A kit and method for extracting cell-free DNA

Abstract

This invention relates to the field of cell-free DNA extraction technology, and provides a kit and method for extracting cell-free DNA. The kit includes a lysis binding buffer A, a first magnetic bead, a lysis binding buffer B, a washing buffer A, a washing buffer B, and an elution buffer. The lysis binding buffer B includes a second magnetic bead. In the environment of the lysis binding buffer A, the first magnetic bead tends to adsorb large nucleic acid fragments, while in the lysis binding buffer B, the second magnetic bead fully adsorbs small nucleic acid fragments. Combined with the use of a magnetic rod module of the instrument or other forms of external magnetic field, the purification and elution process of cell-free DNA is completed. This achieves the goal of increasing the distribution ratio of cell-free DNA in the nucleic acid separation product, reducing background contamination from large nucleic acid fragments, and minimizing the loss of small nucleic acid fragments. It can separate cell-free DNA that meets the requirements of downstream applications under different sample quality conditions.

Description

Technical Field

[0001] This invention relates to the field of cell-free DNA extraction technology, and in particular to a kit and method for extracting cell-free DNA. Background Technology

[0002] Cell-free DNA (cfDNA) is a type of DNA that exists outside of cells in blood or other bodily fluids (urine, tissue fluid, saliva, follicular fluid, etc.). Since Mandel and Metais first reported the existence of cell-free DNA in human plasma in 1948, it has played an increasingly important role as an ideal biomarker in the fields of genetic and disease diagnosis and screening. It is widely used in scenarios such as tumor screening and diagnosis, prenatal screening, cardiovascular disease detection, transplant medicine monitoring, autoimmune disease detection, and infection detection, providing new methods for the diagnosis, decision support, and prognosis of clinically related diseases.

[0003] Cell-free DNA is characterized by small fragments and low concentrations. Sufficient yield and low background nucleic acid contamination are key to ensuring accurate and reliable cell-free DNA detection results. According to current research progress, different release pathways of cell-free DNA may be the reason for the varying fragment lengths, but overall, cell-free DNA is mainly distributed at 166 bp.

[0004] Currently, commercially available cell-free DNA extraction reagents primarily employ two methodologies: silica-based membrane centrifugation column extraction and magnetic bead adsorption. Silica-based membrane centrifugation column extraction yields high-purity nucleic acid, but requires multiple uses of high-speed centrifugation equipment, making high-throughput and fully automated nucleic acid extraction difficult. The repeated tube transfers also introduce the risk of contamination. Magnetic bead adsorption yields lower than centrifugation column extraction, but its magnetic separation-based process can be perfectly implemented on automated equipment, improving experimental efficiency, reducing human error, ensuring result consistency, and saving costs in terms of manpower, equipment, and consumables. However, regardless of the method used to prepare nucleic acid extraction reagents, the problems of low cell-free DNA yield, large-fragment nucleic acid background contamination, and small-fragment nucleic acid loss cannot be solved simply by choosing a methodology.

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

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a kit and method for increasing the distribution ratio of free DNA in nucleic acid isolation products, thereby solving the problems in the prior art.

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

[0008] One of the objectives of this invention is to protect a kit comprising a lysis binding solution A, wherein the lysis binding solution A comprises a hydrophobic agent, a first surfactant, isopropanol, magnesium lauryl sulfate, and magnesium chloride.

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

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

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

[0012] In some embodiments, the concentration of the first surfactant in the lysis binding solution A is 5–10 w / v.

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

[0014] In some 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 some embodiments, the concentration of isopropanol in the lysis binding solution A is 20–30 v / v.

[0017] In some embodiments, the concentration of magnesium lauryl sulfate in the lysis binder A is 1–5 w / v.

[0018] In some embodiments, the concentration of magnesium chloride in the pyrolysis binding solution A is 2–20 w / v.

[0019] In some embodiments, the cleavage binding solution A comprises 10-30 v / v of hydrophobic agent, 5-10 w / v of 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 some embodiments, the kit further includes a lysis binding solution B, which comprises a guanidine salt, a second surfactant, tris(hydroxymethyl)aminomethane, sodium chloride, polyethylene glycol (PEG), and acetone.

[0021] In some embodiments, the concentration of guanidine salt in the lysis binding solution B is 20–30 w / v.

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

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

[0024] In some embodiments, the concentration of the second surfactant in the lysis binding solution B is 0.1–2 v / v.

[0025] In some embodiments, the second surfactant in the lysis binding solution B is a nonionic surfactant.

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

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

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

[0029] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the lysis binder B is 0.05–3 w / w.

[0030] In some embodiments, the concentration of sodium chloride in the lysis binder B is 10–20 w / w.

[0031] In some embodiments, the concentration of polyethylene glycol in the lysis binder B is 15–30 w / v.

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

[0033] In some embodiments, the concentration of acetone in the lysis binding solution B is 20–40 v / v.

[0034] In some embodiments, the kit includes a first magnetic bead and / or a second magnetic bead.

[0035] In some embodiments, the first magnetic bead is a carboxylated magnetic bead.

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

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

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

[0039] In some implementations, the second magnetic bead is modified differently from the first magnetic bead.

[0040] In some specific embodiments, the second magnetic bead is an aminated magnetic bead.

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

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

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

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

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

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

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

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

[0049] In some embodiments, the concentration of the third surfactant in the washing liquid A is 1–5 v / v%.

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

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

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

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

[0054] In some embodiments, the concentration of alcohols in the washing solution A is 40–60 v / v.

[0055] In some embodiments, the alcohols in the washing solution A include ethanol.

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

[0057] In some embodiments, the washing liquid B comprises tris(hydroxymethyl)aminomethane, potassium chloride, and alcohols.

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

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

[0060] In some embodiments, the concentration of alcohols in the washing solution B is 70–80 v / v.

[0061] In some embodiments, the alcohols in the washing solution B include ethanol.

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

[0063] In some embodiments, the eluent comprises tris(hydroxymethyl)aminomethane.

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

[0065] The second objective of this invention is to protect the use of the kit described above in extracting cell-free DNA from samples or in preparing products containing cell-free DNA extracted from samples.

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

[0067] In some implementations, the product is a reagent, a kit, or a system.

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

[0069] A third objective of this invention is to protect a product comprising: a reagent kit as described above.

[0070] In some embodiments, the product also includes a nucleic acid extractor.

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

[0072] In some embodiments, the product also includes other tools capable of providing an external magnetic field.

[0073] The fourth objective of this invention is to protect a method for extracting cell-free DNA, the method comprising the step of extracting cell-free DNA using the product described above;

[0074] The method described is not for disease diagnosis or treatment purposes.

[0075] In some embodiments, the method includes the following steps:

[0076] S1: Mix proteinase K, lysis binding buffer A and the sample to be tested, lyse, add the first magnetic bead for the first incubation and magnetic adsorption, then discard the first magnetic bead to obtain mixture 1;

[0077] S2: Add lysis binding solution B and / or the second magnetic beads to the mixture 1, perform a second incubation and magnetic adsorption, wash with washing solution A and washing solution B, and then elute with elution solution to obtain free DNA extract.

[0078] In some embodiments, the volume ratios of the test sample, proteinase K, lysis binding buffer A, first magnetic bead, lysis binding buffer B, washing buffer A, washing buffer B, and elution buffer 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, and 0.08–0.2.

[0079] In some embodiments, the pyrolysis is performed by shaking for 4 to 8 minutes.

[0080] In some embodiments, the first incubation period is 2 to 5 minutes.

[0081] In some embodiments, the second incubation period is 5 to 20 minutes.

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

[0083] To separate cell-free DNA that meets the requirements of downstream applications under different sample quality conditions, this invention studies the formulation and usage process of cell-free DNA extraction reagents, resulting in a kit comprising proteinase K, first magnetic beads, second magnetic beads, lysis binding buffer A, lysis binding buffer B, washing buffer A, washing buffer B, and elution buffer. The first magnetic beads in lysis binding buffer A tend to adsorb large nucleic acid fragments, while the second magnetic beads in lysis binding buffer B fully adsorb small nucleic acid fragments. Combined with the use of the instrument's magnetic rod module or other forms of external magnetic field, the purification and elution process of cell-free DNA is completed. This achieves the goal of increasing the distribution ratio of cell-free DNA in the nucleic acid separation product, reducing background contamination from large nucleic acid fragments, and minimizing the loss of small nucleic acid fragments. Attached Figure Description

[0084] Figure 1 The results are from capillary electrophoresis of sample A in Example 6.

[0085] Figure 2 The results are from the capillary electrophoresis of sample B in Example 6.

[0086] Figure 3 The results of capillary electrophoresis of samples 1-8 were detected using the kit and method of Example 1 in Test Example 1.

[0087] Figure 4 The results of capillary electrophoresis of samples 1-8 were obtained using the Tiangen magnetic bead extraction kit in Test Example 1. Detailed Implementation

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

[0089] In some embodiments, the concentration of the hydrophobic agent in the cleavage binding solution A is 10–30 v / v%; it can also be 10–20 v / v%, 20–30 v / v%, 15–25 v / v%, or 20 v / v%.

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

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

[0092] This invention investigated the effects of reagents such as cinnamon oil, liquid paraffin, mineral oil, lemon essential oil, and isopropanol, as well as their dosage, on the adsorption of nucleic acid fragments of different sizes onto magnetic beads. It was found that the adsorption effect was best when the dosage of cinnamon oil was 10–30 v / v%.

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

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

[0095] In some 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, or it may be sodium lauroyl sarcosinate.

[0097] In some embodiments, the concentration of magnesium lauryl sulfate in the lysis binder 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% or 5 w / v%.

[0098] Thorough lysis of the sample to release nucleic acids is essential for obtaining sufficient cell-free DNA. This invention investigated the effects of sodium dodecyl sulfate (SDS), sodium lauroyl sarcosinate, magnesium lauryl sulfate, and dithiothreitol on the yield of cell-free DNA. Preferably, using 5–10 w / v% SDS and 1–5 w / v% magnesium lauryl sulfate in lysis binding buffer A yields the optimal amount of cell-free DNA.

[0099] In some embodiments, the concentration of isopropanol in the lysis 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% or 30 v / v.

[0100] In some embodiments, the concentration of magnesium chloride in the pyrolysis 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 some embodiments, the cleavage binding solution A comprises 10-30 v / v% hydrophobic agent, 5-10 w / v% first surfactant, 20-30 v / v% isopropanol, 1-5 w / v% magnesium lauryl sulfate, and 2-20 w / v magnesium chloride.

[0102] In some specific embodiments, the cleavage binding solution A comprises 20 v / v% hydrophobic agent, 10 w / v% first surfactant, 30 v / v% isopropanol, 5 w / v% magnesium lauryl sulfate, and 20 w / v magnesium chloride.

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

[0104] In some embodiments, the concentration of guanidine salt in the lysis 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 some embodiments, the guanidine salt includes at least one of guanidine isothiocyanate and guanidine hydrochloride.

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

[0107] In some embodiments, the concentration of the second surfactant in the lysis 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% or 1 v / v.

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

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

[0110] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the cleavage binder B is 0.05–3 w / w%; it can also be 0.1–1 w / w%, 1–3 w / w%, 0.5–2 w / w%, or 1 w / w% or 2 w / w%.

[0111] In some embodiments, the concentration of sodium chloride in the lysis binding solution B is 10–20 w / w%; it can also be 10–15 w / w%, 15–20 w / w%, 13–18 w / w%, or 10 w / w%, 15 w / w%, or 20 w / w.

[0112] In some embodiments, the concentration of polyethylene glycol in the pyrolysis binder 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 some embodiments, the polyethylene glycol (PEG) may be PEG4000, PEG6000, PEG8000, etc.

[0114] In some embodiments, the concentration of acetone in the lysis 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% or 30 v / v.

[0115] In some embodiments, the kit further includes a first magnetic bead and / or a second magnetic bead; wherein the first magnetic bead tends to adsorb large nucleic acid fragments, and the second magnetic bead is used to adsorb small free DNA fragments.

[0116] In some embodiments, the first magnetic bead is a carboxylated magnetic bead.

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

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

[0119] In some implementations, the second magnetic bead is modified differently from the first magnetic bead.

[0120] In some embodiments, the second magnetic bead is an aminated magnetic bead.

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

[0122] In some embodiments, the second magnetic bead is an aminated magnetic bead.

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

[0124] It should be noted that in this invention, the lysis binding solution (such as lysis binding solution A and lysis binding solution B) can be mixed with the sample to be extracted before adding magnetic beads (such as first magnetic beads and second magnetic beads) for further mixing; alternatively, the lysis binding solution and magnetic beads can be mixed before adding the sample to be extracted for mixing and reaction; or the lysis binding solution, magnetic beads, and sample to be extracted can be mixed simultaneously. This invention includes two magnetic bead adsorption processes, wherein lysis binding solution A and the first magnetic bead are used together to adsorb large nucleic acid fragments, and lysis binding solution B and the second magnetic bead are used together to adsorb small nucleic acid fragments.

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

[0126] In some preferred embodiments, the lysis binding solution B further includes a second magnetic bead with a working concentration of 0.5 to 0.8 w / w%.

[0127] In some specific embodiments, the cleavage binding solution B comprises: guanidine isothiocyanate 30 w / v%, Triton X-100 1 w / v%, tris(hydroxymethyl)aminomethane 1 w / v%, sodium chloride 15 w / v%, PEG 20 w / v%, and acetone 30 w / v.

[0128] In some preferred embodiments, the lysis binding solution B further includes 0.6 w / w% of a second magnetic bead.

[0129] To address the need for the first magnetic bead used in lysis binding solution A to adsorb large nucleic acid fragments, and the magnetic beads used in lysis binding solution B to fully adsorb small nucleic acid fragments, this invention investigated the effects of the type and amount of magnetic bead surface modification, the incubation time, and the formulation of lysis binding solution A on the adsorption effect of nucleic acid fragments of different sizes. For example, when the amount of the first magnetic bead is 0.03 w / w% to 0.06 w / w, and the surface is modified with carboxyl groups, the incubation time between the first magnetic bead and the sample in lysis binding solution A is 2 to 5 min; when the amount of the second magnetic bead in lysis binding solution B is 0.5 w / w% to 0.8 w / w, and the surface is modified with amino groups, the incubation time with the sample is 5 to 20 min; within this range, it is most favorable for obtaining small nucleic acid fragments.

[0130] This invention also investigated the effects of the types and amounts of inorganic salts used during the extraction process, organic compounds such as alcohols and ketones, and substances such as PEG on the recovery of small nucleic acid fragments. The results showed that the optimal formulation of the lysis binding buffer B was 10-20 w / w of sodium chloride, 15-30 w / v of PEG8000, and 20-40 v / v of acetone; within this range, the recovery of small nucleic acid fragments was the best.

[0131] Using acetone and isopropanol in the lysis binding buffer can protect nucleic acid fragments from nuclease shearing during extraction and also promote the adsorption of nucleic acids to magnetic beads.

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

[0133] In some 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 some embodiments, the washing liquid A includes guanidine salt, tris(hydroxymethyl)aminomethane, a third surfactant, and alcohols, wherein the function of washing liquid A is to remove impurities such as proteins, lipids, acetone, and isopropanol.

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

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

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

[0138] In some embodiments, the concentration of tris(hydroxymethyl)aminomethane in the washing liquid 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 some embodiments, the concentration of the third surfactant in the washing liquid A is 1–5 v / v%; it can also be 2–4 v / v%, or 2 v / v% or 3 v / v%.

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

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

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

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

[0144] In some specific embodiments, the alcohols in the washing solution A include ethanol.

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

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

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

[0148] In some 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% or 10 w / v%.

[0149] In some embodiments, the concentration of alcohol in the washing solution B is 70-80 v / v%; it can also be 70-75 v / v%, 75-80 v / v%, or 70 v / v% or 75 v / v%.

[0150] In some embodiments, the alcohols in the washing solution B include ethanol.

[0151] Using anhydrous ethanol in the washing solution protects nucleic acid fragments from nuclease cleavage during extraction and also promotes the adsorption of nucleic acids onto magnetic beads. Washing solution A has a higher concentration of guanidine hydrochloride, which also prevents desorption of nucleic acids from magnetic beads during washing; therefore, the ethanol concentration in washing solution A is lower than that in washing solution B.

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

[0153] In some 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 some embodiments, the concentration of the tris(hydroxymethyl)aminomethane is 5 mM to 50 mM, or it can be 10 mM to 40 mM, or it can be 20 mM to 30 mM, or it can be 10 mM.

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

[0156] The second objective of this invention is to protect the use of the kit described above in extracting cell-free DNA from samples or in preparing products containing cell-free DNA extracted from samples.

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

[0158] In some implementations, the product is a reagent, a kit, or a system.

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

[0160] A third objective of this invention is to protect a product for extracting cell-free DNA, the product comprising: a kit as described above.

[0161] In some embodiments, the product also includes a nucleic acid extractor.

[0162] In some specific embodiments, the nucleic acid extractor is a fully automated nucleic acid extractor or a manual nucleic acid extractor; for example, it uses the cooperation of an external magnetic field such as a magnetic rod and a magnetic rack to achieve manual extraction of nucleic acids.

[0163] The fourth objective of this invention is to protect a method for extracting cell-free DNA, the method comprising the step of extracting cell-free DNA using the product described above.

[0164] In some embodiments, the method includes the following steps:

[0165] S1: Mix proteinase K, lysis binding buffer A and the sample to be tested, lyse, add the first magnetic bead for the first incubation and magnetic adsorption, then discard the first magnetic bead to obtain mixture 1;

[0166] S2: Add lysis binding solution B and / or the second magnetic beads to the mixture 1, perform a second incubation and magnetic adsorption, wash with washing solution A and washing solution B, and then elute with elution solution to obtain free DNA extract.

[0167] It should be noted that lysis binding solution A is used in conjunction with the first magnetic bead, and lysis binding solution B is used in conjunction with the second magnetic bead. The above operation is not limited to mixing the lysis binding solution (including lysis binding solution A or lysis binding solution B) with the sample to be extracted, and then adding the magnetic beads (including the first magnetic bead or the second magnetic bead) for mixing; it is also possible to mix the lysis binding solution and the magnetic beads first, and then add the sample to be extracted for mixing and reaction; or it is possible to mix the lysis binding solution, the magnetic beads, and the sample to be extracted simultaneously. The first magnetic bead undergoes magnetic adsorption in the environment of lysis binding solution A, and the second magnetic bead undergoes magnetic adsorption in the environment of lysis binding solution B. Lysis binding solution 1 may or may not contain the first magnetic bead, and lysis binding solution B may or may not contain the second magnetic bead.

[0168] In some embodiments, the volume ratios of the test sample, proteinase K, lysis binding buffer A, first magnetic beads, lysis binding buffer B, washing buffer A, washing buffer B, and elution buffer 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, and 0.08–0.2, and the lysis binding buffer B contains second magnetic beads.

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

[0170] In some embodiments, the pyrolysis condition is oscillation for 4 to 8 minutes; it can also be 5 to 7 minutes.

[0171] In some embodiments, the first incubation time is 2 to 5 minutes; it can also be 2 to 4 minutes.

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

[0173] This invention first uses proteinase K and lysis binding solution A to lyse the sample, exposing the nucleic acids. Simultaneously, in the liquid environment constructed by lysis binding solution A, large nucleic acid fragments are more easily adsorbed onto the first magnetic beads than small fragments. Then, using the magnetic rod module of the extractor, the first magnetic beads adsorbing the large nucleic acid fragments are discarded along with the magnetic rod sleeve, thus removing the large nucleic acid fragments. Next, a new magnetic rod sleeve is used, and lysis binding solution B is added to the sample. The use of lysis binding solution B promotes the binding of small, free DNA fragments to the second magnetic beads. Combined with the instrument's magnetic rod module, the purification and elution process of the free DNA is completed.

[0174] In this invention, proteinase K, SDS, and guanidine salts (guanidine isothiocyanate and guanidine sulfate, etc.) digest the protein components in the sample, fully exposing free DNA, while also inhibiting nuclease activity and protecting nucleic acids from damage. Isopropanol and anhydrous ethanol promote the adsorption process of nucleic acids to magnetic beads and also inhibit nuclease activity and protect nucleic acids. Tris(hydroxymethyl)aminomethane maintains the pH of the reaction solution, avoiding the impact of pH fluctuations on the extraction effect. Components such as cinnamon oil and lemon essential oil in lysis binding solution A reduce the binding of small nucleic acid fragments to the first magnetic beads, while magnesium lauryl sulfate and magnesium chloride increase the binding of large nucleic acid fragments to the first magnetic beads. Guanidine isothiocyanate, sodium chloride, PEG8000, and acetone in lysis binding solution B promote the recovery of small nucleic acid fragments. By optimizing the formulation and usage process of various extraction reagents, the distribution ratio of free DNA in the nucleic acid separation product is increased, and the background contamination of large nucleic acid fragments and the loss of small nucleic acid fragments are reduced.

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

[0176] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0177] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0178] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

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

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

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

[0182] First magnetic bead: Carboxylated magnetite beads with a working concentration of 0.03% (w / w) and a particle size of 1 μm.

[0183] The lysis binder A contains the following components at the following concentrations: cinnamon oil 20 v / v, SDS 10 w / v, isopropanol 30 v / v, magnesium lauryl sulfate 5 w / v, and magnesium chloride 20 w / v.

[0184] The lysis binder B contains the following components at the following concentrations: guanidine isothiocyanate 30 w / v, Triton X-100 1 v / v, tris(hydroxymethyl)aminomethane 1 w / v, sodium chloride 15 w / v, PEG8000 20 w / v, and acetone 30 v / v. The lysis binder B also includes a second magnetic bead: the second magnetic bead is an aminated magnetite bead with a working concentration of 0.6% (w / w) and a particle size of 100 nm.

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

[0186] Washing solution B contains the following components at the following concentrations: 5 mM tris(hydroxymethyl)aminomethane, 10 w / v potassium chloride, and 70 v / v anhydrous ethanol.

[0187] Eluent: 10 mM tris(hydroxymethyl)aminomethane.

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

[0189] 2. A method for extracting cell-free DNA

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

[0191] (1) Kit preparation

[0192] The kit can be prepared in a pre-filled state, with each reagent pre-filled into a deep-well plate or reagent strip, sealed, and stored at room temperature. The seal can be removed before use. Kit preparation 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 bead is 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 to 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 sleeve, and place each reagent into the corresponding plate position of the nucleic acid extractor according to Table 1.

[0197] Table 1. Reagent Placement Location

[0198] Instrument plate position reagents 1 First magnetic bead and magnetic rod sleeve 2 First plate of lysis binding buffer A with added good sample

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

[0200] Table 2 Program 1

[0201] step Plate number volume Step Name Fluctuation time Magnetization time 1 1 / Load / / 2 2 700μL Pyrolysis 5min / 3 1 500μL magnetization / 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 bead and magnetic rod sleeve in plate position 1.

[0203] 5) Invert the lysis binding solution B several times to ensure the magnetic beads are thoroughly mixed, and then add 300 μL of lysis binding solution B to each well in the lysis binding solution A plate.

[0204] 6) Open washing solution A, washing solution B, and eluent. Take another magnetic rod sleeve and place each reagent into the corresponding plate position on the extractor according to Table 3. If necessary, the number of washes with 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 plate position Deep hole plate position 1 Second deep hole plate 2 3rd deep hole plate 3 4th deep hole plate 4 5th deep hole plate

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

[0208] Table 4 Program 2

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

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

[0211] Example 2: Optimization of the modification type and combination scheme of the first and second magnetic beads

[0212] Except for the first and second magnetic beads, the remaining reagent components were consistent with those in Example 1, and the specific groupings are shown in Table 5. All magnetic beads were iron oxide (Fe3O4) beads. Self-prepared short and long nucleic acid fragments were mixed together to simulate samples contaminated with genomic nucleic acids. Short nucleic acid fragment 1 was a 166bp fragment prepared by PCR using Staphylococcus aureus as a template, and short nucleic acid fragment 2 was an 80bp fragment prepared by PCR using Escherichia coli as a template, used to simulate cell-free DNA fragments. The long nucleic acid fragment was complete human genomic DNA. Except for the 10-minute incubation time between the first magnetic bead and the sample, the samples were processed according to the procedure in Implementation 1. Then, primers and probes targeting Staphylococcus aureus, Escherichia coli, and human β-actin genes were used to amplify the extracted products. The concentrations of short and long nucleic acid fragments in the extracted products were measured using their respective Ct values ​​(a 2-fold difference in concentration corresponds to one Ct value difference, a 2^n-fold difference in concentration corresponds to n Ct values, and the lower the concentration, the larger the Ct value). The results are shown in the table below.

[0213] Table 5

[0214]

[0215]

[0216] The results show that the modification type and combination of magnetic beads affect the removal efficiency of large nucleic acid fragments and the recovery efficiency of small nucleic acid fragments. In order to achieve the purpose of recovering small nucleic acid fragments and removing large nucleic acid fragments, the combination A4, in which the first magnetic bead is a carboxylated magnetic bead and the second magnetic bead is an aminolated magnetic bead, is preferred.

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

[0218] Using the simulated sample preparation method and detection method in Example 2, while keeping the remaining reagent components and sample processing procedures consistent with Example 1, the amount of the first magnetic bead used and its incubation time with the sample were adjusted to increase the recovery rate of small nucleic acid fragments and remove large nucleic acid fragments; see the table below for specific grouping and results.

[0219] Table 6

[0220]

[0221] The results show that the amount of the first magnetic bead used for large nucleic acid fragments and the incubation time have a significant impact on the results. The amount of the first magnetic bead should be 0.03 w / w% to 0.06 w / w%, and the incubation time with the sample should be 2 to 5 min for the best results.

[0222] Example 4: Optimization of the formulation of lysis binding solution B

[0223] Keeping the remaining reagent components consistent with those in Example 1, the formulation of the lysis binding solution B was adjusted to obtain combinations C1-C6; the specific formulation composition is 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 isothiocyanate (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 Isopropanol (v / v%) 30 Acetone (v / v%) 30 30 30 30 30

[0226] Using the sample preparation method, detection method, and sample processing procedure described 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] Results explanation:

[0230] Comparing the results of combinations C1, C2, and C6, it can be seen that the amount of inorganic salts used affects the recovery rate of small nucleic acid fragments, with sodium chloride being the optimal choice.

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

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

[0233] Example 5: Optimization of the formulation of lysis binding solution A

[0234] Using the sample preparation method and detection method described in Example 2, and keeping the remaining reagent components and sample processing procedures consistent with Example 1, the formulation of lysis binding solution A was adjusted to obtain formulation combinations D1-D11, as shown in the table below:

[0235] Table 9

[0236]

[0237] Using the sample preparation method and detection method described in Example 2, and keeping the usage procedures of the remaining reagent components consistent with those in Example 1, the results are shown in the table below:

[0238] Table 10

[0239]

[0240]

[0241] Results explanation:

[0242] Comparing combinations D1-D3, it can be seen that the optimal effect is achieved with 10%-30% cinnamon oil. Insufficient cinnamon oil reduces the adsorption efficiency of the first magnetic bead for large nucleic acid fragments, while excessive cinnamon oil reduces the adsorption of small nucleic acid fragments by the first magnetic bead.

[0243] Comparing the results of combinations D1, D4, D5, D6 and D7, it can be seen that different hydrophobic agents affect the adsorption specificity of large nucleic acid fragments, with mineral oil and cinnamon oil showing the best effects.

[0244] Comparing the effects of combinations D1, D8, D9, D10, and D11, it can be seen that the amount of isopropanol used affects the differential binding of nucleic acids in the entire system. When the amount used is too low, the first magnetic bead does not adsorb large nucleic acid fragments sufficiently, while when the amount used is too high, the first magnetic bead adsorbs small nucleic acid fragments.

[0245] Example 6 tested the effects of SDS, sodium lauroyl sarcosinate, magnesium lauryl sulfate, and dithiothreitol in lysis binding buffer A on the extraction efficiency of free DNA.

[0246] Keeping the usage procedures of the remaining reagent components and reagents consistent with those in Example 1, the surfactant and denaturant in the lysis binding solution A were adjusted to obtain combinations E1-E8, as shown in the table below:

[0247] Table 11

[0248]

[0249] Cell-free DNA was extracted from plasma samples (Sample A and Sample B). The human β-actin gene was detected using qPCR, with the Ct value reflecting the template content in the extracted product. The double-stranded DNA content in the extracted product was determined using a microfluorometer, and the fragment distribution of the extracted product was reflected by capillary electrophoresis. The results are shown in the table below. Figure 1-2 As shown:

[0250] Table 12

[0251]

[0252] Comparison of the results of combinations E1, E4, and E5 shows that sodium dodecyl sulfate (SDS) played a major role in promoting the full release of nucleic acids. Comparison of the results of E1-E3 shows that sodium dodecyl sulfate (SDS) can be replaced with sodium lauroyl sarcosinate. Comparison of the results of combination E1 with those of E5-E8 shows that the combined use of magnesium lauryl sulfate and cinnamon oil can effectively balance the contradiction between insufficient adsorption of large fragments and excessive adsorption of small nucleic acid fragments by the first magnetic bead, so as to achieve the goal of removing large nucleic acid fragment contamination while ensuring the extraction yield of free DNA.

[0253] Test Example 1

[0254] Plasma samples 1-8 were extracted according to the method described in Example 1 and using the commercially available Tiangen magnetic bead method large-volume free nucleic acid extraction kit (DP710). The extracted products were then analyzed for human β-actin gene expression using qPCR, with the Ct value reflecting the template content. The double-stranded DNA content in the extracted products was determined using a microfluorometer, and the fragment distribution was analyzed using capillary electrophoresis. The results are shown in the table below. Figure 3-4 :

[0255] Table 13

[0256]

[0257]

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

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

[0260] 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 better extraction effect and can detect more obvious main peaks (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 invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A reagent kit, characterized in that, The kit includes: The first magnetic bead, wherein the first magnetic bead is a carboxylated magnetic bead; The pyrolysis binding solution A comprises: cinnamon oil, sodium dodecyl sulfate, isopropanol, magnesium lauryl sulfate, and magnesium chloride; The lysis binding solution B comprises guanidine isothiocyanate, Triton, trimethylolpropane, sodium chloride, polyethylene glycol, acetone, and a second magnetic bead. The second magnetic bead is an aminated magnetic bead.

2. The reagent kit according to claim 1, characterized in that, The concentration of cinnamon oil in the lysis binder A is 10–30 v / v%; and / or, The concentration of sodium dodecyl sulfate in the pyrolysis binding solution A is 5–10 w / v%; and / or, The concentration of isopropanol in the lysis binding solution A is 20–30 v / v%; and / or, The concentration of magnesium lauryl sulfate in the lysis binding solution A is 1–5 w / v%; and / or, The concentration of magnesium chloride in the pyrolysis binding solution A is 2–20 w / v.

3. The reagent kit according to claim 1, characterized in that, The concentration of guanidine isothiocyanate in the lysis binding solution B is 20–30 w / v%; and / or, The concentration of Triton in the lysis binding solution B is 0.1–2 v / v%; and / or, The concentration of tris(hydroxymethyl)aminomethane in the lysis binder B is 0.05–3 w / w%; and / or, The concentration of sodium chloride in the lysis binding solution B is 10–20 w / w%; and / or, The concentration of polyethylene glycol in the pyrolysis binder B is 15–30 w / v%; and / or, The concentration of acetone in the lysis binding solution B is 20–40 v / v.

4. The reagent kit according to claim 1, characterized in that, The working concentration of the first magnetic bead is 0.03–0.06 w / w%; and / or, The working concentration of the second magnetic bead is 0.5–0.8 w / w%.

5. The reagent kit according to any one of claims 1 to 4, characterized in that, The kit also includes one or more of proteinase K, washing solution A, washing solution B, and elution solution.

6. The reagent kit according to claim 5, characterized in that... The kit also includes one or more of the following features: 1) The washing liquid A contains guanidine salt, tris(hydroxymethyl)aminomethane, a third surfactant, and alcohols; 2) The washing solution B contains tris(hydroxymethyl)aminomethane, potassium chloride, and alcohols; 3) The eluent contains tris(hydroxymethyl)aminomethane.

7. A product for extracting cell-free DNA, said product comprising the kit according to any one of claims 1 to 6.

8. The product according to claim 7, characterized in that, The product also includes a nucleic acid extractor; and / or, the nucleic acid extractor is a fully automated nucleic acid extractor or a manual nucleic acid extraction device; and / or The product also includes other tools that provide an external magnetic field.

9. The use of the kit according to any one of claims 1 to 6 or the product according to any one of claims 7 to 8 in extracting cell-free DNA from a sample.

10. The application according to claim 9, characterized in that, The samples are selected from one or more of the following: whole blood, serum, plasma, saliva, urine, pleural and peritoneal fluid, sputum, tissue fluid, and follicular fluid.

11. A method for extracting cell-free DNA, the method comprising the step of extracting cell-free DNA using a kit according to any one of claims 1 to 6 or a product according to any one of claims 7 to 8; The method includes the following steps: S1: Mix proteinase K, lysis binding buffer A and the sample to be tested, lyse, add the first magnetic bead for the first incubation and magnetic adsorption, then discard the first magnetic bead to obtain mixture 1; S2: Add lysis binding solution B to mixture 1 and mix. Perform a second incubation and magnetic adsorption. After washing with washing solution A and washing solution B, elute with elution solution to obtain free DNA extract.

12. The method according to claim 11, characterized in that, The first incubation period is 2–5 minutes; and / or, The second incubation period is 5 to 20 minutes.

Images