Magnetic bead, kit and method for extracting short DNA

Abstract

The invention relates to a magnetic bead, a kit and a method for extracting short-fragment DNA (deoxyribonucleic acid). The magnetic beads are prepared by the following method: (1) taking magnetic beads, and adding a water-soluble carbodiimide activator and an activation synergist for activation; (2) taking the activated magnetic beads, and adding polyethyleneimine for reaction to obtain polyethyleneimine modified magnetic beads; and (3) taking the polyethyleneimine modified magnetic beads, adding poly N-isopropylacrylamide, the water-soluble carbodiimide activator and the activation synergist, and carrying out a reaction so as to obtain the magnetic beads. The surface of the magnetic bead is coated with a double-layer functional polymer to construct double channels, traditional chemical competition is replaced with physical screening, the enrichment efficiency and selectivity of short-fragment DNA with the average length of 166 bp are remarkably improved, and the magnetic bead is very suitable for cffDNA capture. By using the magnetic bead and the method, the usage amount of maternal plasma can be effectively reduced, the fetal fraction is increased, and the detection limit of mutant alleles is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nucleic acid extraction, and relates to cell-free fetal DNA extraction, in particular to a magnetic bead for extracting short fragment DNA, a kit and a method. BACKGROUND

[0002] Cell-free fetal DNA (cffDNA) is a type of DNA fragment originating from a fetus existing in the plasma of a pregnant woman, and belongs to the category of cell-free DNA (cfDNA). It is mainly derived from the apoptosis or necrosis of placental trophoblast cells, and the released DNA enters the maternal blood circulation system through the placenta-maternal circulation to become a free component in the plasma. The length of the DNA is about 165-200 bp, and it is highly fragmented, similar to the DNA fragments released by cells under normal physiological conditions. During pregnancy, the proportion of fetal DNA in the maternal plasma gradually increases in the early stages of pregnancy, and can be stably detected after 8 weeks of pregnancy through high-sensitivity technology (such as next-generation sequencing).

[0003] In 1997, lo et al. first discovered that a small amount of cffDNA exists in the plasma of pregnant women. With the development, by analyzing fetal DNA fragments, chromosomal abnormalities (such as Down syndrome) can be screened without invasive operation, mainly for non-invasive prenatal testing. At the same time, by enriching the cffDNA of fetal origin and performing genetic analysis, it can assist in the diagnosis of genetic diseases, mainly for fetal genetic disease research.

[0004] At present, the method for extracting cell-free fetal DNA from maternal plasma is mainly based on plasma separation, cell lysis and DNA release, and DNA capture and purification. Among them, plasma separation obtains cell-free fetal DNA by twice centrifugal separation of plasma after collecting peripheral blood of pregnant women; cell lysis and DNA release decompose proteins and release DNA by using proteinase K and lysis solution (containing guanidine isothiocyanate, SDS and other components) to improve the recovery rate of small fragment fetal DNA. DNA capture and purification include magnetic bead method and centrifugal column method, wherein the magnetic bead method combines DNA with magnetic beads through magnetic field separation, and removes impurities by ethanol rinsing, and finally obtains high-purity DNA with eluent. The centrifugal column method uses a silica gel membrane to adsorb DNA, which is eluted after multiple washes, but the recovery efficiency of small fragment DNA is low. The magnetic bead method is commonly used in practical application.

[0005] In patent 202410150585.9, a kit for extracting free nucleic acid from the plasma of pregnant women and a method thereof are proposed, which uses a composite lysis solution (containing polyethylene glycol, alkyl glycoside quaternary ammonium salt, etc.) to enhance the binding efficiency of small fragment DNA and reduce the interference of maternal genomic DNA.

[0006] In the patent 201510939670.4, a method for extracting free nucleic acid by magnetic bead method and a kit thereof, by eliminating the long-time incubation step of proteinase K, the extraction time is shortened, and based on large sample processing, the extraction amount of low-concentration cffDNA is improved by two-stage magnetic bead adsorption (firstly enriched by low-volume magnetic beads, and secondly purified by high-volume magnetic beads). However, the application uses a large amount of magnetic beads, and the excessive magnetic beads are not uniformly dispersed, which reduces the adsorption efficiency and may precipitate functional proteins. In addition, the magnetic bead adsorption is not accurate, and maternal DNA may be adsorbed.

[0007] Therefore, there is a need in the art for a method for efficiently and rapidly extracting cffDNA from maternal plasma. SUMMARY

[0008] Based on this, the purpose of the present application is to provide a magnetic bead, a kit and a method for extracting short fragment DNA; the magnetic bead can efficiently and rapidly capture cffDNA from maternal plasma.

[0009] The first aspect of the present application is to provide a preparation method of a magnetic bead, comprising the following steps:

[0010] (1) Take the magnetic beads, add water-soluble carbodiimide activator and activation synergist for reaction, magnetic separation, and obtain the activated magnetic beads;

[0011] (2) Take the activated magnetic beads, add polyethyleneimine for reaction, magnetic separation, and obtain the polyethyleneimine modified magnetic beads;

[0012] (3) Take the polyethyleneimine modified magnetic beads, add poly-N-isopropyl acrylamide, the water-soluble carbodiimide activator and the activation synergist for reaction, magnetic separation, and obtain the product.

[0013] In some embodiments, the magnetic beads are selected from at least one of carboxyl magnetic beads, amino-modified magnetic beads, epoxy-modified magnetic beads, and aldehyde-modified magnetic beads.

[0014] In some embodiments, the water-soluble carbodiimide activator is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate.

[0015] In some embodiments, the activation synergist is selected from at least one of N-hydroxysuccinimide and sulfonated N-hydroxysuccinimide.

[0016] In some embodiments, the relative molecular mass of the polyethyleneimine is 10 kDa to 50 kDa.

[0017] In some embodiments, the poly-N-isopropylacrylamide has a relative molecular mass of 15 kDa to 30 kDa.

[0018] In some embodiments, the magnetic beads are carboxyl magnetic beads.

[0019] In some embodiments, the density of the carboxyl groups on the surface of the carboxyl magnetic beads is 0.3 mmol / g to 0.8 mmol / g.

[0020] In some embodiments, the mass ratio of the polyethyleneimine to the magnetic beads is (1-4):1.

[0021] In some embodiments, the mass ratio of the polyethyleneimine to the magnetic beads is (1-2):1.

[0022] In some embodiments, the mass ratio of the polyethyleneimine to the poly-N-isopropylacrylamide is 1:(1-3).

[0023] In some embodiments, the mass ratio of the polyethyleneimine to the poly-N-isopropylacrylamide is 1:(1-2).

[0024] In some embodiments, the temperature of the reaction in step (1) is 20°C to 30°C, and the time is 15 min to 45 min.

[0025] In some embodiments, the temperature of the reaction in step (2) is 20°C to 30°C, and the time is 1 h to 3 h.

[0026] In some embodiments, the temperature of the reaction in step (3) is 20°C to 30°C, and the time is 3 h to 5 h.

[0027] The second aspect of the present application provides the magnetic beads prepared by the preparation method described above.

[0028] The third aspect of the present application provides the use of the magnetic beads described above in extracting cell-free fetal DNA from maternal plasma.

[0029] The fourth aspect of the present application provides a kit for extracting cell-free fetal DNA from maternal plasma, which comprises the magnetic beads described above.

[0030] In some embodiments, the kit further comprises a binding buffer, which is a PBS buffer to which the following components are added: 1 w / v% to 10 w / v% of a neutral water-soluble crowding polymer, 0.1 M to 1.0 M of NaCl.

[0031] The neutral water-soluble crowding polymer is selected from at least one of polyethylene glycol, polyacrylamide, polyvinyl alcohol, and hydroxypropyl cellulose.

[0032] In some embodiments, the average molecular weight of the polyethylene glycol (PEG) is 6000-20000 Dalton.

[0033] In some embodiments, the average molecular weight of the polyacrylamide (PAM) is 50000-1000000 Dalton.

[0034] In some embodiments, the average molecular weight of the polyvinyl alcohol (PVA) is 25000-300000 Dalton.

[0035] In some embodiments, the average molecular weight of the hydroxypropyl cellulose (HPC) is 50000-100000 Dalton.

[0036] A fourth aspect of the present application provides a method for extracting cell-free fetal DNA from maternal plasma, comprising the following steps:

[0037] S1, separating plasma from peripheral blood of a pregnant woman, adding a lysis buffer to obtain a plasma lysis solution;

[0038] S2, adding the magnetic beads in the kit as described above to the binding buffer to obtain a magnetic bead suspension, and keeping at 25±0.5℃ for 5min-15min;

[0039] S3, adding the plasma lysis solution to the magnetic bead suspension, incubating at 25±0.5℃ for 1min-30min, and then warming to 32℃-37℃ and keeping for 5min-10min;

[0040] S4, magnetic separation and elution to obtain cell-free fetal DNA.

[0041] In some embodiments, the concentration of the magnetic beads in the magnetic bead suspension is 0.1mg / mL-1.0mg / mL.

[0042] In some embodiments, the volume ratio of the magnetic bead suspension to the plasma is (0.5-2):1.

[0043] In some embodiments, the incubation in step S3 is at 25±0.5℃ for 1min-20min.

[0044] In some embodiments, the incubation in step S3 is at 25±0.5℃ for 5min-10min.

[0045] In some embodiments, the elution comprises: pre-elution at 25±0.5℃; and main elution at 65℃-70℃.

[0046] In some embodiments, the incubation in step S3 is performed in a dynamic electromagnetic field.

[0047] In some embodiments, the dynamic electromagnetic field has a frequency of 20-150 Hz and a controlled magnetic bead amplitude of ±20-80 μm.

[0048] The present application provides a magnetic bead for efficiently capturing short DNA fragments (mainly cffDNA) with an average length of 166 bp from maternal plasma. The surface of the magnetic bead is coated with a double-layer functional polymer, wherein the inner layer is coated with a suitable amount of positively charged polyethyleneimine layer, which can form a suitable dynamic network structure for efficient capture of short DNA fragments with an average length of 166 bp; the outer layer is a uniform poly-N-isopropylacrylamide layer (temperature-sensitive layer), which is hydrophilic and relaxed at low temperature, exposing the inner polyethyleneimine layer, and specifically adsorbing short DNA fragments with an average length of 166 bp in maternal plasma; after adsorption, the poly-N-isopropylacrylamide layer shrinks and becomes hydrophobic by increasing the temperature, forming a physical barrier to inhibit the binding of long maternal DNA. By coating the surface of the magnetic bead with a suitable double-layer functional polymer structure, a "double-channel" is formed, and physical screening is used instead of traditional chemical competition, which significantly improves the enrichment efficiency and selectivity of short DNA fragments with an average length of 166 bp, thereby efficiently capturing cffDNA and significantly increasing the fetal fraction.

[0049] Further, the present application also optimizes the conditions for the binding of the magnetic bead to short DNA fragments in maternal plasma, especially obtaining a binding buffer containing a suitable concentration of neutral water-soluble crowding polymer and NaCl, which is more conducive to the adsorption and binding of the polyethyleneimine layer of the magnetic bead of the present application to short DNA fragments with an average length of 166 bp, reduces non-specific adsorption of long DNA, and weakens electrostatic interaction to inhibit the binding of long DNA.

[0050] Using the magnetic bead and binding buffer of the present application, efficient capture of short DNA fragments with an average length of 166 bp can be achieved in a dynamic electromagnetic field, with a capture rate of up to 95% and a long DNA inhibition rate of >85%, which is very suitable for capturing cffDNA in maternal plasma and can increase the fetal fraction to about 28% (traditional magnetic bead capture method is only 8%), thereby effectively reducing the detection limit of mutant alleles (as low as 0.1%) and reducing the use of maternal plasma during detection. DETAILED DESCRIPTION

[0051] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0052] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0053] Unless otherwise defined, all technical and scientific terms used in this invention are consistent with those belonging to this invention.

[0054] The meanings are generally understood to be the same by those skilled in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0055] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0056] Some embodiments of the present invention relate to a method for preparing magnetic beads, comprising the following steps:

[0057] (1) Take magnetic beads, add water-soluble carbodiimide activator and activating synergist to react, and magnetically separate to obtain activated magnetic beads;

[0058] (2) Take the activated magnetic beads, add polyethyleneimine to react, and perform magnetic separation to obtain polyethyleneimine-modified magnetic beads;

[0059] (3) Take the polyethyleneimine-modified magnetic beads, add poly(N-isopropylacrylamide), the water-soluble carbodiimide activator and the activating synergist to react, and then magnetically separate to obtain the product.

[0060] This invention provides magnetic beads for rapidly and efficiently capturing short DNA fragments with an average length of 166 bp (of which cff DNA accounts for a very high proportion and can be considered mainly cff DNA) from maternal plasma. The magnetic beads are coated with a bilayer functionalized polymer. The inner layer is a layer coated with a suitable amount of positively charged polyethyleneimine, which forms a suitable dynamic network structure for efficiently capturing short DNA fragments with an average length of 166 bp. The outer layer is a uniformly covered poly(N-isopropylacrylamide) layer (thermosensitive layer), which hydrophilically expands at low temperatures, exposing the inner polyethyleneimine layer and specifically adsorbing short DNA fragments with an average length of 166 bp from maternal plasma. After adsorption, the poly(N-isopropylacrylamide) layer shrinks and becomes hydrophobic by increasing the temperature, forming a physical barrier to inhibit the binding of long-chain maternal DNA. By constructing a "dual channel" by coating the surface of magnetic beads with a double layer of functionalized polymer, physical sieving is used to replace traditional chemical competition, which significantly improves the enrichment efficiency and selectivity of short DNA fragments with an average length of 166bp, thereby efficiently capturing cffDNA and significantly improving fetal fraction.

[0061] The magnetic beads of this invention are coated with a bilayer functionalized polymer, wherein the inner layer is a layer coated with positively charged polyethyleneimine. By coating the activated magnetic bead surface with polyethyleneimine of appropriate molecular weight (10 kDa~50 kDa) in a specific ratio, a suitable dynamic network structure can be formed, which can efficiently capture short DNA fragments (mainly cffDNA) with an average length of 166 bp through electrostatic interaction, thus enabling efficient and specific capture of cffDNA in maternal plasma. The inventors found that the mass ratio of polyethyleneimine to carboxyl magnetic beads significantly affects the capture effect of the short DNA fragments. Too high a ratio leads to magnetic bead aggregation, while too low a ratio leads to a decrease in the capture efficiency of the short DNA fragments. Studies have shown that a mass ratio of polyethyleneimine to carboxyl magnetic beads of (1~4):1 yields better capture efficiency, especially a mass ratio of (1~2):1, where the capture efficiency is even higher.

[0062] The outer layer of the bilayer functionalized polymer coating on the surface of the magnetic beads in this invention is a poly(N-isopropylacrylamide) layer. Poly(N-isopropylacrylamide) is temperature-responsive, expanding at low temperatures (25±0.5℃) and contracting at high temperatures (32℃~37℃). Utilizing this characteristic, the inventors cleverly further coat the polyethyleneimine layer with a poly(N-isopropylacrylamide) layer, controlling the opening and closing of the "channels" through temperature changes, thereby inhibiting the adsorption of long DNA fragments onto the poly(N-isopropylacrylamide) layer and further improving the capture specificity of short DNA fragments. The inventors discovered that the ratio of poly(N-isopropylacrylamide) to polyethyleneimine significantly affects the temperature sensitivity of the poly(N-isopropylacrylamide) layer. When the mass ratio of PEI to PNIPAM is 1:(1~3), uniform coverage of the temperature-sensitive layer can be ensured. Especially when the mass ratio is 1:(1~2), better temperature responsiveness is achieved, allowing for timely control of the opening and closing of the "channels," forming a physical barrier and inhibiting the binding of long-chain parent DNA.

[0063] Some of these embodiments involve the use of magnetic beads, as described above, in the extraction of cell-free fetal DNA from maternal plasma.

[0064] Some of these embodiments involve a kit for extracting cell-free fetal DNA from maternal plasma, the kit comprising magnetic beads as described above.

[0065] In some embodiments, the kit further comprises a binding buffer, which is a PBS buffer containing the following components at concentrations: 1 w / v% ~ 10 w / v% neutral water-soluble crowding polymer and 0.1 M ~ 1.0 M NaCl;

[0066] The neutral water-soluble crowding polymer is selected from at least one of polyethylene glycol, polyacrylamide, polyvinyl alcohol, and hydroxypropyl cellulose.

[0067] In some embodiments, the polyethylene glycol (PEG) has an average molecular weight of 6,000 to 20,000 Daltons, preferably 6,000 to 10,000 Daltons.

[0068] In some embodiments, the polyacrylamide (PAM) has an average molecular weight of 50,000 to 1,000,000 Daltons, preferably 50,000 to 80,000 Daltons.

[0069] In some embodiments, the average molecular weight of the polyvinyl alcohol (PVA) is 25,000 to 300,000 Daltons, preferably 25,000 to 100,000 Daltons.

[0070] In some embodiments, the hydroxypropyl cellulose (HPC) has an average molecular weight of 50,000 to 100,000 Daltons, preferably 50,000 to 80,000 Daltons.

[0071] Through further research, the inventors discovered that the incubation environment of the magnetic beads with maternal plasma affects the capture effect of the magnetic beads on target short DNA fragments. They also optimized and obtained a suitable binding buffer containing appropriate concentrations of PEG and NaCl. This buffer can reduce non-specific adsorption by enhancing the steric hindrance effect and preferentially exclude long DNA chains. It can also moderately shield the charge to reduce the binding of long DNA chains (short DNA chains can still be adsorbed due to the high charge density at their ends).

[0072] In some embodiments, the pH of the PBS buffer is 6.0–8.0, preferably 7.0–8.0, and more preferably 7.2–7.6. A suitable pH value can ensure the degree of protonation and enhance the electrostatic adsorption of short DNA fragments with an average length of 166 bp.

[0073] Some of these embodiments involve a method for extracting cell-free fetal DNA from maternal plasma, comprising the following steps:

[0074] S1. Separate plasma from the peripheral blood of pregnant women, add lysis buffer to obtain plasma lysis buffer;

[0075] S2. Add the magnetic beads as described above to the binding buffer solution as described above to obtain a magnetic bead suspension, and keep it at 25±0.5℃ for 5min~15min;

[0076] S3. Add the plasma lysis buffer to the magnetic bead suspension, incubate at 25±0.5℃ for 1 min to 30 min, and then raise the temperature to 32℃ to 37℃.

[0077] S4. Magnetic separation, elution, to obtain cell-free fetal DNA.

[0078] In some embodiments, incubation at 25±0.5°C for 1 min to 20 min is preferred, and incubation for 5 min to 10 min is more preferred. Because short chains diffuse rapidly, equilibrium can be reached in a short time (about 10 min); long chains require a longer time, so selective enrichment can be achieved in a short time.

[0079] In some embodiments, the magnetic bead concentration in the magnetic bead suspension is 0.1 mg / mL to 1.0 mg / mL, preferably 0.1 mg / mL to 0.5 mg / mL. Too high a concentration will cause the magnetic beads to aggregate, while too low a concentration will result in insufficient DNA binding sites.

[0080] In some embodiments, incubation in a dynamic electromagnetic field occurs in step S3. Dynamic electromagnetic field-assisted capture facilitates the micro-movement of magnetic beads, accelerating the diffusion of short-chain cffDNA to the PEI binding site, resulting in a higher capture rate.

[0081] In some embodiments, the frequency of the dynamic electromagnetic field is 20Hz to 150Hz, and the amplitude of the controlled magnetic bead is ±20μm to ±80μm.

[0082] Using the magnetic beads and binding buffer of this invention, efficient capture of short DNA fragments with an average length of 166 bp can be achieved in a dynamic electromagnetic field, with a capture rate of up to 95% and a long DNA inhibition rate of >85%. It is very suitable for capturing cffDNA in maternal plasma, which can increase the fetal fraction to about 28% (compared to only 8% by traditional magnetic bead capture methods), thereby effectively reducing the detection limit of mutant alleles (as low as 0.1%) and reducing the amount of maternal plasma used during testing.

[0083] The present invention will be further described in detail below with reference to specific embodiments.

[0084] Reagents and materials

[0085] Carboxyl magnetic beads, 1.0 μm in diameter, 10 mg / mL solid content, and ~0.5 mmol / g surface carboxyl density;

[0086] Polyethyleneimine (PEI), Mw=25 kDa; 5 mg / mL (dissolved in PBS, pH 7.4);

[0087] Poly(N-isopropylacrylamide) (PNIPAM), Mw = 20 kDa, 10 mg / mL (dissolved in 0.1 M MES buffer, pH 5.5).

[0088] N-hydroxysuccinimide (NHS), 50 mM;

[0089] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 100 mM;

[0090] 0.1% Tween 20, for washing;

[0091] 0.1 M PBS, pH 7.4, for buffering;

[0092] 0.1 M MES buffer, pH 5.5;

[0093] 100uL plasma lysis buffer formulation: Tris-HCl 10mM (pH 8.0), EDTA 10mM, SDS (sodium dodecyl sulfate) 1% (w / v), proteinase K 200ug / mL, and enzyme-free water to 100uL.

[0094] In the following embodiments, the magnetic beads used in this invention for extracting cell-free fetal DNA from maternal plasma are named PEI-PNIPAM magnetic beads.

[0095] Example 1

[0096] This embodiment provides magnetic beads for extracting cell-free fetal DNA from maternal plasma, the magnetic beads being prepared by the following method:

[0097] Step 1: Activation of carboxyl magnetic beads

[0098] Take 1 mL of carboxyl magnetic beads (10 mg / mL) and wash 3 times with 0.1 M MES buffer (pH 5.5) (5000 rpm, 5 min).

[0099] Resuspend in 1 mL of 0.1 M MES buffer (pH 5.5), add 1 mL of 50 mM NHS and 0.5 mL of 100 mM MEDC, and shake at room temperature for 30 min to activate the carboxyl group to form NHS ester.

[0100] Magnetic separation was performed, the supernatant was discarded, and the mixture was washed three times with PBS (pH 7.4) to remove unreacted EDC / NHS.

[0101] Step 2: PEI modification (inner layer positive charge modification)

[0102] The activated carboxyl magnetic beads were resuspended in 1 mL PBS (pH 7.4) and 1 mL 5 mg / mL PEI solution was added (final concentration 0.5 mg PEI / mg carboxyl magnetic beads).

[0103] The reaction was carried out at room temperature with shaking for 2 h, which allowed PEI to be covalently coupled to NHS ester via amino groups.

[0104] Magnetic separation was performed, followed by washing three times with 0.1% Tween 20 to remove free PEI.

[0105] Step 3: PNIPAM modification (outer temperature-sensitive layer)

[0106] Resuspend the PEI-modified magnetic beads in 1 mL of MES buffer (pH 5.5) and add 1 mL of 10 mg / mL PNIPAM solution (final concentration 1 mg PNIPAM / mg carboxylated magnetic beads).

[0107] Add 0.5 mL of 100 mM EDC and 1.0 mL of 50 mM NHS, and shake at room temperature for 4 h to allow PNIPAM to couple with the unreacted amino groups of PEI via the carboxyl group.

[0108] Magnetic separation was performed, and the beads were washed three times with PBS (pH 7.4) to obtain the PEI-PNIPAM magnetic beads, which were then stored at 4°C for later use.

[0109] Example 2

[0110] This embodiment provides magnetic beads for extracting cell-free fetal DNA from maternal plasma, the magnetic beads being prepared by the following method:

[0111] Step 1: Activation of carboxyl magnetic beads

[0112] Same as Example 1.

[0113] Step 2: PEI modification (inner layer positive charge modification)

[0114] The activated carboxyl magnetic beads were resuspended in 1 mL PBS (pH 7.4), and 2 mL of 5 mg / mL PEI solution was added (final concentration 1 mg PEI / mg carboxyl magnetic beads).

[0115] The reaction was carried out at room temperature with shaking for 2 h, which allowed PEI to be covalently coupled to NHS ester via amino groups.

[0116] Magnetic separation was performed, followed by washing three times with 0.1% Tween 20 to remove free PEI.

[0117] Step 3: PNIPAM modification (outer temperature-sensitive layer)

[0118] Resuspend the PEI-modified magnetic beads in 1 mL of MES buffer (pH 5.5) and add 2 mL of 10 mg / mL PNIPAM solution (final concentration 2 mg PNIPAM / mg carboxylated magnetic beads).

[0119] Add 0.5 mL of 100 mM EDC and 1.0 mL of 50 mM NHS, and shake at room temperature for 4 h to allow PNIPAM to couple with the unreacted amino groups of PEI via the carboxyl group.

[0120] Magnetic separation was performed, and the beads were washed three times with PBS (pH 7.4) to obtain the PEI-PNIPAM magnetic beads, which were then stored at 4°C for later use.

[0121] Example 3

[0122] This embodiment provides magnetic beads for extracting cell-free fetal DNA from maternal plasma, the magnetic beads being prepared by the following method:

[0123] Step 1: Activation of carboxyl magnetic beads

[0124] Same as Example 1.

[0125] Step 2: PEI modification (inner layer positive charge modification)

[0126] The activated carboxyl magnetic beads were resuspended in 1 mL PBS (pH 7.4), and 4 mL of 5 mg / mL PEI solution was added (final concentration 2 mg PEI / mg carboxyl magnetic beads).

[0127] The reaction was carried out at room temperature with shaking for 2 h, which allowed PEI to be covalently coupled to NHS ester via amino groups.

[0128] Magnetic separation was performed, followed by washing three times with 0.1% Tween 20 to remove free PEI.

[0129] Step 3: PNIPAM modification (outer temperature-sensitive layer)

[0130] Resuspend the PEI-modified magnetic beads in 1 mL of MES buffer (pH 5.5) and add 4 mL of 10 mg / mL PNIPAM solution (final concentration 4 mg PNIPAM / mg carboxylated magnetic beads).

[0131] Add 0.5 mL of 100 mM EDC and 1.0 mL of 50 mM NHS, and shake at room temperature for 4 h to allow PNIPAM to couple with the unreacted amino groups of PEI via the carboxyl group.

[0132] Magnetic separation was performed, and the beads were washed three times with PBS (pH 7.4) to obtain the PEI-PNIPAM magnetic beads, which were then stored at 4°C for later use.

[0133] Example 4

[0134] This embodiment provides magnetic beads for extracting cell-free fetal DNA from maternal plasma, the magnetic beads being prepared by the following method:

[0135] Step 1: Activation of carboxyl magnetic beads

[0136] Same as Example 1.

[0137] Step 2: PEI modification (inner layer positive charge modification)

[0138] The activated carboxyl magnetic beads were resuspended in 1 mL PBS (pH 7.4) and 8 mL 5 mg / mL PEI solution was added (final concentration 4 mg PEI / mg carboxyl magnetic beads).

[0139] The reaction was carried out at room temperature with shaking for 2 h, which allowed PEI to be covalently coupled to NHS ester via amino groups.

[0140] Magnetic separation was performed, followed by washing three times with 0.1% Tween 20 to remove free PEI.

[0141] Step 3: PNIPAM modification (outer temperature-sensitive layer)

[0142] Resuspend the PEI-modified magnetic beads in 1 mL of MES buffer (pH 5.5) and add 8 mL of 10 mg / mL PNIPAM solution (final concentration 8 mg PNIPAM / mg carboxylated magnetic beads).

[0143] Add 0.5 mL of 100 mM EDC and 1.0 mL of 50 mM NHS, and shake at room temperature for 4 h to allow PNIPAM to couple with the unreacted amino groups of PEI via the carboxyl group.

[0144] Magnetic separation was performed, and the beads were washed three times with PBS (pH 7.4) to obtain the PEI-PNIPAM magnetic beads, which were then stored at 4°C for later use.

[0145] Example 5

[0146] This embodiment provides magnetic beads for extracting cell-free fetal DNA from maternal plasma, the magnetic beads being prepared by the following method:

[0147] Step 1: Activation of carboxyl magnetic beads

[0148] Same as Example 1.

[0149] Step 2: PEI modification (inner layer positive charge modification)

[0150] The activated carboxyl magnetic beads were resuspended in 1 mL PBS (pH 7.4), and 2 mL of 5 mg / mL PEI solution was added (final concentration 1 mg PEI / mg carboxyl magnetic beads).

[0151] The reaction was carried out at room temperature with shaking for 2 h, which allowed PEI to be covalently coupled to NHS ester via amino groups.

[0152] Magnetic separation was performed, followed by washing three times with 0.1% Tween 20 to remove free PEI.

[0153] Step 3: PNIPAM modification (outer temperature-sensitive layer)

[0154] Resuspend the PEI-modified magnetic beads in 1 mL of MES buffer (pH 5.5) and add 0.5 mL of 10 mg / mL PNIPAM solution (final concentration 2 mg PNIPAM / mg carboxylated magnetic beads).

[0155] Add 0.5 mL of 100 mM EDC and 1.0 mL of 50 mM NHS, and shake at room temperature for 4 h to allow PNIPAM to couple with the unreacted amino groups of PEI via the carboxyl group.

[0156] Magnetic separation was performed, and the beads were washed three times with PBS (pH 7.4) to obtain the PEI-PNIPAM magnetic beads, which were then stored at 4°C for later use.

[0157] Example 6

[0158] This embodiment provides magnetic beads for extracting cell-free fetal DNA from maternal plasma, the magnetic beads being prepared by the following method:

[0159] Step 1: Activation of carboxyl magnetic beads

[0160] Same as Example 1.

[0161] Step 2: PEI modification (inner layer positive charge modification)

[0162] The activated carboxyl magnetic beads were resuspended in 1 mL PBS (pH 7.4), and 2 mL of 5 mg / mL PEI solution was added (final concentration 1 mg PEI / mg carboxyl magnetic beads).

[0163] The reaction was carried out at room temperature with shaking for 2 h, which allowed PEI to be covalently coupled to NHS ester via amino groups.

[0164] Magnetic separation was performed, followed by washing three times with 0.1% Tween 20 to remove free PEI.

[0165] Step 3: PNIPAM modification (outer temperature-sensitive layer)

[0166] Resuspend the PEI-modified magnetic beads in 1 mL of MES buffer (pH 5.5) and add 3 mL of 10 mg / mL PNIPAM solution (final concentration 3 mg PNIPAM / mg carboxylated magnetic beads).

[0167] Add 0.5 mL of 100 mM EDC and 1.0 mL of 50 mM NHS, and shake at room temperature for 4 h to allow PNIPAM to couple with the unreacted amino groups of PEI via the carboxyl group.

[0168] Magnetic separation was performed, and the beads were washed three times with PBS (pH 7.4) to obtain the PEI-PNIPAM magnetic beads, which were then stored at 4°C for later use.

[0169] Example 7

[0170] Using the PEI-PNIPAM magnetic beads prepared in the above examples, cell-free fetal DNA was extracted from maternal plasma.

[0171] Using EWOD technology, precise droplet positioning (10-100nL) is achieved through the electrowetting effect. An electromagnetic matrix is ​​designed, with a programmable electromagnet array integrated at the bottom of the microfluidic chip. The electromagnets can be activated in sections to guide the magnetic beads to specific droplet regions, and it has the characteristic of high-frequency switching magnetic field.

[0172] The specific steps are as follows:

[0173] 1. Plasma pretreatment

[0174] Collect 1 mL of peripheral blood from the pregnant woman (early pregnancy, 10 weeks of gestation) and use EDTA or Streck blood collection tubes for anticoagulation.

[0175] The plasma is then separated by double centrifugation, involving two steps:

[0176] Step 1: Centrifuge at 1600-2000×g for 10 minutes at 4°C to separate the upper plasma layer.

[0177] Step 2: Transfer the supernatant to a new tube and centrifuge at 16,000×g for 10 minutes at 4°C. Collect approximately 500uL of the supernatant plasma. This step removes residual cell debris and platelets (containing maternal genomic DNA) from the supernatant, preventing contamination with cffDNA (mainly short fragments <200bp).

[0178] Plasma lysis and DNA release

[0179] Take 100 μL of plasma and add 100 μL of lysis buffer containing proteinase K. The lysis buffer needs to disrupt the lipid membrane structure and inactivate nucleases (such as DNase I) in the plasma. Mix thoroughly and incubate at 55-60°C for 3-6 minutes (4 minutes at 56°C in this example) to obtain plasma lysis buffer, ensuring complete lysis of vesicle / protein complexes and release cfDNA (including maternal and fetal origins).

[0180] In this embodiment, specific concentrations of 166 BP DNA and 1000 BP DNA standards were added to the plasma lysate to test the performance of the magnetic beads.

[0181] 2. Incubation combined

[0182] Magnetic bead loading: PEI-PNIPAM magnetic beads were suspended in PBS buffer (pH 7.4) containing 5% PEG 8000 and 0.3 M NaCl to a concentration of 0.5 mg / mL.

[0183] Temperature control: 50 μL of magnetic bead suspension was precisely transferred to the reaction zone using electrowetting (EWOD) technology, cooled to 25°C, and kept for 10 min to allow the PNIPAM hydrophilic layer to expand.

[0184] Sample incubation: Add 100 μL of plasma lysis buffer, mix gently, and maintain the temperature at 25°C.

[0185] Electromagnetic field assistance: Maintain the temperature at 25°C, activate the bottom electromagnetic matrix (50 Hz, ±50 μm), incubate for 10 min, raise the temperature to 37°C, hold for 5 min, and obtain magnetic bead-DNA complex.

[0186] 3. Magnetic separation and washing

[0187] Place the centrifuge tube on a magnetic rack and let it stand to allow the magnetic bead-DNA complex to aggregate on the tube wall. Carefully aspirate the supernatant (containing impurities), keep the tube on the magnetic rack, add washing buffer (usually a salt solution containing ethanol to maintain the DNA binding state), and wash 1-2 times to thoroughly remove impurities such as salt ions, proteins, and lipids.

[0188] DNA elution:

[0189] Remove any residual washing solution (to avoid ethanol inhibiting downstream reactions) and remove the tube from the magnetic rack.

[0190] Performing two-phase elution includes:

[0191] Pre-elution (short fragment recovery): Loosely bound short cfDNA was eluted with low-salt buffer (5 mM Tris-HCl) at low temperature (25°C).

[0192] Main elution (residual DNA cleared): Completely dissociate the remaining DNA at high temperature (60°C) using Tris-NaOH buffer (pH 9.0) to avoid cross-contamination.

[0193] Mix thoroughly, then incubate at room temperature or with appropriate heating (e.g., 65-70°C) for 2-10 minutes. This condition disrupts the binding force between the DNA and the magnetic beads, releasing the DNA into the solution. Place the tube back on the magnetic rack and allow it to stand until the magnetic beads aggregate. Carefully aspirate the supernatant into a new tube. This supernatant is the purified cfDNA solution (containing cffDNA).

[0194] The 166BP DNA capture rate (%), 1000BP DNA capture rate (%), selectivity (166BP / 1000BP), and fetal fraction of the PEI-PNIPAM magnetic beads prepared in each example were calculated by comparing the initial amount with the amount after elution.

[0195] 166 BP DNA capture rate (%): Amount of 166 BP DNA after elution / Initial amount of 166 BP DNA × 100%.

[0196] 1000 BP DNA capture rate (%): Amount of 1000 BP DNA after elution / Initial amount of 1000 BP DNA × 100%.

[0197] Selectivity (166 BP / 1000 BP): Amount of 166 BP DNA eluted / Amount of 1000 BP DNA eluted × 100%.

[0198] Fetal fraction (%): (amount of 166-BP DNA after elution) ÷ (amount of 166-BP DNA after elution + amount of 1000-BP DNA after elution) × 100%.

[0199] The calculation results are shown in Table 1:

[0200] Table 1

[0201]

[0202] The above results show that the PEI-PNIPAM magnetic beads of the present invention can extract short-chain DNA with an average length of 166 bp from the mother with high specificity and high efficiency, with a capture rate of up to 95% and good selectivity (166 BP / 1000 BP).

[0203] Comparative studies of Examples 1-4 show that the mass ratio of PEI to carboxyl magnetic beads significantly affects the capture efficiency of short-chain DNA with an average length of 166 bp. A mass ratio of (1-4):1 yields a better capture efficiency for 166 bp DNA; more preferably, a mass ratio of (1-2):1, at which point the capture rate of 166 bp DNA can reach over 92%, with minimal interference from long DNA fragments. A high mass ratio leads to bead aggregation, while a low mass ratio results in decreased DNA capture efficiency.

[0204] Comparative studies of Examples 2, 5, and 6 show that the mass ratio of PEI to PNIPAM significantly affects the 166 BP DNA capture rate. A ratio of 1:(1~2) ensures uniform coverage of the thermosensitive layer, thereby efficiently and specifically capturing short-chain 166 BP DNA while inhibiting the binding of long-chain DNA, significantly improving the selectivity of cffDNA. When the mass ratio is too high, it leads to excessive PNIPAM and an overly thick thermosensitive layer, hindering the electrostatic adsorption of short-chain DNA by PEI, resulting in a decrease in the 166 BP DNA capture rate and an increase in long-chain DNA interference. When the mass ratio is too low, it leads to insufficient PNIPAM, uneven coverage of the thermosensitive layer, and an inability to effectively form a physical barrier to inhibit long-chain DNA, resulting in increased non-specific binding of long-chain DNA and a decrease in the selectivity of 166 BP DNA.

[0205] Example 8

[0206] This embodiment investigates the effect of the binding buffer composition on the extraction of cffDNA from maternal plasma using the PEI-PNIPAM magnetic beads of the present invention.

[0207] Following the method described in Example 7, cffDNA was extracted from the plasma of pregnant women at 10 weeks of gestation using PEI-PNIPAM magnetic beads prepared in Example 2 and binding buffers of different compositions. Each binding buffer was a PBS buffer (pH 7.4) containing the following components at different concentrations:

[0208] Group 1: 5% (w / v) PEG 8000, 0.3M NaCl;

[0209] Group 2: 1% (w / v) PEG 8000, 0.1M NaCl;

[0210] Group 3: 5% (w / v) PEG 8000, 0.5M NaCl;

[0211] Group 4: 5% (w / v) PEG 8000, 0.05M NaCl;

[0212] Group 5: 5% (w / v) PEG 8000, 1.2M NaCl.

[0213] The 166 BP DNA capture rate (%), 1000 BP DNA capture rate (%), selectivity (166 BP / 1000 BP), and fetal fraction were calculated using the same method as described in Example 7. The results are shown in Table 2.

[0214] Table 2

[0215]

[0216]

[0217] The above results indicate that the composition of the binding buffer affects the extraction efficiency of cffDNA. A suitable concentration of PEG creates a stronger steric hindrance effect, reducing non-specific adsorption and preferentially excluding long-chain DNA. A suitable concentration of NaCl can shield the charge, reducing the binding of long-chain DNA, while short-chain cffDNA can still be adsorbed due to its high terminal charge density. The preferred binding buffer of this invention contains: 1% (w / v)~10% (w / v) PEG and 0.1M~1.0 M NaCl; more preferably, it contains: 1% (w / v)~5% (w / v) PEG and 0.1M~0.3M NaCl, which yields higher 166 BP DNA capture rates and lower 1000 BP DNA capture rates. Changes in the composition of the binding buffer significantly affect the extraction efficiency of cffDNA.

[0218] This embodiment also compares the effects of dynamic electromagnetic field assistance and static adsorption on cffDNA capture efficiency. Dynamic electromagnetic field assistance is group 1 mentioned above, while static adsorption differs from group 1 in that it does not activate the bottom electromagnetic matrix of the EWOD device. The capture efficiency of cffDNA under the two conditions is shown in Table 3:

[0219] Table 3

[0220]

[0221] The results showed that dynamic electromagnetic field-assisted capture was more effective in driving the micro-movement of magnetic beads, accelerating the diffusion of short-chain cffDNA to the PEI binding site, and achieving a higher capture rate.

[0222] Example 9

[0223] This embodiment is based on the detection of fetal single-gene diseases by extracting cell-free fetal DNA nucleic acid.

[0224] The paternal mutation (FGFR3 c.1138G>A) was enriched from the maternal background. This mutation site is located in a short cffDNA fragment.

[0225] Maternal plasma source: peripheral blood from 30 healthy pregnant women in early pregnancy (10-12 weeks of gestation), anticoagulated with EDTA.

[0226] The effectiveness of extracting cffDNA from maternal plasma was compared between traditional methods and the method of this invention.

[0227] Traditional method: Refer to the magnetic bead method and results in patent document with application number 201510939670.4.

[0228] The method of this invention is the same as in Example 7, using the PEI-PNIPAM magnetic beads prepared in Example 2, except that:

[0229] The magnetic beads were driven by a high-frequency magnetic field switching (100Hz) to compress the cffDNA binding time to 1 minute at 25℃ (the traditional method requires 10 minutes).

[0230] By activating the PNIPAM hydrophobic layer through temperature control (37℃), the binding of maternal DNA >300bp was inhibited (experiments showed that the adsorption rate of long fragments decreased by 82%).

[0231] Pre-elution buffer (containing enriched cff DNA) was directly introduced into the ddPCR system. Probe design: wild-type (HEX marker) / mutant (FAM marker) FGFR3 gene. Simultaneous master elution (60℃ alkaline buffer) was performed to remove residual DNA from the microarray and avoid cross-contamination.

[0232] The primer and probe sequences for ddPCR are as follows:

[0233] FGFR3-F: 5'-GCTGGTGGTGCTGGTGCTG-3' (SEQ ID NO: 1, covering 15-20 bp upstream of the mutation site to ensure amplification specificity);

[0234] FGFR3-R: 5'-ACGACGACGACGACGACA-3' (SEQ ID NO: 2, covering 15-20 bp downstream of the mutation site, forming an amplicon of about 200 bp with the upstream primer, suitable for short-chain cffDNA detection);

[0235] FGFR3-P: Divided into wild-type and mutant dual probes

[0236] Wild-type probe (HEX label): 5'-HEX-CTGCTGCTGGCTGCTGCT-BHQ1-3' (SEQ ID NO: 3, containing wild-type base G, 3' end-terminated quencher BHQ1);

[0237] Mutant probe (FAM tag): 5'-FAM-CTGCTGCTGACTGCTGCT-BHQ1-3' (SEQ ID NO: 4, containing mutant base A, differing from wild type by only 1 base, ensuring specificity of mutation detection).

[0238] II. ddPCR reaction system (20 μL total volume)

[0239] The components of the ddPCR reaction system are shown in Table 4 below:

[0240] Table 4

[0241]

[0242] III. ddPCR Reaction Procedure

[0243] The ddPCR reaction procedure is as follows:

[0244] 1. Pre-denaturation: 95℃, 10 min (to completely denature the short cffDNA strand and avoid secondary structure affecting amplification);

[0245] 2. Cyclic Phase (40 cycles):

[0246] Denaturation: 94℃, 30s (rapid denaturation of short-strand DNA, reducing primer non-specific binding);

[0247] Annealing extension: 58℃, 1min (adapt primer Tm value to ensure specific primer binding and extension, while ensuring probe hybridization efficiency);

[0248] 3. Inactivation: 98℃, 10min (inactivate DNA polymerase, terminate the reaction, and avoid subsequent contamination);

[0249] 4. Temperature control: 4℃, ∞ (for short-term preservation of reaction products to facilitate subsequent data analysis).

[0250] IV. Maternal DNA inhibition rate and cross-contamination rate between samples

[0251] Table 5 shows a comparison of the maternal DNA inhibition rate, mutation allele detection limit, and cross-contamination rate between the present invention and the traditional magnetic bead method.

[0252] Table 5

[0253]

[0254] The above results indicate that the method of the present invention can effectively improve the maternal DNA inhibition rate, reduce the detection limit of mutant alleles, and reduce the cross-contamination rate between samples.

[0255] Compared with traditional methods, the method of this invention also has the advantages shown in Table 6 below:

[0256] Table 6

[0257]

[0258] Table 7 below shows a technical comparison between the PEI-PNIPAM magnetic beads of the present invention and traditional magnetic beads used in the method of the present invention:

[0259] Table 7

[0260]

[0261] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing magnetic beads, characterized by, The method comprises the following steps: (1) taking magnetic beads, adding a water-soluble carbodiimide-based activator and an activation synergist for reaction, magnetic separation, and obtaining activated magnetic beads; (2) taking the activated magnetic beads, adding polyethyleneimine for reaction, magnetic separation, and obtaining polyethyleneimine-modified magnetic beads; (3) taking the polyethyleneimine-modified magnetic beads, adding poly-N-isopropyl acrylamide, the water-soluble carbodiimide-based activator, and the activation synergist for reaction, and magnetic separation.

2. The production method according to claim 1, wherein The magnetic beads are selected from at least one of carboxyl magnetic beads, amino-modified magnetic beads, epoxy-modified magnetic beads, and aldehyde-modified magnetic beads; and / or, The water-soluble carbodiimide-based activator is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate; and / or, The activation synergist is selected from at least one of N-hydroxysuccinimide and sulfonated N-hydroxysuccinimide; and / or, The relative molecular mass of the polyethyleneimine is 10 kDa to 50 kDa; and / or, The relative molecular mass of the poly-N-isopropyl acrylamide is 15 kDa to 30 kDa.

3. The production method according to claim 2, wherein The magnetic beads are carboxyl magnetic beads; preferably, the density of carboxyl groups on the surface of the carboxyl magnetic beads is 0.3 mmol / g to 0.8 mmol / g.

4. The production method according to claim 1 or 2, wherein The mass ratio of the polyethyleneimine to the magnetic beads is (1 to 4):1, preferably (1 to 2):1; and / or, The mass ratio of the polyethyleneimine to the poly-N-isopropyl acrylamide is 1:(1 to 3), preferably 1:(1 to 2).

5. The production method according to claim 1 or 2, wherein The temperature of the reaction in step (1) is 20°C to 30°C, and the time is 15 min to 45 min; and / or, The temperature of the reaction in step (2) is 20°C to 30°C, and the time is 1 h to 3 h; and / or, The temperature of the reaction in step (3) is 20°C to 30°C, and the time is 3 h to 5 h.

6. The magnetic beads obtained by the preparation method in any one of claims 1 to 5.

7. The use of the magnetic beads in claim 6 in extracting cell-free fetal DNA in maternal plasma.

8. A kit for extracting cell-free fetal DNA from maternal plasma, characterized in that, The kit comprises the magnetic beads in claim 6.

9. The kit of claim 8, wherein It further comprises a binding buffer, which is a PBS buffer to which the following components are added: 1 w / v% to 10 w / v% of a neutral water-soluble crowding polymer, 0.1 M to 1.0 M of NaCl; The neutral water-soluble crowding polymer is selected from at least one of polyethylene glycol, polyacrylamide, polyvinyl alcohol, and hydroxypropyl cellulose.

10. The kit of claim 9, wherein The average molecular weight of the polyethylene glycol is 6000 to 20000 Daltons; and / or, The average molecular weight of the polyacrylamide is 50000 to 1000000 Daltons; and / or, The average molecular weight of the polyvinyl alcohol is 25000 to 300000 Daltons; and / or, The average molecular weight of the hydroxypropyl cellulose is 50000 to 100000 Daltons.

11. A method of extracting cell-free fetal DNA from maternal plasma, characterized in that, The method comprises the following steps: S1, isolating plasma from peripheral blood of a pregnant woman, adding a lysis buffer to obtain a plasma lysis solution; S2, adding the magnetic beads in the kit of any one of claims 7-9 to the binding buffer to obtain a magnetic bead suspension, and keeping at 25±0.5℃ for 5-15 min; S3, adding the plasma lysis solution to the magnetic bead suspension, incubating at 25±0.5℃ for 1-30 min, and then warming to 32-37℃ for 5-10 min; S4, magnetic separation and elution to obtain cell-free fetal DNA.

12. The method of claim 11, wherein, The concentration of the magnetic beads in the magnetic bead suspension is 0.1-1.0 mg / mL; and / or, The volume ratio of the magnetic bead suspension to the plasma is (0.5-2):1; and / or, In step S3, the incubation at 25±0.5℃ is for 1-20 min, preferably 5-10 min; and / or, The elution comprises: pre-elution at 25±0.5℃; and main elution at 65-70℃.

13. The method of claim 11 or 12, wherein, In step S3, the incubation is performed in a dynamic electromagnetic field; Preferably, the frequency of the dynamic electromagnetic field is 20-150 Hz, and the control magnetic bead amplitude is ±20-±80 μm.

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