A kit and method for extracting free DNA from plasma

Abstract

The present invention provides a kit for extracting plasma free DNA, comprising a plurality of extraction solutions, at least one of which contains 0.01-0.5 mmol / L of glycogen and 0.5%-1.5% of cetyl polydimethylsiloxane in a volume / volume ratio. Conventionally, a phenol / chloroform extraction kit is required for extracting plasma free DNA, and most of them use phenol, chloroform and isoamyl alcohol to denature proteins to make them precipitate, and to separate the liquids, and then use ethanol or the like to precipitate and centrifuge to achieve the purpose of extraction, but the operation is cumbersome. The present invention uses glycogen and cetyl polydimethylsiloxane to directly achieve cleavage and binding simultaneously through the extraction solution, without the need to add a binding solution separately, and has the advantages of simple operation steps, shortened extraction time, and improved nucleic acid extraction efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of molecular diagnostic DNA / RNA extraction, and particularly relates to a kit for extracting cell-free DNA from a large volume of plasma by a magnetic bead method and a method for extracting cell-free DNA from plasma. Background Art

[0002] Cell-free DNA, abbreviated as cfDNA (circulating free DNA), refers to nucleic acid substances in circulating blood. It includes nucleic acids in cells in circulating blood, free endogenous deoxyribonucleic acid, and exogenous DNA and RNA, such as exogenous nucleic acids in blood under pathological conditions such as fungemia, bacteremia, and viremia. CfDNA has important potential value in the early diagnosis, prognosis, and monitoring of diseases, and has great application value in the analysis of the condition and observation of the curative effect of non-tumor diseases such as prenatal diagnosis and immune diseases, as well as in tumor-related analysis. Due to the lack of experimental methods with high sensitivity and high specificity, the research on the correlation between cfDNA and diseases has progressed slowly for a long time. It was not until the emergence of effective cfDNA separation technologies and the application of detection technologies combining special fluorescent dyes and PCR technologies that the research in this field has developed relatively rapidly in the past twenty-odd years.

[0003] With the proposal of the concept of precision medicine, precision medicine has gradually been accepted by people, and tumors are one of the primary tasks to be solved by precision medicine. Liquid biopsy, as a branch of in vitro diagnosis, is a method for monitoring circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) fragments released into the blood by tumors or metastatic lesions through a non-invasive blood test. Recently, the relatively popular liquid biopsies include the detection of cfDNA, ctDNA, and CTCs. The detection of cfDNA has become a sensitive biomarker for cancer diagnosis and monitoring, and the extraction of cfDNA has received increasing attention.

[0004] Currently, the solutions in the free DNA extraction and enrichment kits on the market are designed accordingly based on different extraction methods. For example, in the kits based on phenol / chloroform extraction, most of them use phenol, chloroform, and isoamyl alcohol to denature proteins so that they settle, and make the liquid layer, and then precipitate and centrifuge with ethanol, etc. to achieve the extraction purpose. However, the above organic solvents are somewhat toxic, which is not conducive to the operation of technicians, and the loss rate of cfDNA during the extraction process reaches 70% - 80%. The centrifugal column method and the magnetic bead method mostly use the principle of high salt and low pH to prepare the binding solution, so that nucleic acids bind to the silica gel membrane or magnetic beads of the centrifugal column under this condition; then use the condition of low salt and high pH to prepare the elution solution for easy nucleic acid elution. Although the centrifugal column method is convenient and fast to operate and can effectively remove various impurities in the sample, it has a high cost, requires multiple high-speed centrifugations, needs to replace centrifugal tubes, and is not suitable for large-scale clinical sample detection without high-throughput instrument support. The magnetic bead method is a perfect combination of nanotechnology and biotechnology, with advantages that cannot be compared by other DNA extraction methods, mainly reflected in: it can achieve automated and high-throughput operations; the operation is simple and time-consuming; the magnetic bead method has the highest extraction efficiency for plasma-free DNA, the smallest loss rate, and good repeatability. This kit further improves the method of separating cfDNA by magnetic beads, simplifies the experimental operation steps, is simple and fast to operate, obtains high-purity DNA, and is suitable for the extraction of small-fragment DNA. Summary of the Invention

[0005] The present invention provides a kit and method for extracting plasma-free DNA to simplify the extraction process and improve the extraction efficiency.

[0006] The present invention provides a kit for extracting plasma-free DNA, including a variety of extraction solutions, and at least one of the extraction solutions contains glycogen and cetyl polydimethylsiloxane.

[0007] Furthermore, the at least one extraction solution contains 0.01 - 0.5 mmol / L of glycogen and cetyl polydimethylsiloxane with a volume / volume ratio of 0.5% - 1.5%; preferably, the volume / volume ratio of cetyl polydimethylsiloxane is 0.8% - 1.2%; more preferably, the volume / volume ratio of cetyl polydimethylsiloxane is 1%. For example, in the present invention, extraction solutions with a volume / volume ratio of cetyl polydimethylsiloxane of 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5% can all efficiently extract plasma-free DNA.

[0008] Further, the multiple extraction solutions include a first extraction solution, which includes 3 - 4 mol / L guanidine thiocyanate, 0.01 - 0.5 mmol / L glycogen, 1% - 3% (volume / volume) ethyl phenyl polyethylene glycol, 0.25% - 1.0% (volume / volume) Tween 20, 0.5% - 1.5% (volume / volume) cetyl polydimethylsiloxane, 0.5% - 1.5% (mass / volume) Triton X-100, 30% - 45% (volume / volume) isopropanol, and 5 - 15 mmol / L Tris-HCl.

[0009] Even further, the multiple extraction solutions further include a second extraction solution, which includes 0.2 - 1.0 mg / ml magnetic beads, 5 - 15 mmol / L Tris-HCl, and 0.5 - 1.5 mmol / L EDTA.

[0010] Even further, the multiple extraction solutions further include a third extraction solution, which includes 10 - 30 mg / ml proteinase K.

[0011] Even further, the extraction kit further includes a washing solution, which at least includes a first washing solution. The first washing solution includes: 1 - 2 mol / L guanidine thiocyanate, 100 - 200 mmol / ml NaAc-HAc, 10 - 50 mmol / L Tris-HCl, 0.5% - 5% (volume / volume) ethyl phenyl polyethylene glycol, 1% - 5% (volume / volume) Tween 20, 1% - 5% (mass / volume) Triton X-100, and 10% - 30% (volume / volume) isopropanol.

[0012] Even further, the washing solution further includes a second washing solution, which includes: ethanol and 5 - 15 mmol / L Tris-HCl.

[0013] Even further, the extraction kit further includes an elution solution, which includes 5 - 15 mmol / L Tris-HCl, 0.5 - 1.5 mmol / L EDTA, and 0.02% - 0.1% Tween 20.

[0014] The second aspect of the present invention discloses a method for using the extraction kit as described in any one of the above, including an extraction process, and the extraction process is: mixing 1 volume of the sample with a total of 1.5 - 3 volumes of the multiple extraction solutions and performing extraction;

[0015] Among them, the extraction solution containing glycogen and cetyl polydimethylsiloxane accounts for 80% - 95% of the total volume of the multiple extraction solutions.

[0016] The third aspect of the present invention discloses a method for using any of the above extraction kits, including the following steps:

[0017] S1. Mix 1 volume of sample, 1.2 - 2.5 volumes of the first extraction solution, 0.05 - 0.25 volumes of the second extraction solution, and 0.05 - 0.25 volumes of the third extraction solution; shake and heat.

[0018] S2. Let it stand at room temperature and then centrifuge, perform magnetic separation, and remove the waste liquid.

[0019] S3. Add the washing solution for washing.

[0020] S4. Let it stand at room temperature.

[0021] S5. Add 0.05 - 1 volume of the elution solution, shake and mix well, elute the magnetic beads, and after centrifugation, magnetically attract to obtain the eluted nucleic acid, and obtain the extracted nucleic acid.

[0022] Further, the washing solution includes the first washing solution and the second washing solution, and step S3. adding the washing solution for washing includes:

[0023] S31. Add 0.5 - 2 volumes of the first washing solution, shake and mix well, and after centrifugation, magnetically attract to remove the liquid.

[0024] S32. Add 0.5 - 2 volumes of the second washing solution, shake and mix well, and after centrifugation, magnetically attract to remove the liquid.

[0025] S33. Add 0.2 - 1 volume of the second washing solution, shake and mix well, and after centrifugation, magnetically attract to remove the liquid.

[0026] Compared with the conventional free DNA extraction method, by using glycogen and cetyl polydimethylsiloxane, the present invention can directly achieve lysis and binding simultaneously through the extraction solution without the need to separately add a binding solution, and has the advantages of simple operation steps, shortened extraction time, and improved nucleic acid extraction efficiency. Description of the Drawings

[0027] Figure 1 It is the capillary electrophoresis detection result diagram of Example 1 of the present invention using LabChip GX.

[0028] Figure 2 It is the PCR amplification result diagram of the ACTB normal internal standard amplification reagent in Example 1 of the present invention.

[0029] Figure 3 It is the capillary electrophoresis detection result diagram of the DNA Ladder containing DNA fragments of different sizes extracted in Example 1 of the present invention using LabChip GX.

[0030] Figure 4 This is the result graph of fluorescence quantification using the ACTB amplification reagent for Example 1 of the present invention and Comparative Examples 1 and 2. Detailed implementation manners

[0031] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0032] Currently, most of the kits for extracting cell-free DNA are imported from abroad. Although they have good extraction effects, they are expensive, bringing a heavy economic burden to consumers. Moreover, after most commercial kits lyse the samples, they need to be cooled to room temperature, then add the binding solution and the magnetic bead suspension and mix well, and stand for 5 - 10 minutes before washing and eluting. The cumbersome steps result in low automation and poor instrument universality. Therefore, there is an urgent need to develop a magnetic bead-based cell-free DNA extraction kit that is economical, efficient, stable and easy to operate.

[0033] The embodiment of the present invention discloses a kit for extracting plasma cell-free DNA, including a variety of extraction solutions, and at least one of the extraction solutions contains 0.01 - 0.5 mmol / L of glycogen and cetyl polydimethylsiloxane with a volume / volume ratio of 0.5% - 1.5%.

[0034] Optionally, among the variety of extraction solutions, there is a first extraction solution, and the first extraction solution includes 3 - 4 mol / L of guanidine thiocyanate, 0.01 - 0.5 mmol / L of glycogen, ethyl phenyl polyethylene glycol with a volume / volume ratio of 1% - 3%, Tween 20 with a volume / volume ratio of 0.25% - 1.0%, cetyl polydimethylsiloxane with a volume / volume ratio of 0.5% - 1.5%, Triton X-100 with a mass / volume ratio of 0.5% - 1.5%, isopropanol with a volume / volume ratio of 30% - 45%, and 5 - 15 mmol / L of Tris-HCl.

[0035] Among them, the kit of the embodiment of the present invention adds cetyl polydimethylsiloxane to the first nucleic acid extraction solution, realizing large-volume extraction. When specifically extracting plasma cell-free DNA subsequently, the volume of the sample can reach 2 mL.

[0036] The embodiment of the present invention uses glycogen and cetyl polydimethylsiloxane in the first extraction solution, improving the concentration of cell-free DNA obtained during the plasma extraction process and meeting the requirements of the next-step PCR amplification reagent; and the nucleic acid extracted using the extraction solution of the embodiment of the present invention has good amplification effects in PCR reactions.

[0037] Specifically, the multiple extraction solutions further include a second extraction solution, and the second extraction solution includes 0.2 - 1.0 mg / ml magnetic beads, 5 - 15 mmol / L Tris-HCl, and 0.5 - 1.5 mmol / L EDTA.

[0038] Specifically, the multiple extraction solutions further include a third extraction solution, and the third extraction solution includes 10 - 30 mg / ml proteinase K.

[0039] Specifically, the extraction kit further includes a washing solution, and the washing solution at least includes a first washing solution, and the first washing solution includes: 1 - 2 mol / L guanidine thiocyanate, 100 - 200 mmol / ml NaAc-HAc, 10 - 50 mmol / L Tris-HCl, 0.5% - 5% (v / v) ethyl phenyl polyethylene glycol, 1% - 5% (v / v) Tween 20, 1% - 5% (w / v) Triton X-100, 10% - 30% (v / v) isopropanol.

[0040] Specifically, the washing solution further includes a second washing solution, and the second washing solution includes: 75% ethanol, 5 - 15 mmol / L Tris-HCl.

[0041] Specifically, the extraction kit further includes an elution solution, and the elution solution includes 5 - 15 mmol / L Tris-HCl, 0.5 - 1.5 mmol / L EDTA, and 0.02% - 0.1% Tween 20.

[0042] Among them, the components of the first washing solution, the second washing solution, and the elution solution are not limited to the above components, and the staff can replace the appropriate washing solution and elution solution according to the reagent requirements.

[0043] The second aspect of the present invention discloses a method for using any one of the above extraction kits, including an extraction process, and the extraction process is: mixing 1 volume of the sample with a total of 1.5 - 3 volumes of multiple extraction solutions and performing extraction;

[0044] Among them, the extraction solution containing glycogen and cetyl dimethyl silicone accounts for 80% - 95% of the total volume of the multiple extraction solutions.

[0045] The third aspect of the present invention discloses a method for using any one of the above extraction kits, including the following steps:

[0046] S1. Mix 1 volume of the sample, 1.2 - 2.5 volumes of the first extraction solution, 0.05 - 0.25 volumes of the second extraction solution, and 0.05 - 0.25 volumes of the third extraction solution; shake and heat;

[0047] Among them, the volume of the sample can reach 2 mL or more per unit volume;

[0048] S2. After standing at room temperature and then centrifuging, magnetic separation is carried out, and the waste liquid is removed;

[0049] S3. Add washing solution for washing;

[0050] S4. Stand at room temperature;

[0051] S5. Add 0.05 - 1 volume of elution solution, shake and mix evenly, then elute the magnetic beads, and after centrifugation, perform magnetic absorption to obtain the eluted nucleic acid, and obtain the extracted nucleic acid.

[0052] Among them, starting from simplifying the operation steps, the kit of the embodiment of the present invention adopts the method of performing lysis and binding simultaneously for nucleic acid extraction, reducing the extraction steps. It can not only be manually extracted, but also be paired with semi-automatic nucleic acid extraction instruments and fully automatic extraction to achieve automated and high-throughput operations; the operation is simple and the time used is short.

[0053] Optionally, the washing solution includes a first washing solution and a second washing solution, and the step S3. adding the washing solution for washing includes:

[0054] S31. Add 0.5 - 2 volumes of the first washing solution, shake and mix evenly, and after centrifugation, perform magnetic absorption to remove the liquid;

[0055] S32. Add 0.5 - 2 volumes of the second washing solution, shake and mix evenly, and after centrifugation, perform magnetic absorption to remove the liquid;

[0056] S33. Add 0.2 - 1 volume of the second washing solution, shake and mix evenly, and after centrifugation, perform magnetic absorption to remove the liquid.

[0057] To elaborate on the beneficial effects of the embodiments of the present invention in detail, specific embodiments are described as follows:

[0058] Example 1

[0059] The component composition of the nucleic acid extraction kit of Example 1 of the present invention is as follows:

[0060] The first extraction solution includes: 4 mol / L guanidine thiocyanate, 0.1 mmol / L glycogen, 2% (v / v) ethyl phenyl polyethylene glycol (NP-40), 0.5% (v / v) Tween 20, 1% (v / v) cetyl polydimethylsiloxane, 1% (m / v) Triton X-100, 38% (v / v) isopropanol, 10 mmol / L Tris-HCl (pH 6.0).

[0061] The second extraction solution includes: 0.5 mg / ml magnetic beads, 10 mmol / L Tris-HCl (pH 8.0), 1 mmol / L EDTA (pH 8.0).

[0062] The third extraction solution includes: 20 mg / ml proteinase K.

[0063] The first washing solution includes: 1 mol / L guanidine thiocyanate, 200 mmol / ml NaAc-HAc, 10 mmol / L Tris-HCl, 0.5% (v / v) nonylphenoxypolyethoxylethanol (NP-40), 1.5% (v / v) Tween 20, 1% (m / v) Triton X-100, 18% (v / v) isopropanol.

[0064] The second washing solution includes: 75% ethanol, 10 mmol / L Tris-HCl (pH 7.5).

[0065] The elution solution includes: 10 mmol / L Tris-HCl (pH 8.0), 1 mmol / L EDTA (pH 8.0), 0.05% Tween 20.

[0066] Comparative Example 1

[0067] Compared with Example 1 of the present invention, only the components of the first extraction solution are different, specifically as follows:

[0068] The first extraction solution of Comparative Example 1 of the present invention contains: 4 mol / L guanidine thiocyanate, 0.1 mmol / L glycogen, 2% (v / v) nonylphenoxypolyethoxylethanol (NP-40), 0.5% (v / v) Tween 20, 1% (m / v) Triton X-100, 38% (v / v) isopropanol, 10 mmol / L Tris-HCl (pH 6.0).

[0069] Comparative Example 2

[0070] Compared with Example 1 of the present invention, only the components of the first extraction solution are different, specifically as follows:

[0071] The first extraction solution of Comparative Example 2 of the present invention contains: 4 mol / L guanidine thiocyanate, 2% (v / v) nonylphenoxypolyethoxylethanol (NP-40), 0.5% (v / v) Tween 20, 1% (m / v) Triton X-100, 38% (v / v) isopropanol, 10 mmol / L Tris-HCl (pH 6.0).

[0072] In Example 1 of the present invention, Comparative Example 1, and Comparative Example 2, the same operating steps were used for extracting large-volume plasma-free DNA, specifically including:

[0073] 1. Nucleic acid extraction operation process

[0074] Step 1. Take a number of 10 mL centrifuge tubes according to the number of test samples, and add 2 mL of sample to each tube;

[0075] Step 2. Add 3 mL of the first extraction solution, 100 μL of the second extraction solution, and 200 μL of the third extraction solution; cover the tube cap, shake and mix well for 30 s, and heat at 60 °C for 15 min.

[0076] Step 3. Let it stand at room temperature for 5 min, perform low-speed instantaneous centrifugation, place the centrifuge tube on the magnetic separator, and slowly aspirate and discard the waste liquid after 2 min (*note not to touch the magnetic beads adsorbed on the inner wall of the tube);

[0077] Step 4. Add 2 mL of the first washing solution, shake and mix well for 30 s, after low-speed instantaneous centrifugation, place the centrifuge tube on the magnetic separator again. Magnetically attract for 2 min, and completely aspirate and discard the liquid (*note not to touch the magnetic beads adsorbed on the inner wall of the tube);

[0078] Step 5. Add 2 mL of the second washing solution, shake and mix well for 30 s, after low-speed instantaneous centrifugation, place the centrifuge tube on the magnetic separator again. Magnetically attract for 2 min, and completely aspirate and discard the liquid. (*note not to touch the magnetic beads adsorbed on the inner wall of the tube);

[0079] Step 6. Add 1 mL of the third washing solution, shake and mix well for 30 s, after low-speed instantaneous centrifugation, place the centrifuge tube on the magnetic separator again. Magnetically attract for 3 min, completely aspirate and discard the liquid, and continue to stand on the magnetic separator for 2 min to completely aspirate the liquid. (*note not to touch the magnetic beads adsorbed on the inner wall of the tube);

[0080] Step 7. Open the tube cap and let it stand at room temperature for 10 min.

[0081] Step 8. Add 100 μL of the elution solution, shake and mix well for 10 s, elute the magnetic beads on the centrifuge tube wall to the bottom of the tube; perform low-speed instantaneous centrifugation, place the centrifuge tube on the magnetic separator again and magnetically attract for 30 s, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0082] 2. Nucleic acid detection

[0083] After the nucleic acid extraction is completed, capillary electrophoresis is performed using LabChip GX, or fluorescence quantitative PCR is performed on the nucleic acids obtained in Example 1 of the present invention, Comparative Example 1, and Comparative Example 2 using ACTB normal human internal standard amplification reagents.

[0084] The detection results of extracting plasma-free DNA in Example 1 of the present invention are as follows Figure 1 shown:

[0085] After extracting the plasma sample of adult peripheral blood according to the steps described in Example 1, capillary electrophoresis was performed using LabChip GX, and the detection results are as follows:

[0086] From Figure 1 the results, it can be seen that the size of the plasma-free DNA fragments extracted by the extraction reagent is concentrated around 160 bp, which is consistent with the size of the target fragment.

[0087] The plasma-free DNA extracted by the reagent in Example 1 was used for PCR amplification using the ACTB normal human internal standard amplification reagent. As Figure 2 shown, the curve is straight and concentrated, indicating that the extracted nucleic acid can be used for downstream PCR detection.

[0088] The DNA Ladder containing DNA fragments of different sizes was extracted according to the experimental scheme of Example 1 and detected by capillary electrophoresis using LabChip GX. The results are as Figure 3 shown. From the results in the figure, it can be seen that the extraction reagent can extract nucleic acids with fragment sizes ranging from 50 to 1000 bp.

[0089] From Figures 1-3 it can be seen that the extraction kit and method for plasma-free DNA in the embodiments of the present invention can effectively extract plasma-free DNA, and the size of the extracted plasma-free DNA is about 166 bp. In addition, the method provided in Example 1 of the present invention can extract DNA fragments of different sizes. Currently, it can be seen that the method of the embodiments of the present invention can extract DNA as small as 50 bp.

[0090] 3. Comparison of the test results of Example 1, Comparative Example 1, and Comparative Example 2 of the present invention

[0091] 3.1 Content and purity of DNA in the eluate

[0092] The extraction reagents in Example 1 and Comparative Examples 1-2 were used to extract the plasma samples of adult peripheral blood. After extraction, a micro ultraviolet spectrophotometer was used to measure the content and purity of DNA in the eluate as shown in the following table.

[0093] Extraction reagent OD260 / 280 OD260 / 280 Concentration (ng / μL) Example 1 1.82 1.36 3.08 Comparative Example 1 1.86 1.03 1.25 Comparative Example 2 1.79 0.93 0.62

[0094] As can be seen from the above table, the method for extracting free DNA from plasma provided by the present invention has a large extraction amount and high purity of free DNA; moreover, the concentration of free DNA extracted by the methods for extracting free DNA from serum plasma provided in Comparative Examples 1 and 2 has dropped significantly, and the purity of Comparative Example 2 is also worse than that of Example 1.

[0095] 3.2. PCR Amplification Test Results

[0096] After extraction, the extracted nucleic acid was used as a template, and a fluorescence quantitative PCR was performed using the ACTB amplification reagent for detection. The test results are as Figure 4 shown in the following table:

[0097]

[0098]

[0099] It can be seen that the first extraction solution of Example 1 of the present invention contains glycogen and cetyl polydimethylsiloxane; the first extraction solution of Comparative Example 1 contains only glycogen and does not contain cetyl polydimethylsiloxane; the first extraction solution of Comparative Example 2 does not contain glycogen and cetyl polydimethylsiloxane; during the test, the first extraction solution of Example 1 has the best extraction effect on plasma-free DNA, followed by the first extraction solution of Comparative Example 1. By adopting the two components of glycogen and cetyl polydimethylsiloxane, Example 1 of the present invention has an obvious extraction effect on plasma-free DNA, and the addition of glycogen and cetyl polydimethylsiloxane significantly increases the concentration of the extracted plasma-free DNA.

[0100] In summary, compared with the prior art, the embodiments of the present invention have the following advantages:

[0101] 1. The nucleic acid extraction is carried out by a method of simultaneous lysis and binding, reducing the extraction steps;

[0102] 2. When extracting plasma-free DNA from plasma samples, the volume of the extracted sample can reach 2 ml, increasing the concentration of the extracted plasma-free DNA and meeting the requirements of the next-step PCR amplification reagent;

[0103] 3. By adopting the two components of glycogen and cetyl polydimethylsiloxane, the extraction effect on plasma-free DNA is obvious, and the addition of glycogen and cetyl polydimethylsiloxane significantly increases the concentration of the extracted plasma-free DNA.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that after reading the specification of this application, they can still modify the specific implementation manners of the present invention or make equivalent replacements, but these modifications or changes do not depart from the protection scope of the pending claims of the present invention application.

Claims

1. A kit for extracting plasma free DNA, characterized in that: The extraction kit includes a plurality of extraction solutions, wherein the plurality of extraction solutions include a first extraction solution, a second extraction solution and a third extraction solution; The first extraction solution comprises 3-4 mol / L guanidine thiocyanate, 0.01-0.5 mmol / L glycogen, 1%-3% ethylphenyl polyethylene glycol in a volume / volume ratio, 0.25%-1.0% Tween 20 in a volume / volume ratio, 0.5%-1.5% cetyl polydimethylsiloxane in a volume / volume ratio, 0.5%-1.5% Triton X-100 in a mass / volume ratio, 30%-45% isopropanol in a volume / volume ratio, and 5-15 mmol / L Tris-HCl; The second extraction solution includes 0.2-1.0 mg / ml magnetic beads, 5-15 mmol / L Tris-HCl, and 0.5-1.5 mmol / L EDTA; The third extraction solution includes 10-30 mg / ml of proteinase K.

2. A kit for extracting plasma free DNA according to claim 1, characterized in that: The extraction kit also includes a washing solution.

3. A kit for extracting plasma free DNA according to claim 2, characterized in that: The washing solution includes a first washing solution, which includes 1-2 mol / L guanidine thiocyanate, 100-200 mmol / ml NaAc-HAc, 10-50 mmol / L Tris-HCl, 0.5%-5% ethylphenyl polyethylene glycol in a volume / volume ratio, 1%-5% Tween 20 in a volume / volume ratio, 1%-5% Triton X-100 in a mass / volume ratio, and 10%-30% isopropanol in a volume / volume ratio; and / or, The second washing solution comprises ethanol and 5-15 mmol / L Tris-HCl.

4. A kit for extracting plasma free DNA according to any one of claims 2-3, characterized in that: The extraction kit also includes an eluent, which includes 5-15 mmol / L Tris-HCl, 0.5-1.5 mmol / L EDTA, and 0.02%-0.1% Tween 20.

5. A method for using the kit for extracting plasma free DNA according to any one of claims 1 to 4 for non-diagnostic purposes, characterized in that: The method comprises an extraction process, wherein: 1 volume of sample is mixed with a total volume of 1.5 to 3 volumes of a plurality of extraction solutions, and extraction is performed; The extraction solution containing glycogen and cetyl polydimethylsiloxane accounts for 80% to 95% of the total volume of the multiple extraction solutions.

6. A method for using the kit for extracting plasma free DNA according to claim 5 for non-diagnostic purposes, characterized in that: The steps include: S1. Mix 1 volume of sample, 1.2-2.5 volumes of the first extraction solution, 0.05-0.25 volumes of the second extraction solution, and 0.05-0.25 volumes of the third extraction solution; shake and heat; S2. After standing at room temperature, centrifuge, perform magnetic separation, and remove waste liquid; S3. Add washing solution for washing; S4. Let stand at room temperature; S5. Add 0.05 to 1 volume of elution buffer, shake and mix, elute the magnetic beads, centrifuge, and magnetically absorb to obtain the eluted nucleic acid to obtain the extracted nucleic acid.

7. The method for using the kit for extracting plasma free DNA for non-diagnostic purposes according to claim 6, characterized in that: The washing liquid includes a first washing liquid and a second washing liquid, and the step S3 of adding the washing liquid for washing includes: S31. Add 0.5 to 2 volumes of the first washing solution, shake and mix, centrifuge, and remove the liquid by magnetic suction; S32. Add 0.5 to 2 volumes of the second washing solution, shake and mix, centrifuge, and remove the liquid by magnetic suction; S33. Add 0.2 to 1 volume of the second washing solution, shake to mix, centrifuge, and remove the liquid by magnetic suction.

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