CN113930512A - Plasma circulation tumor free DNA standard substance and preparation method and application thereof - Google Patents
Plasma circulation tumor free DNA standard substance and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to biotechnology, and particularly relates to a plasma circulation-simulated tumor free DNA standard substance, and a preparation method and application thereof. The invention prepares fragmented DNA by combining nucleosome enzyme cutting method with column purification, and the obtained DNA fragment has very high yield. The plasma circulating tumor free DNA standard substance is close to the natural and real state of ctDNA existing in a human body, a series of experimental operations including blood collection, transportation, ctDNA separation and extraction, library building and sequencing can be carried out on the detection of the ctDNA for quality control, the reliability of a detection result is enhanced, and the plasma circulating tumor free DNA standard substance is stable, high in safety and capable of being repeatedly produced.
Description
Technical Field
The invention relates to the technical field of circulating tumor free DNA gene mutation detection, in particular to a plasma circulating tumor free DNA simulation standard substance, a preparation method and application thereof, which are used for circulating tumor free DNA detection.
Background
Fluid biopsies are the diagnosis and monitoring of diseases such as tumors by capturing and detecting biomarkers (e.g., cells, DNA, RNA, proteins, etc.) in body fluids (e.g., blood, urine, saliva, ascites, pleural effusion, etc.). Compared with tissue biopsy, the liquid biopsy has the advantages that a tumor sample is obtained in a non-invasive and repeatable manner, the side effect is small, the operation is simple and convenient, the cost is low, the detection speed is high, the deviation of tumor heterogeneity to diagnosis is reduced, dynamic changes of tumor development can be reflected timely, the clinical indications are wide, and common tumors such as lung cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer and the like can be diagnosed and monitored by liquid biopsy. At present, a great deal of research in the scientific research field discovers that compared with the traditional means such as tissue biopsy, serum tumor markers, medical imaging and the like, the liquid biopsy can discover early tumors in advance, and further can treat the early tumors in time, so that the prognosis of a patient is obviously improved.
Circulating tumor free DNA (ctDNA) is one of the classic targets for liquid biopsy of tumors. Tumor cells can release broken, incomplete single-or double-stranded ctDNA into the blood by apoptosis, necrosis, and secretion. Since ctDNA itself is derived from DNA inside tumor cells, it naturally carries information on mutations in tumor DNA. ctDNA is widely distributed and is detected in blood of different tumor patients successively, and the more advanced the tumor is, the higher the malignancy degree of the tumor is, the higher the frequency of specific mutation of the ctDNA is, so that the ctDNA can be used for monitoring the development of the tumor in real time.
The main difficulty of the detection of the ctDNA is that the sensitivity is difficult to improve, the ctDNA content is low, the ctDNA is easy to degrade in the processes of extracting and treating a sample in the previous stage, and the half-life period of the ctDNA is very short and is only 0.5-1 hour, so that the false negative rate of the detection can be increased; in addition, when the sample is not handled properly, leukocyte breakage is likely to occur, and the proportion of the cfDNA is further decreased by mixing the released genomic DNA with the cfDNA, which means that the false negative rate of detection is further increased. About 4-5 ml of plasma can be isolated from 10 ml of blood from asymptomatic human, ideally 15-30ng cfDNA can be obtained, which is equivalent to 5000 genome equivalents of template from 2500-. But only about one-ten-thousandth of cfDNA in asymptomatic individuals is ctDNA. Overall, there is a risk that the accuracy of the result is influenced in a series of experimental operations from blood collection, transportation, ctDNA isolation and extraction, and library construction and sequencing.
Disclosure of Invention
The conventional ctDNA standard substance taking TE as a matrix cannot well control the extraction link of ctDNA. The invention aims to solve the technical problems that the preparation process of the plasma circulating tumor free DNA standard substance is simulated, the natural and real state of the ctDNA in a human body is approached to the maximum extent, a series of experimental operations of blood collection, transportation, ctDNA separation and extraction, and library building and sequencing for the detection of the ctDNA can be controlled, the reliability of the detection result is enhanced, and the product is stable, high in safety and capable of being produced repeatedly.
The invention adopts the following technical scheme:
a plasma circulation simulating tumor free DNA standard comprises a plasma simulating and fragmented DNA mixture; the fragmented DNA mixture is a background cell DNA fragmentation product, or the fragmented DNA mixture is a background cell DNA fragmentation product and a tumor cell DNA fragmentation product; the fragmentation product is prepared by adopting a nucleosome enzyme digestion combined column purification method. The method prepares the wild type background cell fragmentation product and the tumor cell fragmentation product of multiple cancer gene mutations by combining a nucleosome enzyme digestion and column purification method for the first time, mixes the two products to obtain the fragmentation DNA mixture with different mutation frequencies, and conventionally designs the mutation frequencies in a gradient manner according to the requirements of standard products.
In the invention, the simulated plasma comprises sodium chloride, sodium bicarbonate, sodium carbonate, potassium chloride, dipotassium hydrogen phosphate trihydrate, magnesium chloride hexahydrate, sodium hydroxide, hydroxyethyl piperazine ethanethiosulfonic acid, calcium chloride, sodium sulfate, Ethylene Diamine Tetraacetic Acid (EDTA), human serum albumin and ultrapure water. The simulated plasma is prepared by adding various components according to a certain proportion by using a cell culture-grade reagent to ensure that the ion concentration and the protein content in the obtained solution are close to those of real human plasma, and adding EDTA (ethylene diamine tetraacetic acid) to simulate an anticoagulant component in the human plasma for gene detection.
Adding the mixed solution into cells, incubating with ice after oscillation, adding a non-ionic surfactant solution, incubating with ice after oscillation, centrifuging, removing the supernatant, adding a micrococcal nuclease buffer solution, centrifuging after blowing, removing the supernatant, adding the micrococcal nuclease buffer solution for resuspension, adding the micrococcal nuclease, performing water bath treatment after oscillation, adding an ethylenediamine tetraacetic acid solution and a ribonuclease A solution, performing water bath treatment after oscillation, adding a sodium dodecyl sulfate solution and a protease K solution, performing water bath treatment after oscillation, and purifying and recycling to obtain DNA fragments; the mixed solution comprises 4-hydroxyethyl piperazine ethanesulfonic acid solution, inorganic magnesium salt solution, inorganic potassium salt solution, organic reducing agent solution and benzyl sulfonyl fluoride solution; the micrococcus nuclease buffer solution comprises Tris-HCl buffer solution and CaCl2、BSA。
In the invention, the mixed solution consists of 4-hydroxyethyl piperazine ethanesulfonic acid solution, inorganic magnesium salt solution, inorganic potassium salt solution, organic reducing agent solution, phenylmethylsulfonyl fluoride solution and water; preferably, the volume ratio of the 4-hydroxyethyl piperazine ethanesulfonic acid solution to the inorganic magnesium salt solution to the inorganic potassium salt solution to the organic reducing agent solution to the phenylmethylsulfonyl fluoride solution to water is (8-12) to (1-2) to (8-12) to (0.3-0.8) to (8-12) to (900-1050). The concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid solution is 0.08-0.12M, the pH value is 7.5-8, and the solution is a conventional reagent; the concentration of the inorganic magnesium salt solution is 0.8-1.2M, the concentration of the inorganic sylvite solution is 0.8-1.2M, the concentration of the organic reducing agent solution is 0.8-1.2M, and the concentration of the phenylmethylsulfonyl fluoride solution is 0.08-0.12M. Wherein the inorganic magnesium salt comprises magnesium chloride, the inorganic potassium salt comprises potassium chloride, and the organic reducing agent is dithiothreitol DTT; the nonionic surfactant is CA-630.
In the invention, the volume ratio of the mixed solution, the nonionic surfactant solution, the ethylene diamine tetraacetic acid solution, the ribonuclease A solution, the sodium dodecyl sulfate solution and the protease K solution is 1000 to (5-15) to (30-50) to (3-18) to (70-150) to (10-30), preferably 1000 to (8-12) to (35-45) to (8-13) to (90-110) to (15-25).
In the invention, the using amount of the micrococcus nuclease buffer solution is 0.8-1.2 times, preferably 0.9-1.1 times of the volume of the mixed solution; the volume of the buffer solution for the two uses of the micrococcal nuclease may be the same or different. The composition of the micrococcal nuclease buffer is exemplified as follows: 50 mM Tris-HCl (pH = 8.0), 5 mM CaCl20.1 mg/mL BSA (bovine serum albumin), and water.
In the invention, specific operations of shaking, ice incubation, water bath, centrifugation and whipping are conventional techniques, wherein the shaking time is not particularly limited, and the purpose is conventional and is to mix uniformly; the temperature of the centrifugal treatment is 0-5 ℃, the centrifugal force is 700-1000 g, and the time is 3-10 minutes; the ice incubation time is 3-15 minutes; the blow beating is carried out for 2-5 times by a liquid transfer device; the temperature of the water bath treatment is 35-60 ℃, and the time is 5-45 minutes.
In the present invention, the number of cells is 1X 107～1×109The cell is a wild background cell or a tumor cell with multiple cancer gene mutations, and the tumor cell has gene mutations and can carry a single gene mutation site or multiple gene mutation sites and is selected according to requirements. Selecting GM12878 cell as background cell of multiple cancer gene mutation wild background cell; tumor cells and mutation are selected according to requirements, a multi-cancer gene mutation wild type background cell fragmentation product and a tumor cell fragmentation product are obtained respectively, and the multi-cancer gene mutation wild type background cell fragmentation product and the tumor cell fragmentation product are added into simulated plasma according to different proportions according to the actual gradient requirement of a quality control product to form a gradient product.
The invention prepares fragmented DNA by combining nucleosome enzyme cutting method with column purification, the process of the obtained DNA fragment is similar to the process of the DNA fragment naturally generated in human body, the size distribution structure of the DNA is more consistent than that of the DNA fragment obtained by ultrasonic breaking, and the biological characteristics of the tail end of the obtained DNA fragment are similar to those of natural ctDNA: the fragmented DNA generated in human body is mainly generated by in vivo enzymolysis, the tail ends of DNA molecules are 5 'end phosphoric acid and 3' end hydroxyl, the tail ends of the fragmented DNA molecules obtained by the invention are 5 'end phosphoric acid and 3' end hydroxyl, and the operation flows of the fragmented DNA extracted from human plasma and the fragmented DNA samples obtained by nucleosome enzyme digestion are consistent in the process of detecting gene mutation by using PCR or NGS sequencing; the complexity of the DNA fragment obtained by the invention is similar to that of a natural ctDNA sequence, the size distribution of the DNA fragment is highly consistent with that of the natural ctDNA, and mutation positions are randomly distributed on the DNA fragment, so that the sensitivity and accuracy of the detection equipment and the detection method can be reflected and evaluated more truly.
Drawings
FIG. 1 shows the mutation frequency of BRAF _ V600E gene of CCL-238 fragmented DNA;
FIG. 2 shows the size distribution of DNA fragments obtained by extraction;
FIG. 3 is the verification of the mutation frequency of ctDNA gene obtained by extraction.
Detailed Description
The raw materials adopted by the invention are conventional in the field, the pretreatment of the cells is also conventional in the field, and the specific preparation operation method and the test method are conventional in the field. The creativity of the invention lies in that a nucleosome enzyme digestion combined column purification method is adopted to prepare a multiple cancer gene mutation wild type background cell fragmentation product and a tumor cell fragmentation product; mixing the two substances, adding the mixture into simulated plasma to obtain a fragmented DNA mixture with different mutation frequencies, and conventionally performing gradient design on the mutation frequencies according to the requirements of standard products, wherein the mutation frequencies are 0-50% in the fragmented DNA mixture; according to the actual requirement, the input amount of the fragmented DNA mixture can be designed into gradient products such as 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL and the like, but is not limited thereto.
Purchasing a target cell line from a cell bank, downloading a cell culture instruction on a cell bank website, and preparing a complete culture medium suitable for cell growth for later use; the purchased cell line arrives at a cell culture room in a frozen form, the cells are put into a water bath kettle at 37 ℃ for fast thawing, the freezing tube is continuously and gently shaken, after the cell suspension in the freezing tube is melted, the freezing tube is sprayed with 75% alcohol for disinfection, and the cell suspension is transferred into a biosafety roomOpening the freezing tube in the cabinet, sucking out the cell suspension, adding into 5ml of complete culture medium, transferring into a centrifuge, centrifuging at 1000rpm for 8min, removing supernatant after centrifugation, mixing the tube bottom precipitate with 1ml of complete culture medium, mixing the sample with trypan blue at a volume of 1:1, calculating cell number and activity, adding complete culture medium according to the counting result, and adjusting cell density to 1 × 106mL, for transfer to cell culture 6-well plates, 2 mL/well, cell culture plates were transferred to 37 ℃ with 5% CO2Culturing in an incubator with 95% humidity; sampling every 48 hours, counting the number of cells, adding complete culture medium to maintain the cell density at 0.5-1 × 106and/mL. The cell amount is larger than 1 × 108Collect cells into 1.5mL centrifuge tubes, 1X 108Tube for storage at-20 ℃ while collecting a tube of cells at a cell count of 1X 106And performing STR identification of the cells. Comparing the obtained STR typing result with a professional cell STR database, confirming that the purchased cell line is correct and is not polluted by other cells, performing cell cracking and enzyme digestion, and according to ANSI standard, the matching degree of the cells used in the invention is more than or equal to 80%.
Examples
In preparing a standard substance simulating plasma circulating tumor free DNA, a standard substance comprising one or more gene mutation sites can be designed according to needs, and the gene mutation frequency can be designed into gradient products of 0%, 10%, 20%, 30%, 40%, 50% and the like according to needs, and the method is common knowledge in the field. In the specific example, a background cell GM12878 cell is used as a wild-type background cell line with multiple oncogene mutations, the obtained fragmented DNA is used as a wild-type background, the fragmented DNA obtained from CCL-238 cell is used as a BRAF _ V600E gene mutation, and the obtained fragmented DNA and the wild-type background cell line are mixed to prepare a plasma circulating tumor free DNA mimic standard with a mutation frequency of 1%.
Preparing simulated plasma by mixing the components according to the following ratio:
electrolyte concentration and protein content in the prepared simulated plasma are compared with real plasma:
the prepared simulated plasma is filtered by a 0.22 mu m filter membrane and collected into a serum bottle for later use.
2. The product of cell fragmentation. Taking GM12878 cells out of a refrigerator at-20 ℃, putting on ice for 20min, and waiting for the cells to melt; 1X 108To the cells were added 10. mu.L of 0.1M Hepes (4-hydroxyethylpiperazine ethanesulfonic acid) aqueous solution and 1.5. mu.L of 1M MgCl2Aqueous solution, 10. mu.L of 1M KCl aqueous solution, 0.5. mu.L of 1M DTT aqueous solution, 10. mu.L of 0.1M PMSF (phenylmethylsulfonyl fluoride) aqueous solution and 970. mu.L of water, shaking for 15s, and incubating for 10min on ice; then adding 10uL 10% IGEPAL CA-630 aqueous solution, vibrating for 10s, and incubating for 5min with ice; centrifuging at 4 deg.C and 900g for 5min, and removing supernatant; then adding 1000 mu L micrococcal nuclease buffer solution for cleaning, blowing and beating for 3 times by a pipette at 4 ℃ and 900g, centrifuging for 5min, removing supernatant, adding 1000 mu L micrococcal nuclease buffer solution for resuspending cell nucleus, adding 100uL micrococcal nuclease (100U/uL), shaking and uniformly mixing, and carrying out water bath at 42 ℃ for 10 min; then adding 40uL EDTA (Thermo R1021), shaking and mixing uniformly; adding 10uL of RNaseA (ribonuclease A) Tris-HCL buffer solution (Tris (hydroxymethyl) aminomethane hydrochloride buffer solution) with the concentration of 20mg/mL, and shaking and mixing uniformly; water bath at 37 deg.C for 30 min; then 100uL of 10% SDS (sodium dodecyl sulfate) aqueous solution is added, and the mixture is shaken and mixed evenly; then 20uL of protease K (Proteinase K, 200 mg/mL) is added, shaken and mixed evenly; water bath at 56 deg.C for 30 min; and finally, purifying and recovering the DNA fragment to obtain fragmented DNA, wherein the micrococcus nuclease buffer solution comprises the following components: 50 mM Tris-HCl (pH = 8.0), 5 mM CaCl20.1 mg/mL BSA and water. The digested DNA fragment was purified and recovered using QIAQuick PCR purification Kit, according to the instructions, as follows:
the samples were transferred to a 15mL centrifuge tube, and Buffer PB of 5 sample volumes in the QIAQuick PCR purification Kit, i.e., V sample: VBuffer PB = 1: 5, shaking and uniformly mixing;
taking 10 adsorption columns and collection tubes provided by QIAQuick PCR purification Kit (cat # 28106), and transferring the mixed samples to 10 adsorption columns on average;
centrifuging each adsorption column and the collection tube at 14000rpm for 30s, discarding the liquid in the collection tube, and repeating the steps until the sample is transferred;
adding 750 mu L of Buffer PE into each adsorption column, 14000rpm, centrifuging for 30s, and discarding liquid in the collection tube;
centrifuging each adsorption column at 14000rpm for 2min, and placing the adsorption column in a new EP tube;
adding 55 μ L Buffer EB into each adsorption column, keeping for 3min, centrifuging at 14000rpm for 1 min; obtaining a product DNA fragment.
The fragmented DNA obtained above was quantitatively detected using the Qubit dsDNA HS Assay kit (cat # Q32854) according to the instructions, and the concentration of the fragmented DNA solution was found to be 39.4 ng/. mu.L, and the total amount of DNA was found to be: 21670ng, by the formula: concentration 1 × volume 1= concentration 2 × volume 2, the volume of the newly added EB was calculated to be 172.3 μ L, and the concentration of the obtained fragmented DNA solution was adjusted to 30ng/μ L.
Using CCL-238 cells as an extraction sample, obtaining a fragmented DNA solution according to the method, using a Qubit dsDNA HS Assay kit (cat number: Q32854) and quantitatively detecting the obtained fragmented DNA according to an instruction, and determining that the concentration of the fragmented DNA solution is 48.1 ng/muL, the total amount of DNA is: 26455ng, by the formula: concentration 1 × volume 1= concentration 2 × volume 2, the volume of the newly added EB was calculated to be 331.8 μ L, and the concentration of the obtained fragmented DNA solution was adjusted to 30ng/μ L.
Size analysis of the obtained DNA fragments using agilent 2100 Bioanalyzer, the DNA fragment size distribution was similar to that of natural cfDNA.
For comparison, in the method for extracting the sample from the CCL-238 cells, the column purification was replaced by magnetic bead purification, and the rest was unchanged, and the concentration of the fragmented DNA solution was measured to be 21.8ng/μ L, that is, the total amount of the obtained DNA was: 11990 ng. In addition, changing the composition of the micrococcal nuclease buffer also reduces the yield of fragmented DNA product or changes the size distribution of DNA fragments, see in particular another application filed by the applicant on the same day: a high-yield DNA fragment and a preparation method thereof.
3. Mixing of fragmented DNA. And (3) detecting the two fragmented DNA by using ddPCR (double-stranded polymerase chain reaction) by using the fragmented DNA prepared by GM12878 cells as a wild background and the fragmented DNA prepared by CCL-238 cells as a gene mutation template, and determining the mutation frequency of the BRAF _ V600E gene of the two fragmented DNA.
ddPCR primer Probe sequence:
preparing a PCR reaction system:
the loading amount is 10ng, the sample amount is less than 8 mu L, the nuclease-free water is used for complementing, and the final concentration of the primer is as follows: 960nM, final probe concentration: 250 nM;
the reaction procedure was set up in a PCR instrument according to the following table, with a total reaction system of 40 μ L:
the mutation frequency of the BRAF _ V600E gene of the obtained GM12878 fragmented DNA is 0%, and the mutation frequency of the BRAF _ V600E gene of the CCL-238 fragmented DNA is 52.7% (FIG. 1).
According to the formula: (mass of GM12878 fragmented DNA + mass of CCL-238 fragmented DNA)/mass of CCL-238 fragmented DNA =52.7 calculation, mixing was performed according to the following table sampling to obtain a mixture of fragmented DNA of BRAF _ V600E mutation with a theoretical mutation frequency of 1%:
4. plasma circulating tumor free DNA standard is simulated. And (3) putting the fragmented DNA mixture into prepared simulated plasma, wherein the input amount is 100ng/mL, and preparing a BRAF _ V600E mutant simulated plasma circulating tumor free DNA standard substance with the theoretical concentration of 1%.
5. And (6) verifying.
The free DNA standard of BRAF _ V600E mutant mimic plasma Circulating tumor with the theoretical content of 1% was subjected to ctDNA extraction according to the Kit instruction of QIAamp Circulating Nucleic Acid Kit (cat # 55114), the extraction amount was 4mL, and the DNA recovery rate was determined by using Qubit3.0, the DNA recovery amount was 303.5ng, and the DNA recovery rate was 75.86%.
The size of the extracted DNA fragment is checked by using an Agilent 2100 Bioanalyzer, the result shows that the size distribution of the DNA fragment meets 144bp-176bp which is more than or equal to 92 percent (figure 2), the result shows that the mutation frequency of the ctDNA obtained by extraction is verified by ddPCR (polymerase chain reaction), the result shows that the mutation frequency of BRAF _ V600E is within the range of 0.95-1.05 percent and meets the requirement of a standard product (figure 3), and the sequence of a ddPCR primer probe, a PCR reaction system and a reaction program are consistent with the step 3.
In conclusion, the tumor cell line is used for obtaining the fragmented DNA, the fragmented DNA can be mixed with wild type background cell fragmentation products of multiple cancer gene mutations according to needs, the preset mutation frequency is designed, the ion concentration, the protein content and the anticoagulation environment which are similar to those of real human plasma can be obtained by preparing simulated plasma, and the mixed fragmented DNA is put into the simulated plasma to obtain the circulating tumor free DNA standard product of the simulated plasma; through reextraction verification, the size distribution of DNA fragments, gene mutation sites and gene mutation frequency show good stability. The plasma circulating tumor free DNA standard substance is close to the natural and real state of ctDNA in a human body to the maximum extent, a series of experimental operations of blood collection, transportation, ctDNA separation and extraction, library building and sequencing for the detection of the ctDNA can be controlled, the product is stable and reliable, the safety is high, and the repeated production can be realized.
The above-described embodiments are not intended to limit the present invention, and the present invention is not limited to the above-described examples. Those of ordinary skill in the art will understand that: the combination, change, modification, addition or substitution made within the spirit of the present invention shall also fall within the protection scope of the present invention.
Sequence listing
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Claims (10)
1. A plasma circulation simulating tumor free DNA standard comprises a plasma simulating and fragmented DNA mixture; the method is characterized in that the fragmented DNA mixture is a background cell DNA fragmented product, or the fragmented DNA mixture is a background cell DNA fragmented product and a tumor cell DNA fragmented product, and the fragmented product is prepared by adopting a nucleosome enzyme digestion combined column purification method.
2. The plasma circulating tumor free DNA standard according to claim 1, wherein the simulated plasma comprises sodium chloride, sodium bicarbonate, sodium carbonate, potassium chloride, dipotassium hydrogen phosphate trihydrate, magnesium chloride hexahydrate, sodium hydroxide, hydroxyethylpiperazine ethanethiosulfonic acid, calcium chloride, sodium sulfate, ethylenediamine tetraacetic acid, human serum albumin, and ultrapure water.
3. The free DNA standard substance for simulating circulating tumor in plasma according to claim 2, wherein the amounts of EDTA and human albumin in 1000mL of the simulated plasma are 1.5g and 50g, respectively.
4. The free DNA standard substance for simulating plasma circulating tumor according to claim 1, wherein the mixed solution is added into cells, ice is incubated after shaking, a non-ionic surfactant solution is added, ice is incubated after shaking, then centrifugal treatment is performed, the supernatant is discarded, then a micrococcal nuclease buffer solution is added, centrifugal treatment is performed after blowing, the supernatant is discarded, then a micrococcal nuclease buffer solution is added for resuspension, then micrococcal nuclease is added, water bath treatment is performed after shaking, an ethylene diamine tetraacetic acid solution and a ribonuclease A solution are added, water bath treatment is performed after shaking, then a sodium dodecyl sulfate solution and a protease K solution are added, water bath treatment is performed after shaking, and then purification and recovery are performed to obtain DNA fragments; the mixed solution comprises 4-hydroxyethyl piperazine ethanesulfonic acid solution, inorganic magnesium salt solution, inorganic potassium salt solution, organic reducing agent solution and benzyl sulfonyl fluoride solution; the micrococcus nuclease buffer solution comprises Tris-HCl buffer solution and CaCl2、BSA。
5. The plasma circulating tumor free DNA standard substance as claimed in claim 4, wherein the mixed solution is composed of 4-hydroxyethylpiperazine ethanesulfonic acid solution, inorganic magnesium salt solution, inorganic potassium salt solution, organic reducing agent solution, phenylmethylsulfonyl fluoride solution and water.
6. The plasma circulating tumor free DNA standard substance as claimed in claim 5, wherein the volume ratio of the 4-hydroxyethyl piperazine ethanesulfonic acid solution, the inorganic magnesium salt solution, the inorganic potassium salt solution, the organic reducing agent solution, the phenylmethylsulfonyl fluoride solution and the water is (8-12) to (1-2) to (8-12) to (0.3-0.8) to (8-12) to (900-1050).
7. The plasma circulating tumor free DNA standard substance as claimed in claim 4, wherein the volume ratio of the mixed solution, the nonionic surfactant solution, the ethylene diamine tetraacetic acid solution, the ribonuclease A solution, the sodium dodecyl sulfate solution and the proteinase K solution is 1000: 5-15: 30-50: 3-18: 70-150: 10-30.
8. The plasma circulating tumor-free DNA standard according to claim 4, wherein the background cells are wild-type background cells with multiple oncogenes mutated.
9. The method for preparing the plasma circulating tumor-simulating free DNA standard substance according to claim 1, wherein the plasma circulating tumor-simulating free DNA standard substance is obtained by mixing the plasma simulating free DNA standard substance with the fragmented DNA mixture; the fragmented DNA mixture is a background cell DNA fragmentation product, or the fragmented DNA mixture is a background cell DNA fragmentation product and a tumor cell DNA fragmentation product; preparing a background cell DNA fragmentation product by adopting a nucleosome enzyme digestion combined column purification method; preparing a tumor cell DNA fragmentation product by adopting a nucleosome enzyme digestion combined column purification method.
10. Use of the fragmented DNA mixture of claim 1 for the preparation of a plasma circulating tumor-free DNA mimetic standard or use of the plasma circulating tumor-free DNA mimetic standard of claim 1 for the detection of gene mutations.
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