Extraction reagent, kit and extraction method for free DNA (Deoxyribose Nucleic Acid) of plasma

Abstract

The invention discloses a plasma free DNA extraction reagent, a kit and an extraction method, the extraction reagent comprises protease K, a lysis solution, a binding solution, a washing solution 1, a washing solution 2, a washing solution 3 and an eluent, the extraction reagents contain more common and less-dosage common reagents such as Tris-HCl (pH 8.0), EDTA.2Na. 2H2O, NaCl, glycerol, SDS, isopropanol, ethanol, Tween-20 and the like. The method has the advantages that the problems that the reagent is easy to separate out and needs to be heated and redissolved for use during storage can be solved without traditional high-concentration guanidine salts such as guanidine hydrochloride and guanidinium isothiocyanate, the stability and the operation convenience of the extraction reagent are increased, a better extraction effect is achieved, and the eluent can effectively protect the extracted cfDNA from being degraded.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a reagent, kit, and extraction method for cell-free DNA from plasma. Background Technology

[0002] Cell-free DNA (cfDNA) refers to DNA fragments that exist outside of cells in the human circulatory system (including blood, lymph, etc.). These fragments mainly originate from chromatin degradation products released during programmed cell death (apoptosis) or pathological necrosis. In healthy individuals, cfDNA primarily originates from the normal metabolic renewal of blood cells (especially white blood cells) in the hematopoietic system, and its concentration is typically maintained below 100 ng / mL.

[0003] In cancer patients, cfDNA exhibits unique biological characteristics and clinical value. cfDNA derived from tumor cells is called circulating tumor DNA (ctDNA), and its concentration is significantly higher than in healthy individuals. This is mainly due to the abnormally high proliferation rate of tumor tissue, leakage caused by abnormal tumor vascular structure, and the killing effect of immune cells in the tumor microenvironment. This elevated concentration is directly related to multiple factors: tumor burden, tumor invasiveness, and tumor type. ctDNA carries complete tumor genomic information, including somatic mutations (such as point mutations, insertions / deletions, and copy number variations), epigenetic modifications (such as methylation patterns), and nucleosome footprint characteristics. These characteristics make ctDNA a core target for liquid biopsy, with significant value in early tumor screening, molecular subtyping, efficacy assessment, drug resistance monitoring, and prognosis. Compared to traditional tissue biopsy, ctDNA detection has significant advantages such as being non-invasive, highly reproducible, and overcoming tumor heterogeneity.

[0004] Currently, the commonly used cfDNA extraction techniques in clinical practice and research mainly include two types of methods: column chromatography (silica gel membrane adsorption method) and magnetic bead method (magnetic bead covalent binding method). Existing methods still face several technical bottlenecks, especially in the efficient enrichment of tumor ctDNA, which presents significant challenges: fragment loss, background interference, sample preservation and stability, extraction efficiency and complexity, and standardization and automation bottlenecks. Therefore, there is an urgent need for a highly efficient and versatile cfDNA extraction kit to address these issues. Summary of the Invention

[0005] To address at least one of the above problems, the present invention provides a reagent and kit for extracting cell-free DNA from plasma.

[0006] To achieve the above objectives, the present invention employs the following technical means: A first aspect of the present invention provides an extraction reagent for cell-free DNA in plasma, the extraction reagent comprising: proteinase K, lysis buffer, binding buffer, washing buffer 1, washing buffer 2, washing buffer 3, and elution buffer; The lysis buffer includes Tris-HCl (pH 8.0), EDTA·2Na·2H2O, NaCl, glycerol, and SDS; The binding solution comprises Tris-HCl (pH 8.0), EDTA·2Na·2H2O, NaCl, glycerol, and isopropanol; The washing solution 1 includes Tris-HCl (pH 8.0), EDTA·2Na·2H2O, NaCl, glycerol, and isopropanol; The washing solution 2 includes Tris-HCl (pH 8.0), EDTA·2Na·2H2O, glycerol, and ethanol; The eluent includes Tris-HCl (pH 8.0), EDTA·2Na·2H2O, and Tween-20.

[0007] In some embodiments of the present invention, the extraction reagent includes: (1) Proteinase K: 20 mg / mL; (2) Lysis buffer: 10 mM~1M Tris-HCl (pH 8.0), 1 mM~0.2M EDTA·2Na·2H2O, 50 mM~5M NaCl, 1%~30% glycerol (volume concentration), and 0.5%~20% SDS (volume concentration). (3) Binding solution: 10 mM~1M Tris-HCl (pH 8.0), 1 mM~0.2M EDTA·2Na·2H2O, 50 mM~5M NaCl, 1%~30% glycerol (volume concentration), and 30%~80% isopropanol (volume concentration). (4) Washing solution 1: 50mM Tris-HCl (pH 8.0), 10mM EDTA·2Na·2H2O, 50mM~5M NaCl, 1%~30% glycerol (volume concentration), 30%~80% isopropanol (volume concentration). (5) Washing solution 2: 20 mM Tris-HCl (pH 8.0), 1 mM EDTA·2Na·2H2O, 10% glycerol (volume concentration), and 70% ethanol (volume concentration); (6) Washing solution 3: anhydrous ethanol; (7) Eluent: 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA·2Na·2H2O, and Tween-20 with a volume concentration of 0.01%~1%.

[0008] A second aspect of the present invention provides a kit for extracting cell-free DNA from plasma, comprising the extraction reagent described in the first aspect.

[0009] The application of the extraction reagent described in the first aspect or the extraction kit described in the second aspect in the extraction of cell-free DNA from plasma.

[0010] A third aspect of the present invention provides a method for extracting cell-free DNA from plasma, comprising the following steps: S1. Take a plasma sample using a sample tube and add a certain volume of proteinase K and sample lysis buffer. Perform lysis in a water bath and cool after lysis. S2. Add the binding solution, mix thoroughly, and incubate at room temperature. After incubation, place the solution on a magnetic rack and wait until the solution is completely clear before discarding the supernatant. S3. Add washing solution 1, mix thoroughly, transfer all liquid to the sample tube, place the sample tube on the magnetic rack and magnetically attract it. After the solution becomes clear, take the supernatant to rinse the sample tube, then transfer the resuspension to the sample tube, magnetically attract it, and discard the supernatant. S4. Add washing solution 2, mix thoroughly, centrifuge briefly, place on a magnetic rack for magnetic attraction, and discard the supernatant; S5. Add washing solution 3, place it on a magnetic rack and discard the supernatant after magnetic attraction, centrifuge briefly and then put it back on the magnetic rack to fully remove residual liquid; S6. Open the tube cover and dry the magnetic beads at room temperature; S7. Add elution buffer, mix thoroughly, and incubate at room temperature; S8. After the sample tube is centrifuged briefly, it is placed on a magnetic rack and magnetically attracted. The supernatant is the extracted cfDNA.

[0011] In some embodiments of the present invention, the conditions for water bath pyrolysis in step S1 are: pyrolysis in a 60°C water bath for 25 minutes, with the mixture being inverted and mixed once every 10 minutes.

[0012] In some embodiments of the present invention, step S4 is repeated 1-2 times.

[0013] In some embodiments of the present invention, the washing liquid 3 in step S5 is anhydrous ethanol.

[0014] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: 1. The lysis buffer and binding buffer in this invention do not require traditional high-concentration guanidine salts, such as guanidine hydrochloride and guanidine isothiocyanate. Instead, they use more common and less-used ordinary reagents, which achieve better extraction results. 2. The lysis buffer and binding solution in this invention can be stored for more than 6 months without crystallization at storage temperatures of 4℃, 15-25℃, and 37℃, which solves the problem that traditional high-concentration guanidine salts are prone to precipitation at low temperatures and require heating to reconstitute before use, thus increasing the stability and ease of operation of the extraction reagent. 3. The reagent components in the lysis buffer and binding buffer of this invention can effectively solve the problem of mechanical shearing force and nuclease degradation of cfDNA in plasma extraction, and at the same time have great advantages in the recovery of short fragments in ctDNA; 4. The reagent components of the elution solution in this invention can effectively protect the extracted cfDNA from degradation, allowing for longer storage time and more stable cfDNA fragments at 4°C. Attached Figure Description

[0015] Figure 1 The quality control results of the distribution of cfDNA fragments extracted in Example 1, Comparative Example 1, and Comparative Example 2 are shown. Figure 2 The quality control results of the distribution of cfDNA fragments extracted in Examples 3-5 and Examples 5-8 are shown. Figure 3 The quality control results of the distribution of cfDNA fragments extracted in Examples 6-8 are shown. Figure 4 The quality control results of the distribution of cfDNA fragments extracted in Examples 9-11 are shown; Figure 5 The quality control results of the distribution of cfDNA fragments extracted from Examples 9-14 are shown in Example 12. Figure 6 The results of qPCR detection of cfDNA extracted from Examples 12 and Comparative Examples 15-19 after being stored at 4°C for one month are shown. Figure 7 The quality control results of the distribution of cfDNA fragments extracted in Example 13, Comparative Examples 20 and 21 are shown. Figure 8 The library quality control results of the cfDNA extracted in Example 14, Comparative Examples 22 and 23 are shown. Detailed Implementation

[0016] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0018] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0019] Example 1 Take the same plasma sample, use 2 mL of plasma each time, and extract cfDNA from the plasma.

[0020] The concentrations of each component in the extraction reagent are as follows: (1) Proteinase K: 20 mg / mL; (2) Lysis buffer: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), 0.5% SDS (v / v); (3) Binding solution: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (volume concentration), 30% isopropanol (volume concentration); (4) Magnetic beads: 200nm silicon hydroxyl magnetic beads; (5) Washing solution 1: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (volume concentration), 30% isopropanol (volume concentration); (6) Washing solution 2: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 10% glycerol (volume concentration), and 70% anhydrous ethanol (volume concentration); (7) Washing solution 3: anhydrous ethanol; (8) Eluent: 10mM Tris-HCl pH8.0, 0.1mM EDTA·2Na·2H2O, Tween-20 (volume concentration 0.01%).

[0021] The extraction steps are as follows: 1. Take 2 mL of plasma in a 15 mL centrifuge tube and add a certain volume of proteinase K and sample lysis buffer. Place it in a 60 °C water bath for 25 min, inverting and mixing once every 10 min. After the lysis is complete, place it in a 4 °C water bath for 5 min. 2. Add 4 mL of binding solution, shake thoroughly to mix, and incubate at room temperature for 10 min. After incubation, place on a magnetic rack for 5 min. Once the solution is completely clear, discard the supernatant. 3. Add 1 mL of washing solution 1, shake thoroughly to mix, and then transfer all the liquid to a 1.5 mL centrifuge tube. Place the 1.5 mL centrifuge tube on a magnetic rack and magnetically aspirate for 5 min. After the solution has clarified, take 500 μL of the supernatant to rinse the 15 mL centrifuge tube. Then transfer the resuspension to a 1.5 mL centrifuge tube, magnetically aspirate for 2 min, and then discard the supernatant. 4. Add 1 mL of washing solution 2, shake well to mix, centrifuge briefly, place on a magnetic rack and magnetically absorb for 2 min, then discard the supernatant; 5. Repeat step 4 once; 6. Add 600 μL of anhydrous ethanol, place on a magnetic rack for 2 min, discard the supernatant, centrifuge briefly, return to the magnetic rack, and use a small pipette tip to remove the residual liquid completely. 7. Open the tube cap and let it dry at room temperature for 5 minutes until the magnetic beads are matte. 8. Add 50 μL of elution buffer, shake thoroughly to mix, and incubate at room temperature for 5 min; 9. After briefly centrifuging the centrifuge tube, place it on a magnetic rack and magnetically aspirate for 3 minutes. Carefully transfer the supernatant to a new 1.5mL centrifuge tube. The supernatant is the extracted cfDNA, which can be stored at -20℃ for a long time.

[0022] Comparative Example 1 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), 0.5% SDS (v / v), 6M guanidine hydrochloride; Binding solution: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), 30% isopropanol (v / v), 8M guanidine hydrochloride.

[0023] Comparative Example 2 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), 0.5% SDS (v / v), 5M guanidine isothiocyanate; Binding solution: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), 30% isopropanol (v / v), 6M guanidine isothiocyanate.

[0024] The cfDNA extracted from Example 1, Comparative Example 1, and Comparative Example 2 was analyzed for concentration using Novizan dsDNA Qubit quantitative reagent on a Qubit 4.0 instrument. The concentration results are shown in Table 1. The distribution of the extracted cfDNA fragments was analyzed using Qsep400, and the quality control results are as follows: Figure 1 As shown.

[0025] Table 1. Concentration results of cfDNA extracted in Example 1 and Comparative Examples 1-2

[0026] The results showed that cfDNA could still be effectively extracted when the sample was lysed and bound using an extraction reagent that did not contain traditional guanidine salts.

[0027] Example 2 The reagents are prepared as follows: Lysis buffer: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), 0.5% SDS (v / v); Binding solution: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 1M NaCl, 1% glycerol (v / v), 50% isopropanol (v / v).

[0028] Comparative Example 3 The reagents are prepared as follows: Lysis buffer: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), 0.5% SDS (v / v), 6M guanidine hydrochloride; Binding solution: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 1M NaCl, 1% glycerol (v / v), 50% isopropanol (v / v), 8M guanidine hydrochloride.

[0029] Comparative Example 4 The reagents are prepared as follows: Lysis buffer: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), 0.5% SDS (v / v), 5M guanidine isothiocyanate; Binding solution: 10mM Tris-HCl pH8.0, 1mM EDTA·2Na·2H2O, 1M NaCl, 1% glycerol (v / v), 50% isopropanol (v / v), 6M guanidine isothiocyanate.

[0030] The lysis buffer and binding buffer from Example 2 and Comparative Examples 3-4 were each dispensed into 10 bottles. The reagents were stored in environments of 4°C, 15-25°C, and 37°C for 6 months for a comparative experiment. The stability of the reagents was determined by observing whether crystals precipitated.

[0031] The test results are shown in Table 2.

[0032] Table 2. Long-term storage stability results of the lysis buffer and binding solution in Example 2 and Comparative Examples 3-4.

[0033] The results showed that the use of lysis buffers and binding buffers without traditional guanidine salts overcame the reagent instability caused by the high concentration of guanidine salts, the main component of traditional extraction reagents, and both could be stably preserved under normal storage conditions.

[0034] Example 3 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% glycerol (v / v), 10% SDS (v / v).

[0035] Example 4 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 1M Tris-HCl (pH 8.0), 0.2M EDTA·2Na·2H2O, 5M NaCl, 15% glycerol (v / v), 0.5% SDS (v / v).

[0036] Example 5 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA·2Na·2H2O, 50 mM NaCl, 1% glycerol (v / v), 20% SDS (v / v).

[0037] Comparative Example 5 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% glycerol (v / v), 10% Brij 35 (v / v).

[0038] Comparative Example 6 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% glycerol (v / v), 10% Triton X-100 (v / v).

[0039] Comparative Example 7 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% glycerol (v / v), 10% NP-40 (v / v).

[0040] Comparative Example 8 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% glycerol (v / v), 10% Tween-20 (v / v).

[0041] The concentration and fragment distribution of the cfDNA extracted in Examples 3-5 and Comparative Examples 5-8 were detected, and the results are shown in Table 3 and 4. Figure 2 As shown.

[0042] Table 3 shows the concentration results of cfDNA extracted in Examples 3-5 and Examples 5-8.

[0043] The results showed that the components of the extraction reagent lysis buffer of the present invention can effectively lyse proteins and membrane substances in plasma, releasing more cfDNA.

[0044] Example 6 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 3, with the only difference being: Binding solution: 1M Tris-HCl (pH 8.0), 0.2M EDTA·2Na·2H2O, 5M NaCl, 15% glycerol (v / v), and 30% isopropanol (v / v).

[0045] Example 7 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 3, with the only difference being: Binding solution: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% glycerol (v / v), and 50% isopropanol (v / v).

[0046] Example 8 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 3, with the only difference being: Binding solution: 10mM Tris-HCl (pH 8.0), 1mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), and 80% isopropanol (v / v).

[0047] The concentration and fragment distribution of the cfDNA extracted in Examples 6-8 were detected, and the results are shown in Table 4 and 5. Figure 3 As shown.

[0048] Table 4. Results of cfDNA concentrations extracted in Examples 6-8

[0049] The results showed that the composition of the extraction reagent binding solution of the present invention can provide a hydrophobic environment for the magnetic beads to bind efficiently with cfDNA, which greatly improves the extraction efficiency.

[0050] Example 9 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 7, with the only difference being: Washing solution 1: 50mM Tris-HCl (pH 8.0), 10mM EDTA·2Na·2H2O, 5M NaCl, 30% glycerol (v / v), and 30% isopropanol (v / v).

[0051] Example 10 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 7, with the only difference being: Washing solution 1: 50mM Tris-HCl (pH 8.0), 10mM EDTA·2Na·2H2O, 2.5M NaCl, 15% glycerol (v / v), and 50% isopropanol (v / v).

[0052] Example 11 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 7, with the only difference being: Washing solution 1: 50mM Tris-HCl (pH 8.0), 10mM EDTA·2Na·2H2O, 50mM NaCl, 1% glycerol (v / v), and 80% isopropanol (v / v).

[0053] The concentration and fragment distribution of the cfDNA extracted in Examples 9-11 were detected, and the results are shown in Table 5. Figure 4 As shown.

[0054] Table 5. Results of cfDNA concentrations extracted in Examples 9-11

[0055] The results showed that the composition of the extraction reagent washing solution 1 of the present invention can provide a hydrophobic environment for the magnetic beads to bind efficiently with cfDNA, effectively avoiding the loss of cfDNA during the washing process and thus improving the extraction efficiency.

[0056] Example 12 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 1, with the only difference being: Lysis buffer: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% glycerol (v / v), 10% SDS (v / v); Binding solution: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% glycerol (v / v), 50% isopropanol (v / v); Washing solution 1: 50mM Tris-HCl (pH 8.0), 10mM EDTA·2Na·2H2O, 2.5M NaCl, 15% glycerol (v / v), and 50% isopropanol (v / v).

[0057] Comparative Example 9 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Lysis buffer: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% propylene glycol (v / v), 10% SDS (v / v).

[0058] Comparative Example 10 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Lysis buffer: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 30% glycerol (v / v), 10% SDS (v / v).

[0059] Comparative Example 11 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Binding solution: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 2.5M NaCl, 30% ethylene glycol (v / v), and 50% isopropanol (v / v).

[0060] Comparative Example 12 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Binding solution: 500mM Tris-HCl (pH 8.0), 0.1M EDTA·2Na·2H2O, 30% glycerol (v / v), and 50% isopropanol (v / v).

[0061] Comparative Example 13 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Washing solution 1: 50mM Tris-HCl (pH 8.0), 10mM EDTA·2Na·2H2O, 2.5M NaCl, 15% polyethylene glycol (v / v), and 50% isopropanol (v / v).

[0062] Comparative Example 14 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Washing solution 1: 50mM Tris-HCl (pH 8.0), 10mM EDTA·2Na·2H2O, 15% glycerol (v / v), and 50% isopropanol (v / v).

[0063] The concentration and fragment distribution of the cfDNA extracted in Example 12 and Comparative Examples 9-14 were detected, and the results are shown in Table 6. Figure 5 As shown.

[0064] Table 6. Concentration results of cfDNA extracted in Example 12 and Comparative Examples 9-14

[0065] The results showed that the composition of the extraction reagent of the present invention can effectively prevent the extracted cfDNA from being degraded by mechanical shearing force and nucleases. The final extracted cfDNA concentration was the highest and the main peak of the extracted fragment was the smallest, indicating that the extraction reagent of the present invention has great advantages in the recovery of short fragments in ctDNA.

[0066] Comparative Example 15 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Eluent: 10mM Tris-HCl pH8.0, 0.1mM EDTA·2Na·2H2O, 0.1% Tween-20 (v / v).

[0067] Comparative Example 16 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Eluent: 10mM Tris-HCl pH 8.0, 0.1mM EDTA·2Na·2H2O, 1% Tween-20 (v / v).

[0068] Comparative Example 17 The test samples, other reagents used for extraction, and the extraction process were all the same as in Example 12, with the only difference being: Eluent: 10mM Tris-HCl pH8.0, 0.1mM EDTA·2Na·2H2O.

[0069] Comparative Example 18 The test samples and extraction process were the same as in Example 12, and the reagents used for extraction were the beaver cfDN extraction kit.

[0070] Comparative Example 19 The test samples and extraction process were the same as in Example 12, and the reagents used for extraction were the Yisheng cfDN extraction kit.

[0071] The cfDNA extracted from Example 12 and Comparative Examples 15-19 was incubated at 4°C for one month before being subjected to qPCR detection. The detection results are as follows: Figure 6 As shown.

[0072] The results showed that the extraction reagent elution buffer of the present invention can provide strong protection for the extracted cfDNA, and there is no degradation after storage at 4°C for one month.

[0073] Example 13 Take the same plasma sample and extract cfDNA from it separately.

[0074] The extraction reagents used in this embodiment are the same as in Example 12. Extraction was performed using an Ausen automated extraction instrument. First, 2 mL of plasma was taken from a 15 mL centrifuge tube, and a certain volume of proteinase K and sample lysis buffer were added. The mixture was placed in a 60°C water bath for 25 min, inverted and mixed every 10 min. After lysis, it was cooled at 4°C for 5 min. Then, the automated instrument was used. Well 1 contained 4 mL of binding buffer, well 2 contained 1 mL of washing buffer 1, well 3 contained 1 mL of washing buffer 2, well 4 contained 1 mL of washing buffer 2, well 5 contained 1 mL of washing buffer 3, and well 6 contained elution buffer. First, 40 μL of magnetic beads were added to well 2, and then the lysed and cooled sample was added to well 1. The program was then run. After the operation was completed, the liquid in well 6 was the extracted cfDNA.

[0075] Comparative Example 20 The test sample was the same as in Example 12, and the extraction reagent was the Beaver Automated Nucleic Acid Extraction Kit and its supporting instruments.

[0076] Comparative Example 21 The test sample was the same as in Example 12, and the extraction reagent was the Yisheng Automated Nucleic Acid Extraction Kit and its supporting instruments.

[0077] The cfDNA extracted in Example 13, Comparative Examples 20 and 21 was quantified using Novizan dsDNA quantification reagent with Qubit 4.0. 1 μl was taken for Qsep quality control. The concentration and fragment distribution results are shown in Table 7 below. Figure 7 As shown.

[0078] Table 7 shows the detection results of cfDNA extracted in Example 13 and Comparative Examples 20 and 21.

[0079] The results show that the extraction reagent of the present invention can be effectively integrated with automated instruments, making it very convenient to carry out large-scale extraction operations, greatly reducing extraction time and labor costs. Moreover, compared with competing extraction kits, the reagent composition of the present invention has a higher extraction efficiency for short fragments (such as ctDNA).

[0080] Example 14 Take the same healthy plasma sample, mix it with 1% lung cancer SNV detection standard, and extract cfDNA from the mixed plasma. The extraction reagents and process used in this example are the same as in Example 12.

[0081] Comparative Example 22 The test sample was the same as in Example 14, and the extraction reagent was the beaver cfDN extraction kit.

[0082] Comparative Example 23 The test sample was the same as in Example 14, and the extraction reagent was the Yisheng cfDN extraction kit.

[0083] The cfDNA extracted in Example 14, Comparative Examples 22 and 23 was quantitatively analyzed using Novizan dsDNA quantification reagent with Qubit 4.0 to determine its concentration and fragment distribution. All cfDNA was used for NGS DNA library construction. Hybridization capture was performed using a lung cancer-customized panel. The captured libraries were then sent to the Illumina platform for sequencing. Bioinformatics analysis of the sequencing results is shown in Table 8 below. Figure 8 As shown.

[0084] Table 8 shows the detection results of cfDNA extracted in Example 14 and Comparative Examples 22 and 23.

[0085] The results showed that the extraction reagent of the present invention extracted shorter fragments, resulting in higher library output and better sequencing quality. Furthermore, based on the detection frequency of 1% SNV mutation standards, the extraction efficiency of the reagent of the present invention is higher than that of competing reagent kits.

[0086] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A reagent for extracting cell-free DNA from blood plasma, characterized in that, The extraction reagents include: proteinase K, lysis buffer, binding buffer, washing buffer 1, washing buffer 2, washing buffer 3, and elution buffer. The lysis buffer includes Tris-HCl (pH 8.0), EDTA·2Na·2H2O, NaCl, glycerol, and SDS; The binding solution comprises Tris-HCl (pH 8.0), EDTA·2Na·2H2O, NaCl, glycerol, and isopropanol; The washing solution 1 includes Tris-HCl (pH 8.0), EDTA·2Na·2H2O, NaCl, glycerol, and isopropanol; The washing solution 2 includes Tris-HCl (pH 8.0), EDTA·2Na·2H2O, glycerol, and ethanol; The eluent includes Tris-HCl (pH 8.0), EDTA·2Na·2H2O, and Tween-20.

2. The plasma cell-free DNA extraction reagent according to claim 1, characterized in that, In the extraction reagent: (1) Proteinase K: 20 mg / mL; (2) Lysis buffer: 10 mM~1M Tris-HCl (pH 8.0), 1 mM~0.2M EDTA·2Na·2H2O, 50 mM~5M NaCl, 1%~30% glycerol (volume concentration), and 0.5%~20% SDS (volume concentration). (3) Binding solution: 10 mM~1M Tris-HCl (pH 8.0), 1 mM~0.2M EDTA·2Na·2H2O, 50 mM~5M NaCl, 1%~30% glycerol (volume concentration), and 30%~80% isopropanol (volume concentration). (4) Washing solution 1: 50mM Tris-HCl (pH 8.0), 10mM EDTA·2Na·2H2O, 50mM~5M NaCl, 1%~30% glycerol (volume concentration), 30%~80% isopropanol (volume concentration). (5) Washing solution 2: 20 mM Tris-HCl (pH 8.0), 1 mM EDTA·2Na·2H2O, 10% glycerol (volume concentration), and 70% ethanol (volume concentration); (6) Washing solution 3: anhydrous ethanol; (7) Eluent: 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA·2Na·2H2O, and Tween-20 with a volume concentration of 0.01%~1%.

3. A reagent kit for extracting cell-free DNA from plasma, characterized in that, Includes the extraction reagent as described in any one of claims 1-2.

4. The use of the extraction reagent of claim 2 or the extraction kit of claim 3 in the extraction of cell-free DNA from plasma.

5. A method for extracting cell-free DNA from blood plasma, characterized in that, Includes the following steps: S1. Take a plasma sample in a sample tube and add a certain volume of proteinase K and sample lysis buffer. Perform lysis in a water bath and cool after lysis. S2. Add the binding solution, mix thoroughly, and incubate at room temperature. After incubation, place the solution on a magnetic rack and wait until the solution is completely clear before discarding the supernatant. S3. Add washing solution 1, mix thoroughly, transfer all liquid to the sample tube, place the sample tube on the magnetic rack and magnetically attract it. After the solution becomes clear, take the supernatant to rinse the sample tube, then transfer the resuspension to the sample tube, magnetically attract it, and discard the supernatant. S4. Add washing solution 2, mix thoroughly, centrifuge briefly, place on a magnetic rack for magnetic attraction, and discard the supernatant; S5. Add washing solution 3, place it on a magnetic rack and discard the supernatant after magnetic attraction, centrifuge briefly and then put it back on the magnetic rack to fully remove residual liquid; S6. Open the tube cover and dry the magnetic beads at room temperature; S7. Add elution buffer, mix thoroughly, and incubate at room temperature; S8. After the sample tube is centrifuged briefly, it is placed on a magnetic rack and magnetically attracted. The supernatant is the extracted cfDNA.

6. The method for extracting cell-free DNA from plasma according to claim 5, characterized in that, The conditions for water bath pyrolysis in step S1 are: pyrolysis in a 60℃ water bath for 25 minutes, with the mixture being inverted and mixed once every 10 minutes.

7. The method for extracting cell-free DNA from plasma according to claim 5, characterized in that, Repeat step S4 1-2 times.

8. The method for extracting cell-free DNA from plasma according to claim 5, characterized in that, The washing solution 3 in step S5 is anhydrous ethanol.

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