydrophobic agent includes one or more of cinnamon oil and mineral oil.

[0091] In some embodiments, the hydrophobic agent is cinnamon oil.

[0092] The invention studies the influence of reagents such as cinnamon oil, liquid paraffin, mineral oil, lemon essential oil, isopropanol and the like and the usage amount on the adsorption of nucleic acid fragments of different sizes to magnetic beads, and finds that the adsorption effect is best when the usage amount of cinnamon oil is 10-30 v / v%.

[0093] In certain embodiments, the concentration of the first surfactant in the lysis and binding solution A is 5-10 w / v%; it can also be 5-7 w / v%, or 8-10 w / v%; it can also be 6-8 w / v%, or it can be 8 w / v%, 10 w / v%.

[0094] In certain embodiments, the first surfactant is an anionic surfactant.

[0095] In certain embodiments, the anionic surfactant includes one or more of sodium dodecyl sulfate (SDS), sodium lauroyl sarcosinate, sodium alkylbenzene sulfonate, and sodium dodecyl sulfonate.

[0096] In some specific embodiments, the anionic surfactant is SDS, and may also be sodium lauroyl sarcosinate.

[0097] In certain embodiments, the concentration of magnesium lauryl sulfate in the lysis and binding solution A is 1-5 w / v%; it can also be 1-3 w / v%, 3-5 w / v%, 2-4 w / v%, or 3 w / v%, 5 w / v%.

[0098] Fully lysing the sample to release nucleic acids is a necessary condition for obtaining sufficient free DNA. The present invention studies the effects of reagents such as sodium dodecyl sulfate (SDS), sodium lauroyl sarcosinate, magnesium lauryl sulfate, and dithiothreitol on the yield of free DNA. Preferably, using 5-10 w / v% SDS and 1-5 w / v% magnesium lauryl sulfate in the lysis and binding solution A can obtain the best yield of free DNA.

[0099] In certain embodiments, the concentration of isopropanol in the lysis and binding solution A is 20-30 v / v%; it can also be 20-25 v / v%, 25-30 v / v%, 23-28 v / v%, or 25 v / v%, 30 v / v%.

[0100] In certain embodiments, the concentration of magnesium chloride in the lysis and binding solution A is 2-20 w / v%; it can also be 2-10 w / v%, 10-20 w / v%, 5-25 w / v%, or 10 w / v%, 15 w / v, or 20 w / v%.

[0101] In certain embodiments, the lysis and binding solution A includes 10-30 v / v% of a hydrophobic agent, 5-10 w / v% of a first surfactant, 20-30 v / v% of isopropanol, 1-5 w / v% of magnesium lauryl sulfate, and 2-20 w / v% of magnesium chloride.

[0102] In certain specific embodiments, the lysis and binding solution A includes 20 v / v% of a hydrophobic agent, 10 w / v% of a first surfactant, 30 v / v% of isopropanol, 5 w / v% of magnesium lauryl sulfate, and 20 w / v% of magnesium chloride.

[0103] In certain embodiments, the kit further comprises a lysis and binding solution B, wherein the lysis and binding solution B comprises a guanidine salt, a second surfactant, tris(hydroxymethyl)aminomethane, sodium chloride, polyethylene glycol (PEG) and acetone.

[0104] In certain embodiments, the concentration of guanidine salt in the lysis and binding solution B is 20-30 w / v%; it can also be 20-25 w / v%, 25-30 w / v%, 23-27 w / v%, or 20 w / v%, 25 w / v%, or 30 w / v%.

[0105] In certain embodiments, the guanidine salt includes at least one of guanidine isothiocyanate and guanidine hydrochloride.

[0106] In certain embodiments, the guanidine salt is guanidine isothiocyanate.

[0107] In certain embodiments, the concentration of the second surfactant in the lysis and binding solution B is 0.1-2 v / v%; it can also be 0.5-1 v / v%, 1-2 v / v%, 0.5-1.5 v / v%, or 0.5 v / v%, 1 v / v%.

[0108] In certain embodiments, the second surfactant is a nonionic surfactant.

[0109] In certain embodiments, the nonionic surfactant is at least one of TritonX-100, Tween-20, Tween-80, and NP40.

[0110] In certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the lysis and binding solution B is 0.05-3w / w%, 0.1-1w / w%, 1-3w / w%, 0.5-2w / w%, 1w / w% or 2w / w%.

[0111] In certain embodiments, the concentration of sodium chloride in the lysis and binding solution B is 10-20w / w%, or 10-15w / w%, or 15-20w / w%, or 13-18w / w%, or 10w / w%, 15w / w%, or 20w / w%.

[0112] In certain embodiments, the concentration of polyethylene glycol in the lysis and binding solution B is 15-30 w / v%; it can also be 15-25 w / v%, 25-30 w / v%, 20-27 w / v%, or 20 w / v%, 25 w / v%, or 30 w / v%.

[0113] In certain embodiments, the polyethylene glycol (PEG) may be PEG4000, PEG6000, PEG8000, or the like.

[0114] In certain embodiments, the concentration of acetone in the lysis and binding solution B is 20-40 v / v%; it can also be 20-30 v / v%, 30-40 v / v%, 25-35 v / v%, or 25 v / v%, 30 v / v%.

[0115] In certain embodiments, the kit further comprises first magnetic beads and / or second magnetic beads; wherein the first magnetic beads tend to adsorb large fragments of nucleic acid, and the second magnetic beads are used to adsorb small fragments of free DNA.

[0116] In certain embodiments, the first magnetic beads are carboxylated magnetic beads.

[0117] In certain embodiments, the working concentration of the first magnetic beads is 0.03w / w% to 0.06w / w%; it can also be 0.03w / w% to 0.04w / w%, it can also be 0.04w / w% to 0.06w / w%, it can also be 0.04w / w% to 0.05w / w%; it can also be 0.03w / w% or 0.04w / w%.

[0118] In certain embodiments, the particle size of the first magnetic beads is 500nm-1μm; it can also be 500nm-700nm, 800nm-1μm, or 600nm-900nm.

[0119] In certain embodiments, the second magnetic beads are modified differently from the first magnetic beads.

[0120] In certain embodiments, the second magnetic beads are amino-modified magnetic beads.

[0121] In certain embodiments, the working concentration of the second magnetic beads is 0.5 w / w% to 0.8 w / w%; it can also be 0.5 w / w% to 0.7 w / w%, it can also be 0.6 w / w% to 0.8 w / w%; it can also be 0.6 w / w%.

[0122] In certain embodiments, the second magnetic beads are amino-modified magnetic beads.

[0123] In some embodiments, the particle size of the second magnetic beads is 50 nm to 200 nm; it can also be 50-100 nm, 100-150 nm, or 150-200 nm.

[0124] It should be noted that in the present invention, the lysis binding solution (such as lysis binding solution A, lysis binding solution B) can be mixed with the sample to be extracted, and then magnetic beads (such as first magnetic beads, second magnetic beads) can be added for mixing; the lysis binding solution and magnetic beads can also be mixed, and then the sample to be extracted can be added for mixing and reaction; the lysis solution binding solution, magnetic beads and sample to be extracted can also be mixed at the same time; the present invention includes two magnetic bead adsorptions, wherein the lysis binding solution A and the first magnetic beads are used in combination to adsorb large-fragment nucleic acids, and the lysis binding solution B and the second magnetic beads are used in combination to adsorb small-fragment nucleic acids.

[0125] In certain embodiments, the lysis and binding solution B comprises: 20-30 w / v% guanidine salt, 0.1-2 v / v% second surfactant, 0.05-3 w / w% tris(hydroxymethyl)aminomethane, 10-20 w / w% sodium chloride, 15-30 w / v% PEG, and 20-40 v / v% acetone.

[0126] In certain preferred embodiments, the lysis and binding solution B further comprises second magnetic beads at a working concentration of 0.5 to 0.8 w / w%.

[0127] In some specific embodiments, the lysis and binding solution B includes: guanidine thiocyanate 30w / v%, TritonX-100 1w / v%, tris(hydroxymethyl)aminomethane 1w / w%, sodium chloride 15w / w%, PEG 20w / v%, and acetone 30v / v%.

[0128] In certain preferred embodiments, the lysis and binding solution B further comprises 0.6 w / w% second magnetic beads.

[0129] Aiming at the requirement that the first magnetic beads used in the lysis binding solution A environment should tend to adsorb large-fragment nucleic acids, and the magnetic beads in the lysis binding solution B should fully adsorb small-fragment nucleic acids; the present invention studies the effects of the type of surface modification of the magnetic beads, the amount used and the incubation time, and the formula of the lysis binding solution A on the adsorption effect of nucleic acid fragments of different sizes. For example, the dosage of the first magnetic beads is 0.03w / w% to 0.06w / w%, the surface is carboxyl-modified, and the incubation time of the first magnetic beads and the sample in the lysis binding solution A is 2 to 5min; the dosage of the second magnetic beads in the lysis binding solution B environment is 0.5w / w% to 0.8w / w%, the surface is amino-modified, and the incubation time with the sample is 5 to 20min; within this range, it is most conducive to obtaining small-fragment nucleic acids.

[0130] The present invention also studies the effects of the types and usage amounts of inorganic salts, alcohol and ketone organic matter, PEG and other substances on the recovery effect of small nucleic acid fragments during the extraction process; the results show that in the lysis and binding solution B, the best formula combination is sodium chloride 10-20w / w%, PEG8000 15-30w / v%, acetone 20-40v / v%; within this range, the recovery effect of small nucleic acid fragments is the best.

[0131] The use of acetone and isopropanol in the lysis and binding solution can not only protect the nucleic acid fragments from the shearing action of nucleases during the extraction process, but also promote the adsorption process of nucleic acids and magnetic beads.

[0132] In certain embodiments, the kit further comprises one or more of proteinase K, washing solution A, washing solution B, and elution solution.

[0133] In certain embodiments, the working concentration of the proteinase K is 0.5-4 mg / mL; it can also be 0.5-2 mg / mL, 2-3 mg / mL, 3-4 mg / mL, 1.5-2.5 mg / mL, or 1, 1.5, 2, 2.5, 3, 3.5, or 4 mg / mL.

[0134] In certain embodiments, the washing solution A comprises guanidine salt, tris(hydroxymethyl)aminomethane, a third surfactant and alcohols, wherein the washing solution A is used to remove impurities such as proteins, lipids, acetone and isopropanol.

[0135] In certain embodiments, the concentration of the guanidine salt in the washing solution A is 2M to 3M; it can also be 2 to 2.5M, it can also be 2.4 to 3M, or it can be 2M or 3M.

[0136] In certain embodiments, the guanidine salt includes at least one of guanidine isothiocyanate and guanidine hydrochloride.

[0137] In certain embodiments, the guanidine salt is guanidine isothiocyanate.

[0138] In certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the washing solution A is 5 mM to 10 mM; it can also be 5 to 8 mM, 7 to 10 mM, or 5 mM, 6 mM, or 7 mM.

[0139] In certain embodiments, the concentration of the third surfactant in the washing solution A is 1-5 v / v%; it can also be 2-4 v / v%, or it can be 2 v / v% or 3 v / v%.

[0140] In certain embodiments, the third surfactant in the washing solution A is a nonionic surfactant.

[0141] In certain embodiments, the nonionic surfactant is at least one of TritonX-100, Tween-20, Tween-80, and NP40.

[0142] In some embodiments, the nonionic surfactant is NP40.

[0143] In certain embodiments, the concentration of alcohol in the washing solution A is 40-60 v / v%; it can also be 45-55 v / v%, or it can also be 50 v / v%.

[0144] In some specific embodiments, the alcohol in the washing solution A includes ethanol.

[0145] In certain embodiments, the ethanol is anhydrous ethanol.

[0146] In certain embodiments, the washing solution B comprises tris(hydroxymethyl)aminomethane, potassium chloride and alcohols. The washing solution B is used to remove impurities such as sugars and inorganic salts.

[0147] In certain embodiments, the concentration of tris(hydroxymethyl)aminomethane in the washing solution B is 5 mM to 10 mM; it can also be 6 mM to 8 mM, or it can be 5 mM, 6 mM, or 7 mM.

[0148] In certain embodiments, the concentration of potassium chloride in the washing solution B is 1-10 w / v%; it can also be 1-5 w / v%, 5-10 w / v%, 3-7 w / v%, or 8 w / v%, 10 w / v%.

[0149] In certain embodiments, the concentration of alcohol in the washing solution B is 70-80 v / v%, or 70-75 v / v%, or 75-80 v / v%, or 70 v / v%, or 75 v / v%.

[0150] In certain embodiments, the alcohol in the washing solution B includes ethanol.

[0151] The use of anhydrous ethanol in the washing solution can not only protect the nucleic acid fragments from the shearing action of nucleases during the extraction process, but also promote the adsorption process of nucleic acids and magnetic beads. The concentration of guanidine hydrochloride in washing solution A is relatively high, and guanidine hydrochloride can also prevent the desorption of nucleic acids and magnetic beads during the washing process, so the ethanol concentration in washing solution A is lower than that in washing solution B.

[0152] In certain embodiments, the ethanol is anhydrous ethanol.

[0153] In certain embodiments, the eluent is tris(hydroxymethyl)aminomethane; the addition of other components such as EDTA may affect the use of some downstream experiments.

[0154] In certain embodiments, the concentration of the tris(hydroxymethyl)aminomethane is 5 mM to 50 mM, 10 mM to 40 mM, 20 mM to 30 mM, or 10 mM.

[0155] The function of the elution buffer is to elute the free DNA from the magnetic beads and serve as a storage matrix for the free DNA for use in downstream experiments.

[0156] The second object of the present invention is to protect the use of the kit as described above in extracting free DNA from a sample or preparing a product for extracting free DNA from a sample.

[0157] In certain embodiments, the sample comprises: at least one of whole blood, serum, plasma, saliva, urine, pleural effusion, ascites, sputum, tissue fluid, and follicular fluid samples.

[0158] In certain embodiments, the product is a reagent, a kit, or a system.

[0159] In certain embodiments, the system is a nucleic acid extraction and / or detection system.

[0160] The third object of the present invention is to protect a product for extracting free DNA, which comprises: the kit as described above.

[0161] In certain embodiments, the product further comprises a nucleic acid extractor.

[0162] In some specific embodiments, the nucleic acid extractor is a fully automatic nucleic acid extractor or a manual nucleic acid extractor; for example, manual extraction of nucleic acids is achieved by using the cooperation of an external magnetic field such as a magnetic rod and a magnetic frame.

[0163] A fourth object of the present invention is to protect a method for extracting free DNA, which comprises the step of extracting free DNA using the product as described above.

[0164] In certain embodiments, the method comprises the steps of:

[0165] S1: Mix proteinase K, cleavage binding solution A and the sample to be tested, lyse, add the first magnetic beads for the first incubation, magnetic adsorption, and then discard the first magnetic beads to obtain a mixed solution 1;

[0166] S2: adding lysis binding solution B and / or the second magnetic beads to the mixed solution 1, performing a second incubation and magnetic adsorption, and then washing with washing solution A and washing solution B, eluting with elution solution to obtain a free DNA extract.

[0167] It should be noted that the lysis binding solution A and the first magnetic beads are used in combination, and the lysis binding solution B and the second magnetic beads are used in combination. The above operation is not limited to mixing the lysis binding solution (including the lysis binding solution A or the lysis binding solution B) with the sample to be extracted, and then adding the magnetic beads (including the first magnetic beads or the second magnetic beads) for mixing; the lysis binding solution and the magnetic beads can also be mixed, and then the sample to be extracted is added for mixing and reaction; the lysis binding solution, the magnetic beads and the sample to be extracted can also be mixed at the same time. The first magnetic beads are magnetically adsorbed in the environment of the lysis binding solution A, and the second magnetic beads are magnetically adsorbed in the environment of the lysis binding solution B. The lysis binding solution 1 may contain or not contain the first magnetic beads, and the lysis binding solution B may contain or not contain the second magnetic beads.

[0168] In some embodiments, the volume ratios of the test sample, proteinase K, lysis binding solution A, first magnetic beads, lysis binding solution B, washing solution A, washing solution B and eluent are: 1, 0.08-0.12, 0.2-0.6, 0.8-1.2, 0.4-0.8, 1.2-1.8, 1.4-2, 0.08-0.2, and the lysis binding solution B contains second magnetic beads.

[0169] In some specific embodiments, the volume ratios of the test sample, proteinase K, lysis binding solution A, first magnetic beads, lysis binding solution B, washing solution A, washing solution B and eluent are: 1, 0.1, 0.4, 1, 0.6, 1.4, 1.6, 0.1-0.14, and the lysis binding solution B contains second magnetic beads.

[0170] In certain embodiments, the lysis condition is shaking for 4 to 8 minutes; or 5 to 7 minutes.

[0171] In some embodiments, the first incubation time is 2 to 5 minutes; or 2 to 4 minutes.

[0172] In certain embodiments, the second incubation time is 5 to 20 min; it can also be 8 to 15 min, or it can be 10 to 13 min.

[0173] The present invention first uses proteinase K and cleavage binding solution A to cleave the sample to expose the nucleic acid. At the same time, in the liquid environment constructed by the cleavage binding solution A, large-fragment nucleic acid is more easily adsorbed onto the first magnetic bead than small-fragment nucleic acid. Then, the magnetic bar module of the extractor is used to discard the first magnetic bead adsorbing the large-fragment nucleic acid together with the magnetic bar cover to achieve the purpose of removing the large-fragment nucleic acid. After that, a new magnetic bar cover is used to add the cleavage binding solution B to the sample. The use of the cleavage binding solution B can promote the binding of the small-fragment free DNA with the second magnetic bead. Then, the magnetic bar module of the instrument is used in combination to complete the purification and elution process of the free DNA.

[0174] In the present invention, proteinase K, SDS and guanidine salts (guanidine isothiocyanate and guanidine sulfate, etc.) can digest the protein components in the sample, fully expose the free DNA, and also inhibit the activity of nucleases to protect the nucleic acid from being destroyed; isopropanol and anhydrous ethanol can promote the adsorption process of nucleic acid and magnetic beads, and also have the effect of inhibiting the activity of nucleases and protecting nucleic acids; trishydroxymethylaminomethane can maintain the pH value of the reaction solution stable to avoid the influence of the extraction effect due to the fluctuation of pH value; cinnamon oil, lemon essential oil and other ingredients in the lysis and binding solution A can reduce the binding of small fragments of nucleic acid to the first magnetic beads, and at the same time, laurel magnesium sulfate and magnesium chloride can increase the binding of large fragments of nucleic acid to the first magnetic beads; guanidine isothiocyanate, sodium chloride, PEG8000 and acetone in the lysis and binding solution B can promote the recovery of small fragments of nucleic acid. By optimizing the formula and use process of various extraction reagents, the purpose of increasing the distribution ratio of free DNA in nucleic acid separation products and reducing the background contamination of large fragments of nucleic acid and the loss of small fragments of nucleic acid is achieved.

[0175] The unit w / v involved in the present invention refers to the ratio of mass to volume, wherein the unit of mass is g and the unit of volume is mL, expressed in grams per milliliter (g / mL).

[0176] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0177] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0178] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0179] Example 1 A kit and method for extracting free DNA

[0180] 1. A kit for extracting free DNA, the kit comprising:

[0181] Proteinase K: working concentration is 2 mg / mL;

[0182] The first magnetic beads: ferroferric oxide carboxyl magnetic beads with a working concentration of 0.03% (w / w) and a particle size of 1 μm.

[0183] Lysis and binding solution A contains components with the following concentrations: cinnamon oil 20v / v%, SDS 10w / v%, isopropanol 30v / v%, lauryl magnesium sulfate 5w / v%, and magnesium chloride 20w / v%.

[0184] The lysis and binding solution B comprises the following components: guanidine thiocyanate 30w / v%, TritonX-100 1v / v%, tris(hydroxymethyl)aminomethane 1w / w%, sodium chloride 15w / w%, PEG8000 20w / v%, acetone 30v / v%. The lysis and binding solution B also comprises second magnetic beads: the second magnetic beads are ferroferric oxide amino magnetic beads, with a working concentration of 0.6% (w / w) and a particle size of 100nm.

[0185] The washing solution A contained the following components in the following concentrations: 3 M guanidine hydrochloride, 5 mM tris(hydroxymethyl)aminomethane, 2 v / v% NP40, and 50 v / v% anhydrous ethanol.

[0186] The washing solution B contains the following components at the following concentrations: 5 mM Tris, 10 w / v % potassium chloride, 70 v / v % anhydrous ethanol.

[0187] Eluent: 10 mM Tris(OH)2.

[0188] During the extraction process, the amount of lysis binding solution A and lysis binding solution B can be increased or decreased according to the sample volume, and the amount of reagents and samples can be adaptively adjusted according to the volume ratio based on the volume of the deep well plate.

[0189] 2. A method for extracting free DNA

[0190] Taking the extraction of free DNA from 1 mL of plasma using a deep-well plate with a volume of 2.2 mL / well and the kit in Part 1 as an example, the specific steps include:

[0191] (1) Kit preparation

[0192] The test kit can be prepared in a pre-packed state, with each reagent filled into a deep-well plate or reagent strip in advance, sealed and stored at room temperature, and the sealing film can be removed before use. The preparation of the test kit can be completed in advance by the production line.

[0193] Specifically, taking the application scenario of a 12*8 deep-well plate (volume of 2.2 mL / well) with a 96-throughput nucleic acid extractor (Mole 96M) as an example, the first magnetic beads are dispensed into each well of the first deep-well plate at 500 μL / well, the lysis binding solution A is dispensed into each well of the second deep-well plate at 200 μL / well, the washing solution A is dispensed into each well of the third deep-well plate at 700 μL / well, the washing solution B is dispensed into each well of the fourth deep-well plate at 800 μL / well, and the eluent is dispensed into each well of the fifth deep-well plate at 50 μL-70 μL / well.

[0194] (2) Sample processing

[0195] 1) Open the second deep-well plate and add 50 μL of proteinase K (20 mg / mL) and 500 μL of sample to each well. Each well in the same plate corresponds to one sample.

[0196] 2) Open the first magnetic bead and magnetic rod cover, and place each reagent on the corresponding plate position of the nucleic acid extraction instrument according to Table 1.

[0197] Table 1 Reagent placement

[0198] Instrument board position Reagents 1 First magnetic beads and magnetic rods set 2 First plate lysis binding buffer A with added samples

[0199] 3) Set the program on the nucleic acid extraction instrument according to Table 2 or open the program 1 saved on the instrument and start the program.

[0200] Table 2 Program 1

[0201] step Plate number volume Step Name Oscillation time Magnetic attraction time 1 1 / Load / / 2 2 700μL Lysis 5min / 3 1 500μL Magnetic / 15s 4 2 700μL Incubation 2min 20s 5 1 / Unload / /

[0202] 4) After the first procedure is completed, remove all reagents and discard the first magnetic beads and magnetic rod cover in plate position 1.

[0203] 5) Invert the Lysis Binding Solution B several times to mix the magnetic beads thoroughly, and then add 300 μL of Lysis Binding Solution B to each well in the Lysis Binding Solution A plate.

[0204] 6) Open the washing solution A, washing solution B and elution solution, take another magnetic rod cover, and place each reagent on the corresponding plate position of the extractor according to Table 3. If necessary, the washing times of washing solution A and washing solution B can be increased, and the extraction procedure in the next step can be modified accordingly.

[0205] Table 3 Reagent placement

[0206] Instrument board position Deep well plate 1 The second deep well plate 2 The third deep well plate 3 The fourth deep well plate 4 The fifth deep well plate

[0207] 7) Set up the program according to Table 4 or open the program 2 saved in the instrument, and then start the program.

[0208] Table 4 Program 2

[0209] step Plate number volume Step Name Oscillation time Magnetic attraction time 1 1 / Load / / 2 1 900μL Incubation 10min 15s 3 2 700μL washing 30s / 4 3 800μL washing 60s 15s 5 4 50μL Elution 4min 15s 6 2 / Unload

[0210] 8) After the program is completed, transfer the nucleic acid in the eluate plate to a clean nuclease-free centrifuge tube for later use.

[0211] Example 2 Optimization of modification types and combination schemes of the first magnetic beads and the second magnetic beads

[0212] Except for the first magnetic bead and the second magnetic bead, the other reagent components are consistent with Example 1. The specific grouping is shown in Table 5, where the magnetic beads are all ferroferric oxide magnetic beads. The homemade short-fragment nucleic acid and long-fragment nucleic acid are mixed together as a sample simulating contamination by genomic nucleic acid, wherein the short-fragment nucleic acid 1 is a nucleic acid fragment of about 166 bp prepared by PCR method using Staphylococcus aureus as a template, and the short-fragment nucleic acid 2 is a nucleic acid fragment of about 80 bp prepared by PCR method using Escherichia coli as a template, so as to simulate free DNA fragments; the long-fragment nucleic acid is a complete human genomic DNA. Except that the incubation time between the first magnetic bead and the sample was 10 minutes, the rest of the samples were processed according to the operation flow of implementation 1, and then the extracted products were amplified using primer probes for Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and human β-actin gene, and the concentrations of short-fragment nucleic acids and long-fragment nucleic acids in the extracted products were measured by their respective Ct value results (a difference of 2 times the concentration corresponds to a difference of 1 Ct value, a difference of 2 times the concentration corresponds to a difference of n Ct values, and the lower the concentration, the greater the Ct value). The results are as follows;

[0213] Table 5

[0214]

[0215]

[0216] The results show that the modification type and combination of the magnetic beads affect the removal effect of large nucleic acid fragments and the recovery effect of small nucleic acid fragments. In order to achieve the purpose of recovering small nucleic acid fragments and removing large nucleic acid fragments, the preferred combination A4 is that the first magnetic beads are carboxylated magnetic beads and the second magnetic beads are amino magnetic beads.

[0217] Example 3 Optimization of the amount of the first magnetic beads (carboxylated magnetic beads) used and the incubation time with the sample

[0218] The preparation method and detection method of the simulated sample in Example 2 were used, the remaining reagent components and the sample processing flow were kept consistent with Example 1, and the amount of the first magnetic beads used and the incubation time with the sample were adjusted to increase the recovery rate of small nucleic acid fragments and remove large nucleic acid fragments; specific grouping and results are shown in the table below.

[0219] Table 6

[0220]

[0221] The results show that the dosage of the first magnetic beads and the incubation time of large-fragment nucleic acids have a significant impact on the results. The dosage of the first magnetic beads is preferably 0.03w / w% to 0.06w / w%, and the incubation time with the sample is 2 to 5 minutes for the best effect.

[0222] Example 4 Optimization of the formula of lysis and binding solution B

[0223] The remaining reagent components were kept consistent with those in Example 1, and the formula of the lysis and binding solution B was adjusted to obtain combinations C1-C6; the specific formula compositions are shown in Table 7.

[0224] Table 7

[0225] combination Combination C1 Combination C2 Combination C3 Combination C4 Combination C5 Combination C6 Sodium chloride (w / w%) 10 10 5 10 Magnesium chloride (w / w%) 15 Potassium chloride (w / w%) 15 Aminated magnetic beads (w / w%) 0.6 0.6 0.6 0.6 0.6 0.6 Guanidine thiocyanate (w / v%) 30 30 30 30 30 30 TritonX-100(v / v%) 1 1 1 1 1 1 Tris(hydroxymethyl)aminomethane(w / w%) 1 1 1 1 1 1 PEG8000 (w / v%) 20 20 20 20 20 Isopropyl alcohol (v / v%) 30 Acetone (v / v%) 30 30 30 30 30

[0226] Using the preparation method, detection method and sample processing flow of the simulated sample in Example 2, the following results were obtained:

[0227] Table 8

[0228] combination S. aureus E. coli β-actin Combination C1 24.67 25.02 32.72 Combination C2 22.32 23.49 32.12 Combination C3 23.16 24.15 32.10 Combination C4 25.45 26.48 31.43 Combination C5 24.83 25.76 32.64 Combination C6 25.74 26.86 32.09

[0229] Result description:

[0230] Comparing the results of combinations C1, C2, and C6, it can be seen that the amount of inorganic salt used has an impact on the recovery rate of small nucleic acid fragments, and sodium chloride is the best choice;

[0231] Comparing the results of combinations C2 and C5, it can be seen that the use of PEG8000 facilitates the recovery of small nucleic acid fragments;

[0232] Comparing the combinations C2 and C3, it can be seen that the use of acetone is more conducive to the recovery of small nucleic acid fragments than isopropanol.

[0233] Example 5 Optimization of the formula of lysis and binding solution A

[0234] Using the preparation method and detection method of the simulated sample in Example 2, keeping the other reagent components and sample processing procedures consistent with Example 1, the formula of the lysis and binding solution A was adjusted to obtain formula combinations D1-D11, and the specific formulas are shown in the following table:

[0235] Table 9

[0236]

[0237] The preparation method and detection method of the simulated sample in Example 2 were used, and the remaining reagent components and the reagent use process were kept consistent with Example 1. The results are shown in the following table:

[0238] Table 10

[0239]

[0240]

[0241] Result description:

[0242] Comparing the combinations D1-D3, it can be seen that the use of 10%-30% cinnamon oil has the best effect. Insufficient use of cinnamon oil leads to a decrease in the adsorption efficiency of the first magnetic beads for large nucleic acid fragments, and excessive use of cinnamon oil leads to the adsorption of small nucleic acid fragments by the first magnetic beads.

[0243] Comparison of the results of combinations D1, D4, D5, D6 and D7 showed that different hydrophobic agents had an effect on the adsorption specificity of large fragments of nucleic acids, among which mineral oil and cinnamon oil had the best effects;

[0244] By comparing the effects of combinations D1, D8, D9, D10 and D11, it can be seen that the amount of isopropanol used has an impact on the differential binding of nucleic acids in the entire system. When the amount used is too low, the first magnetic beads will not adsorb large nucleic acids sufficiently. When the amount used is too high, the first magnetic beads will adsorb small nucleic acids.

[0245] Example 6 Testing the Effect of SDS, Sodium Lauroyl Sarcosinate, Magnesium Laureate Sulfate and Dithiothreitol in Lysis Binding Solution A on the Extraction of Free DNA

[0246] The remaining reagent components and the reagent use process are kept consistent with Example 1, and the surfactant and denaturant in the lysis and binding solution A are adjusted to obtain combinations E1-E8, as shown in the following table:

[0247] Table 11

[0248]

[0249] Free DNA was extracted from plasma samples (sample A and sample B). The human β-actin gene was detected using the qPCR method, and its Ct value was used to reflect the content of the template in the extracted product. The double-stranded DNA content in the extracted product was determined using a microfluorimeter, and the detection results of capillary electrophoresis were used to reflect the fragment distribution of the extracted product. The results are shown in the following table and Figure 1-2 As shown:

[0250] Table 12

[0251]

[0252] By comparing the results of combinations E1, E4 and E5, it can be seen that sodium dodecyl sulfate (SDS) plays a major role in promoting the full release of nucleic acids; by comparing the results of E1-E3, it can be seen that sodium dodecyl sulfate (SDS) can be replaced by sodium lauroyl sarcosinate; by comparing the results of combinations E1 and E5-E8, it can be seen that the combined use of magnesium laurel sulfate and cinnamon oil can effectively balance the contradiction between the insufficient adsorption of large fragments by the first magnetic beads and the excessive adsorption of small fragments of nucleic acids, so as to achieve the purpose of removing large fragments of nucleic acid contamination while ensuring the extraction rate of free DNA.

[0253] Test Example 1

[0254] Plasma samples 1-8 were extracted according to the method in Example 1 and the commercial reagent Tiangen magnetic bead method large volume free nucleic acid extraction kit (DP710), and the obtained extraction products were respectively detected by qPCR method for human β-actin gene, and its Ct value was used to reflect the content of the template in the extraction product, and the double-stranded DNA content in the extraction product was determined by a microfluorimeter. The detection results of capillary electrophoresis were used to reflect the fragment distribution of the extraction product. The results are shown in the following table and Figure 3-4 :

[0255] Table 13

[0256]

[0257]

[0258] The Ct values ​​and concentration test results of the 8 samples were summarized, and the ΔCt and concentration ratio were calculated for the same sample. ΔCt = Ct value obtained by the method in Example 1 - Ct value obtained by the Tiangen reagent method, and concentration ratio = concentration obtained by the method in Example 1 / concentration obtained by the Tiangen reagent method.

[0259] The results showed that the method of Example 1 had a Ct value that was 0.2-1.56 ahead of that of the commercial reagent, with an average advance of 0.94, and the obtained concentration was 1.75-2.86 times that of the commercial reagent, with an average of 2.28 times.

[0260] By comparing the results of capillary electrophoresis, it can be seen that compared with commercial reagents, the method of Example 1 can reduce the possibility of large-fragment nucleic acid contamination (such as samples 2 and 3); for low-concentration samples, the method of Example 1 has a better extraction effect and can detect a more obvious main peak (such as samples 7 and 8).

[0261] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A kit, characterized in that: The kit comprises a lysis and binding solution A, wherein the lysis and binding solution A comprises: a hydrophobic agent, a first surfactant, isopropanol, lauryl magnesium sulfate and magnesium chloride.

2. The kit according to claim 1, characterized in that The concentration of the hydrophobic agent in the lysis and binding solution A is 10 to 30 v / v%; and / or, The concentration of the first surfactant in the lysis and binding solution A is 5-10 w / v%; and / or, The concentration of isopropanol in the lysis and binding solution A is 20-30 v / v%; and / or, The concentration of lauryl magnesium sulfate in the lysis and binding solution A is 1-5 w / v%; and / or, The concentration of magnesium chloride in the lysis and binding solution A is 2-20 w / v%; The first surfactant is an anionic surfactant; and / or, The hydrophobic agent in the lysis and binding solution A includes one or more of cinnamon oil and mineral oil; Preferably, the anionic surfactant includes one or more of sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium alkylbenzene sulfonate, and sodium dodecyl sulfonate.

3. The kit according to claim 1, characterized in that The kit also includes a lysis and binding solution B, which includes guanidine salt, a second surfactant, tris(hydroxymethyl)aminomethane, sodium chloride, polyethylene glycol and acetone.

4. The kit according to claim 3, characterized in that The concentration of guanidine salt in the lysis and binding solution B is 20-30 w / v%; and / or, The concentration of the second surfactant in the lysis and binding solution B is 0.1-2 v / v%; and / or, The concentration of tris(hydroxymethyl)aminomethane in the lysis and binding solution B is 0.05-3w / w%; and / or, The concentration of sodium chloride in the lysis and binding solution B is 10-20 w / w%; and / or, The concentration of polyethylene glycol in the lysis and binding solution B is 15-30 w / v%; and / or, The concentration of acetone in the lysis and binding solution B is 20-40 v / v%.

5. The kit according to claim 4, characterized in that The guanidine salt in the lysis and binding solution B includes one or both of guanidine isothiocyanate and guanidine hydrochloride; and / or, The second surfactant in the lysis and binding solution B is a non-ionic surfactant; Preferably, the nonionic surfactant includes one or more of Triton, polysorbate, and ethylphenyl polyethylene glycol.

6. The kit according to claim 1, characterized in that The kit further comprises first magnetic beads and / or second magnetic beads; Preferably, the first magnetic beads are carboxylated magnetic beads; Preferably, the modification mode of the second magnetic beads is different from that of the first magnetic beads; Preferably, the second magnetic beads are amino magnetic beads; Preferably, the working concentration of the first magnetic beads is 0.03-0.06 w / w%; Preferably, the working concentration of the second magnetic beads is 0.5-0.8 w / w%.

7. The kit according to any one of claims 1 to 6, characterized in that The kit further comprises one or more of proteinase K, washing solution A, washing solution B, and eluent; Preferably, the kit further comprises one or more of the following features: 1) The washing solution A contains guanidine salt, tris(hydroxymethylaminomethane), a third surfactant and alcohols; 2) the washing liquid B contains tris(hydroxymethyl)aminomethane, potassium chloride and alcohol; 3) The eluent contains tris(hydroxymethyl)aminomethane.

8. A product for extracting free DNA, comprising the kit according to any one of claims 1 to 7; Preferably, the product further comprises a nucleic acid extractor; Preferably, the nucleic acid extractor is a fully automatic nucleic acid extractor or a manual nucleic acid extraction device; Preferably, the product also includes other tools that can provide an external magnetic field.

9. Use of the kit according to any one of claims 1 to 7 or the product according to claim 8 in extracting free DNA from a sample; Preferably, the sample is selected from: one or more of whole blood, serum, plasma, saliva, urine, pleural effusion, ascites, sputum, tissue fluid, and follicular fluid samples.

10. A method for extracting free DNA, comprising the step of extracting free DNA using the kit according to any one of claims 1 to 7 or the product according to claim 8; Preferably, the method comprises the following steps: S1: Mix proteinase K, cleavage binding solution A and the sample to be tested, lyse, add the first magnetic beads for the first incubation, magnetic adsorption, and then discard the first magnetic beads to obtain a mixed solution 1; S2: adding lysis binding solution B and / or the second magnetic beads to the mixed solution 1, performing a second incubation and magnetic adsorption, and then washing with washing solution A and washing solution B, and eluting with an elution solution to obtain a free DNA extract; Preferably, the first incubation time is 2 to 5 minutes; Preferably, the second incubation time is 5 to 20 minutes.

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