A cfDNA extraction kit for prenatal diagnosis

Abstract

The present invention relates to a cfDNA extraction kit for prenatal diagnosis, belonging to the field of bioengineering technology. The kit mainly includes a lysis-binding solution, nanomagnetic beads, and a washing solution. The kit of the present invention uses carboxyl-modified PEG nanomagnetic beads with a multi-layer sandwich structure, which can achieve magnetic bead amplification without elution. The special components of the lysis-binding solution and the washing solution can specifically obtain cfDNA without the need to separately remove genomic DNA. This kit does not require the use of components such as ethanol and isopropanol. This kit can be operated manually or automated on existing nucleic acid automatic extractors on the market without the need to customize special consumables, and has the characteristics of simple operation steps, high cfDNA yield, good sensitivity, and convenient transportation. In particular, by using the kit of the present invention and the operation method provided by the kit, a cfDNA sample that can be directly used for prenatal diagnosis can be obtained simply and quickly, improving the efficiency and sensitivity of prenatal diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a cfDNA extraction kit for prenatal diagnosis. Background Art

[0002] Cell-free DNA in blood, abbreviated as cfDNA (circulating free DNA), refers to nucleic acid substances in circulating blood, including nucleic acids in cells in circulating blood, free endogenous deoxyribonucleic acid, and exogenous DNA and RNA. CfDNA in blood has important potential value in the early diagnosis, prognosis, monitoring, etc. of diseases. Under specific physiological conditions or disease processes, a considerable part of cfDNA is different from that in a healthy state. Based on this result, cfDNA has been used for non-invasive diagnosis in recent years. In pregnant women, about 10%-15% of cfDNA comes from placental trophoblasts. Now cfDNA is mostly used to screen for fetal genetic defects in high-risk pregnant women, which is the basis of the current popular non-invasive Down syndrome screening! In cancer patients, the detection of tumors by quantifying mutations in cfDNA has been recognized by more and more people. In the aspect of transplantation, transplant rejection is also related to the level of cfDNA in the donor transplanted organ.

[0003] CfDNA is a DNA fragment released from human cells into the blood. The amount of this DNA is very small and the fragments are very short, with a length of about 170 bp. The extraction of cfDNA in the prior art has the following difficulties and defects: Since the content of cfDNA is very low, the extraction is difficult, the extraction efficiency is low, resulting in a relatively high non-detection rate in prenatal diagnosis, which limits the application and development of cfDNA as a conventional detection method; the DNA obtained by using conventional traditional extraction methods is genomic DNA, which contains all DNA fragments in the human genome and will generate large and complex background noise in subsequent detections; traditional extraction methods have many extraction steps, take a long time, and require the use of a variety of flammable chemical reagents such as ethanol and isopropanol. Summary of the Invention

[0004] To improve the extraction efficiency of cfDNA, a first object of the present invention is to provide a cfDNA extraction kit for prenatal diagnosis. A second object is to provide a method for extracting cfDNA for prenatal diagnosis using the extraction kit; a third object is the application of the extraction kit in the preparation of prenatal diagnostic drugs.

[0005] The kit has the characteristics of simple operation steps, high cfDNA yield, good sensitivity, and convenient transportation. Using the kit of the present invention and the operation method provided by the kit, the sample pretreatment work for cfDNA detection can be simply and quickly completed.

[0006] To achieve the above object, the specific solution adopted by the present invention is as follows:

[0007] A cfDNA extraction kit for prenatal diagnosis, comprising a lysis binding solution, magnetic nanoparticles, a washing solution, and an elution solution;

[0008] The pH value of the lysis binding solution is 5.7 to 7.9, and it includes 1.2 to 4.8 M sodium diisooctyl sulfosuccinate, 1.5 to 2.6 mM guanidine isothiocyanate, 2.3 to 3.8 mM guanidine hydrochloride, 1.3 to 3.4 mM SDS, 1% to 3.6% MES, and 10% to 20% PEG;

[0009] The magnetic nanoparticles are nano magnetic particles with a polystyrene core, a nano ferric oxide sandwich, a silica or cellulose coating, wrapped with PEG, and then carboxylated; the magnetic nanoparticles are a multi-layer sandwich structure, with a diameter of 400 to 2000 nm and a concentration of 30 to 80 mg / ml;

[0010] The washing solution includes 80 to 160 mM cetyltrimethylammonium bromide, 20 to 38 mM Tris-Hcl with a pH value of 7.5 to 8.5, 5% to 9% ASEA, and 10 to 50 mM FMES;

[0011] The pH of the elution solution is not less than 8.0, and it includes EDTA and Tris-Hcl.

[0012] As a further optimization of the above extraction kit, the PEG selected in the lysis binding solution has a molecular weight greater than 2000.

[0013] As a further optimization of the above extraction kit, the magnetic nanoparticles are prepared by the following method: a: Select 100 g of polystyrene microspheres with a particle size of 400 to 700 nm and disperse them in 1 L of anhydrous ethanol solution; b: Add 4 mol / L Fe prepared according to a molar ratio of 3:2 3+ and Fe 2+400 ml of the mixed salt solution was deoxygenated by introducing an inert gas for 30 min, the temperature of the system was adjusted to 60 - 65 °C, ammonia water was added until the pH value of the system reached 10, and the reaction was carried out for 2 h. 50 ml of a mixture of oleic acid and lauric acid premixed in a volume ratio of 3:2 was added, and the reaction was continued for 30 min. Then, it was rapidly cooled to 30 °C. After magnetic separation, the precipitate was washed repeatedly with 50% ethanol and deionized water several times until the washing solution was neutral, and then dispersed in 800 ml of absolute ethanol; c: tetraethyl orthosilicate and 200 ml of ammonia water or carboxymethyl cellulose and 260 ml of ammonia water were added, and the reaction was carried out for 1 h. Then, magnetic separation was carried out, and the precipitate was washed repeatedly with 30% ethanol and deionized water several times until the washing solution was neutral. Finally, it was dispersed in 800 ml of 30% ethanol solution; d: 200 ml of 20% PEG6000 solution was added, ammonia water was added until the pH value of the system reached 10, and it was incubated at 80 °C for 2 h. 100 ml of citric acid solution was added, and the reaction was continued for 30 min. Then, magnetic separation was carried out, and the precipitate was washed repeatedly with 30% ethanol and deionized water several times until the washing solution was neutral. Finally, it was dispersed in 2 L of the magnetic bead base solution containing PEG and Tween.

[0014] As a further optimization of the above extraction kit, the ZETA potential of the nano - magnetic beads is not greater than - 30. Further, the base solution of the nano - magnetic beads contains 15% - 30% PEG and 1% - 10% Tween 20; the molecular weight of PEG in the nano - magnetic bead base solution is not less than 4000; the surface tension of the nano - magnetic bead base solution is not less than 70 dynes / cm.

[0015] The present invention also provides a method for extracting cfDNA for prenatal diagnosis using the above extraction kit, including the following steps:

[0016] Step S1: Take 500 - 1200 ul of fresh plasma sample in a 5 ml or 10 ml centrifuge tube, add 900 - 2500 ul of lysis - binding solution using a sterile pipette, and incubate and mix well at 50 - 90 °C for 10 - 30 min with shaking;

[0017] Step S2: Add 10 - 50 ul of nano - magnetic beads and mix well by shaking for 5 - 15 min;

[0018] Step S3: Place the centrifuge tube treated in Step S2 on a magnetic rack. After the nano - magnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant solution;

[0019] Step S4: Use a sterile pipette to add 500 - 800 ul of washing solution to the centrifuge tube treated in Step S3, mix well by shaking for 2 min, place it on the magnetic rack. After the nano - magnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant solution;

[0020] Step S5: Use a sterile pipette to add 30 - 50 μl of eluent to the centrifuge tube processed in Step S4, incubate at 65°C with shaking for 3 - 5 min, then place it on a magnetic stand. After the magnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate the supernatant for cfDNA detection.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The extraction kit of the present invention uses carboxyl-modified PEG magnetic beads with a multi-layer sandwich structure, which can achieve amplification with magnetic beads without elution. Special lysis binding solution and washing solution components can specifically obtain cfDNA without the need to separately remove genomic DNA. This kit does not require the use of components such as ethanol and isopropanol. This kit can be operated manually or automated on existing nucleic acid automatic extractors on the market without the need to customize special consumables, and has the characteristics of simple operation steps, high cfDNA yield, good sensitivity, and convenient transportation. In particular, using the kit of the present invention and the operation method provided by the kit, cfDNA samples that can be directly used for prenatal diagnosis can be obtained simply and quickly, improving the efficiency and sensitivity of prenatal diagnosis.

[0023] 2. Using the extraction kit of the present invention, the obtained cfDNA bands are clear and there are no miscellaneous peaks. Compared with existing superior kits, the plasma usage is lower, the purity and quality are higher, and it is more excellent in terms of concentration and total amount, and is very suitable for sample processing of prenatal diagnosis. Description of the Drawings

[0024] Figure 1 is the cfDNA electrophoresis pattern obtained in Example 2 of the present invention;

[0025] Figure 2 is the cfDNA electrophoresis pattern obtained by comparison. Detailed Embodiments

[0026] The present invention provides a cfDNA extraction kit for prenatal diagnosis, including a lysis binding solution, magnetic beads, a washing solution, and an eluent.

[0027] In the above kit, the lysis binding solution includes sodium diisooctyl sulfosuccinate, guanidine isothiocyanate, guanidine hydrochloride, SDS, MES, and PEG; the magnetic beads are nano-magnetic particles with a polystyrene core, a nano-ferroferric oxide sandwich, a silica or cellulose coating, wrapped with PEG, and then carboxylated; the washing solution includes Tris-Hcl, cetyltrimethylammonium bromide, ASEA, FMES, etc.; the pH of the eluent should not be lower than 8.0 and includes EDTA and Tris-Hcl.

[0028] The pH value of the lysis binding solution is 5.7 - 7.9, and it includes sodium diisooctyl sulfosuccinate at 1.2 - 4.8 M, guanidine isothiocyanate at 1.5 - 2.6 mM, guanidine hydrochloride at 2.3 - 3.8 mM, SDS at 1.3 - 3.4 mM, MES at 1% - 3.6%, and PEG at 10% - 20%. The PEG selected for the lysis binding solution should have a molecular weight greater than 2000, and PEG6000 is preferred.

[0029] The nano magnetic beads have a multi-layer sandwich structure. The core is polystyrene, the outermost layer is PEG with carboxyl modification, the diameter is 400 - 2000 nm, the concentration is 30 - 80 mg / ml, and the ZETA potential should not be higher than -30, preferably -45 to -60.

[0030] The washing solution includes cetyltrimethylammonium bromide at 80 - 160 mM, Tris-Hcl at 20 - 38 mM with a pH value of 7.5 - 8.5, ASEA at 5% - 9%, and FMES at 10 - 50 mM.

[0031] The base solution of the nano magnetic beads contains PEG at 15% - 30%, Tween 20 at 1% - 10%, etc. The molecular weight of PEG in the base solution of the nano magnetic beads should not be less than 4000. The surface tension of the base solution of the nano magnetic beads should not be less than 70 dynes / cm.

[0032] The kit includes the following operating steps:

[0033] Step S1: Take 500 - 1200 ul of fresh plasma sample in a 5 ml or 10 ml centrifuge tube, add 900 - 2500 ul of lysis binding solution using a sterile pipette, and incubate and mix well at 50 - 90 °C for 10 - 30 min with shaking;

[0034] Step S2: Add 10 - 50 ul of nano magnetic beads using a sterile pipette, and mix well by shaking for 5 - 15 min;

[0035] Step S3: Place the centrifuge tube in step S2 on a magnetic rack for 50 s. After the nano magnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant solution;

[0036] Step S4: Add 500 - 800 ul of washing solution to the centrifuge tube in step S3 using a sterile pipette, mix the centrifuge tube well by shaking for 2 min, place it on a magnetic rack for 50 s. After the nano magnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant solution;

[0037] Step S5: Use a sterile pipette to add 30 - 50 ul of elution buffer to the centrifuge tube in Step S4, incubate at 65°C with shaking for 3 - 5 min, then place it on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate the supernatant for cfDNA detection.

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention.

[0039] Example 1

[0040] A cfDNA extraction kit for prenatal diagnosis includes the following specific steps:

[0041] Step S1: Take 600 ul of fresh plasma sample in a 5 ml centrifuge tube, use a sterile pipette to add 900 ul of lysis and binding solution, and incubate at 70°C with shaking for 15 min;

[0042] Step S2: Use a sterile pipette to add 25 ul of nanomagnetic beads and mix well by shaking for 5 min;

[0043] Step S3: Place the centrifuge tube in Step S2 on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant;

[0044] Step S4: Use a sterile pipette to add 550 ul of washing solution to the centrifuge tube in Step S3, mix the centrifuge tube well by shaking for 2 min, place it on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant;

[0045] Step S5: Use a sterile pipette to add 550 ul of washing solution to the centrifuge tube in Step S4, mix the centrifuge tube well by shaking for 2 min, place it on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant;

[0046] Step S6: Use a sterile pipette to add 30 ul of elution buffer to the centrifuge tube in Step S4, incubate at 65°C with shaking for 5 min, then place it on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate the supernatant for cfDNA detection.

[0047] Example 2

[0048] A cfDNA extraction kit for prenatal diagnosis includes the following specific steps:

[0049] Step S1: Take 800 ul of fresh plasma sample in a 5 ml centrifuge tube, use a sterile pipette to add 2500 ul of lysis and binding solution, and incubate at 65°C with shaking for 20 min;

[0050] Step S2: Add 40 μl of nanomagnetic beads using a sterile pipette, and mix well by shaking for 10 min;

[0051] Step S3: Place the centrifuge tube in Step S2 on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant solution;

[0052] Step S4: Add 800 μl of washing solution to the centrifuge tube in Step S3 using a sterile pipette. Mix the centrifuge tube well by shaking for 2 min, place it on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant solution;

[0053] Step S5: Add 50 μl of elution solution to the centrifuge tube in Step S4 using a sterile pipette. Incubate at 65 °C with shaking and mixing for 5 min, then place it on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate the supernatant solution for cfDNA detection.

[0054] In the present invention, the control reagent selects QIAseq cfDNA All-in-One Kits to extract 1000 μl of fresh plasma sample, and the elution volume is 50 μl. The results are as Figure 1 and Figure 2 shown.

[0055] Example 3

[0056] A cfDNA extraction kit that can be used for prenatal diagnosis, comprising the following specific steps:

[0057] Step S1: Take 1000 μl of fresh plasma sample in a 5 ml centrifuge tube, add 2000 μl of lysis-binding solution using a sterile pipette, and incubate in a water bath at 65 °C for 20 min;

[0058] Step S2: Add 50 μl of nanomagnetic beads using a sterile pipette, and mix well by shaking for 10 min;

[0059] Step S3: Place the centrifuge tube in Step S2 on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant solution;

[0060] Step S4: Add 500 μl of washing solution to the centrifuge tube in Step S3 using a sterile pipette. Mix the centrifuge tube well by shaking for 2 min, place it on a magnetic stand for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate and discard the supernatant solution;

[0061] Step S5: Use a sterile pipette to add 40 μl of elution buffer to the centrifuge tube in Step S4, incubate at 65°C with shaking for 3 min, then place the centrifuge tube on a magnetic rack for 50 s. After the nanomagnetic beads are completely adsorbed to the tube wall, use a sterile pipette to aspirate the supernatant into a new centrifuge tube for cfDNA detection.

[0062] In the present invention, the control reagent is selected as QIAseq cfDNA All-in-One Kits, and the elution volume is 40 μl. The results are shown in Table 1.

[0063] After performing cfDNA detection on Example 2 and the control reagent respectively, the results are as Figure 1 compared with Figure 2 shown.

[0064] Figure 1 compared with Figure 2 are respectively the schematic diagrams of cfDNA extraction performed on Example 2 of the present invention and QIAseq cfDNA All-in-One Kits, detected by Agilent 2100 electrophoresis. Among them, the 35 bp and 10380 bp are the internal standard indicator bands of the detection reagent, indicating normal electrophoresis detection. The 169 bp ( Figure 2 is 168 bp in

[0065] as Figure 1 compared with Figure 2 shown. Using the cfDNA extraction kit based on multi-layer sandwich carboxyl-modified PEG nanomagnetic beads of the present invention, the extracted cfDNA and the cfDNA obtained from QIAseq cfDNA All-in-One Kits are detected using Agilent 2100. The results show that there is no obvious difference in fragment size between the two, and cfDNA is obtained in both cases. In addition to the cfDNA band at 169 bp, the cfDNA obtained by the present invention has no other miscellaneous peaks, which is significantly better than the control reagent.

[0066] After the extraction of Example 3 and the control reagent is completed, 10 μl of the obtained cfDNA solution is taken respectively, and the concentration is detected using Qubit3.0 of ThermoFisher Scientific. The results are shown in Table 1.

[0067] Table 1: Detection results of cfDNA solution concentration.

[0068] Number 1 2 3 4 5 6 Sample ID Example 3 Example 3 Example 3 Control reagent Control reagent Control reagent Concentration (ng / ul) 0.163 0.158 0.167 0.141 0.152 0.149 Total amount (ng) 6.52 6.32 6.68 5.64 6.08 5.96

[0069] As shown in Table 1, compared with QIAseq cfDNA All-in-One Kits, the cfDNA obtained by the present invention is superior to the control kit in terms of concentration and total amount. The cfDNA obtained by the kit of the present invention is more, and the results are more stable, which is suitable for sample processing of prenatal diagnosis.

[0070] It should be noted that the above-described embodiments should be understood as illustrative and not limiting the scope of protection of the present invention. The scope of protection of the present invention is subject to the claims. For those skilled in the art, without departing from the essence and scope of the present invention, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention.

Claims

1. A cfDNA extraction kit for prenatal diagnosis, characterized in that: It includes a lysis binding solution, nano magnetic beads, a washing solution, and an elution solution; The pH value of the lysis binding solution is 5.7 to 7.9, and it is composed of 1.2 to 4.8 M sodium diisooctyl sulfosuccinate, 1.5 to 2.6 mM guanidine isothiocyanate, 2.3 to 3.8 mM guanidine hydrochloride, 1.3 to 3.4 mM SDS, 1% to 3.6% MES, and 10% to 20% PEG; The nano magnetic beads are nano magnetic particles with a polystyrene core, a nano ferric oxide sandwich, a silica or cellulose coating, wrapped with PEG, and then carboxylated; the nano magnetic beads are a multi-layer sandwich structure, with a diameter of 400 to 2000 nm and a concentration of 30 to 80 mg / ml; The washing solution is composed of 80 to 160 mM cetyltrimethylammonium bromide, 20 to 38 mM Tris-Hcl with a pH value of 7.5 to 8.5, 5% to 9% ASEA, and 10 to 50 mM FMES; The pH of the elution solution is not lower than 8.0, and it includes EDTA and Tris-Hcl.

2. The cfDNA extraction kit for prenatal diagnosis according to claim 1, wherein: The PEG with a molecular weight greater than 2000 is selected in the lysis binding solution.

3. The cfDNA extraction kit for prenatal diagnosis according to claim 1, wherein: The nano magnetic beads are prepared by the following method: a: Select 100 g of polystyrene microspheres with a particle size of 400 - 700 nm and disperse them in 1 L of anhydrous ethanol solution; b: Add 400 ml of a mixed salt solution of Fe 3+ and Fe 2+ prepared according to a molar ratio of 3:2 and with a concentration of 4 mol / L. Pass an inert gas to remove oxygen for 30 min, adjust the system temperature to 60 - 65 °C, add ammonia water until the pH value of the system is 10, react for 2 h, add 50 ml of a mixture of oleic acid and lauric acid premixed according to a volume ratio of 3:2, continue to react for 30 min, then quickly cool to 30 °C. After magnetic separation, repeatedly wash the precipitate with 50% ethanol and deionized water several times until the washing liquid is neutral, and then disperse it in 800 ml of anhydrous ethanol; c: Add 200 ml of tetraethyl orthosilicate and 200 ml of ammonia water or 260 ml of carboxymethyl cellulose and 260 ml of ammonia water, react for 1 h, then perform magnetic separation and repeatedly wash the precipitate with 30% ethanol and deionized water several times until the washing liquid is neutral, and finally disperse it in 800 ml of 30% ethanol solution; d: Add 200 ml of 20% PEG6000 solution, add ammonia water until the pH value of the system is 10, incubate at 80 °C for 2 h, add 100 ml of citric acid solution, continue to react for 30 min, then perform magnetic separation and repeatedly wash the precipitate with 30% ethanol and deionized water several times until the washing liquid is neutral, and finally disperse it in 2 L of a magnetic bead base solution containing PEG and Tween.

4. The cfDNA extraction kit for prenatal diagnosis according to claim 1 or 3, characterized in that: The ZETA potential of the nano magnetic beads is not greater than -30.

5. A cfDNA extraction kit for prenatal diagnosis according to claim 4, characterized in that: The base solution of the nano magnetic beads contains 15% to 30% PEG and 1% to 10% Tween 20; the molecular weight of PEG in the base solution of the nano magnetic beads is not less than 4000; the surface tension of the base solution of the nano magnetic beads is not less than 70 dynes / cm.

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