Circulating tumor DNA (deoxyribonucleic acid) standard substance as well as preparation method and valuing method thereof

Abstract

The invention discloses a circulating tumor DNA (deoxyribonucleic acid) standard substance as well as a preparation method and a valuing method thereof. The preparation method comprises the following steps: (1) culturing cells; (2) cell nucleus extraction; (3) carrying out enzyme digestion on cell nucleuses; (4) purifying the fragmented DNA; (5) sampling a sample, and quantifying the sample by nanodrop measurement; (6) sampling the length of a verification fragment; and (7) mixing different ctDNAs according to the finally obtained theoretical mutation abundance of 1%, 5% and 10% to prepare the standard substance. The valuing method comprises the following steps: carrying out digital PCR amplification on a sample through a double-labeled probe to obtain the copy number concentration of a mutant gene and a wild gene or the copy number concentration of a target gene and a reference gene, and calculating the gene mutation frequency and the amplification multiple. According to the method disclosed by the invention, the fragmented DNA with the length similar to that of the ctDNA is obtained; 330bp and large fragments can be removed through two rounds of purification of a paramagnetic particle method, and a single-fragment standard substance is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA standard substances, and in particular to a circulating tumor DNA standard substance, a preparation method and a determination method. Background Art

[0002] Cancer is one of the increasingly serious problems in global health care. The main source of genetically related information for cancer patients is still tumor tissue samples, but local sampling cannot reflect the heterogeneity of tumors, let alone dynamic monitoring. Therefore, liquid biopsy is gradually used in clinical testing. Liquid biopsy, also known as liquid biopsy or liquid phase biopsy, refers to a detection technology that collects human body fluids as test samples to reflect the molecular characteristics and dynamic changes of tumors. Human body fluids contain circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes released by tumor cells, circulating tumor RNA (ctRNA), and other substances, which carry tumor-related information and can reflect the dynamic progression of tumors.

[0003] ctDNA is a cell-free DNA (cfDNA) derived from tumor somatic cells. cfDNA is a degraded DNA fragment released into body fluids such as plasma. ctDNA is derived from somatic DNA of tumor cells that is released into the circulation system after shedding or when cells undergo apoptosis. It is a characteristic tumor biomarker. Studies have confirmed that ctDNA matches sequencing results from tumor tissues. In various cancers, the consistency of ctDNA and tumor tissue mutations is greater than 60-80%. Through ctDNA testing, tumor traces in the blood can be detected, which can provide accurate information about cell types, gene expression, cell physiology or pathology, or therapeutic effects.

[0004] The length of cfDNA in healthy people is between 120-220bp, centered around 167bp, while studies have shown that ctDNA is more fragmented than cfDNA and 20bp-40bp shorter than cfDNA with non-cancer mutations, at 147bp or even shorter, and ctDNA is more specific for tumor detection. Detection of ctDNA can also reveal a variety of epigenetic changes such as single nucleotide variation, copy number variation, and methylation, which can make up for the shortcomings of imaging examinations and pathological biopsies that cannot obtain comprehensive biological information of tumors, and is therefore increasingly being used to guide clinical decision-making.

[0005] There are two main methods for ctDNA detection. One is targeted sequencing based on PCR methods, such as dPCR, BEAMing, and ARMS-qPCR, and the other is next-generation sequencing (NGS) technology with higher throughput than PCR, such as TEC-Seq and TAm-Seq. When detecting ctDNA with low mutation abundance, deep targeted sequencing of selected genomic regions can be used, but the results may also result in false positives or false negatives due to sequencing depth, signal interruption, etc. When impurities are present in the sample, the measurement results will also be affected. Therefore, it is necessary to develop relevant standard substances as measurement standards for detection method validation, product development and evaluation, and laboratory quality control, so as to improve the accuracy of experimental results and ensure the comparability of test results.

[0006] Standard materials have specific sequences and values ​​(sequence length, copy number, etc.) and have good uniformity and stability. They have the functions of reproducing, preserving and transmitting values. They can calibrate and evaluate equipment and methods, and are the "gold standard" to ensure the accuracy and traceability of results. However, there is still a lack of standard materials that can evaluate the reliability of test results. Ideal quality control materials should be extracted from patients. However, the ctDNA contained in the plasma of cancer patients is diluted by a large amount of DNA from non-cancerous sources in clinical practice. Therefore, ctDNA usually accounts for only a small part of the total free cfDNA. The concentration of ctDNA is much lower in patients with low tumor burden and certain types of cancer (such as glioma and renal cancer), so the ctDNA content in clinical samples is low. During the extraction and processing of clinical samples, ctDNA is extremely easy to degrade and has a short half-life, which will increase the false negative rate of the test. If the sample is not handled properly during processing, it is easy to cause white blood cells to rupture, and the genomic DNA released is mixed with ctDNA, which can further lead to a decrease in the proportion of ctDNA, which means that the false negative rate of the test is further increased. Therefore, clinical ctDNA cannot be used as a long-term source of standard materials, and it is necessary to develop alternative and reliable standard materials.

[0007] At present, there are two methods to meet the needs of ctDNA standard and reference product development, namely ultrasonic shearing and nucleosome enzyme digestion. Ultrasonic shearing is to ultrasonically lyse the cell genome by placing the lysed cells in an ultrasonic disruptor to obtain fragmented DNA; nucleosome enzyme digestion is to use micrococcal nuclease to act on the cell nucleus to fragment the cell DNA.

[0008] (1) The DNA fragments obtained by ultrasonic shearing are significantly different from those of natural ctDNA and cannot simulate real samples. In terms of fragment length distribution, the ctDNA obtained by ultrasonic shearing is in the range of 120bp-200bp, which is a wide range, while the length distribution range of natural ctDNA fragments is more concentrated; in terms of the biological characteristics of the ends of DNA fragments, natural ctDNA is mainly produced by enzymatic hydrolysis in vivo, and the ends of DNA molecules are 5' phosphate and 3' hydroxyl. However, the fragmented DNA obtained by ultrasonic shearing will have bases at the 5' and 3' protruding ends and 5' hydroxyl and 3' phosphate.

[0009] (2) Using the nucleosome enzyme digestion method, the samples prepared by the existing technology are fragments that simulate the length of cfDNA, that is, about 167bp, but the length of ctDNA is shorter, less than 145bp. If standard substances are to be developed, higher requirements will be placed on sample purity and measurement accuracy compared to standard products and reference products to meet the needs of measurement traceability. If this length of fragment DNA is used as a standard substance, it may be impossible to correctly control the quality, calibrate, and trace the measurement of ctDNA detection methods and products.

[0010] First, the PCR-based method uses a pair of primers and a probe to amplify DNA, and the amplified fragment is generally between 80-200bp. Samples of about 167bp cannot fully and correctly control samples below 145bp. If the length of the amplified fragment designed by the detection method is greater than 145bp, it will not be able to correctly amplify samples below 145bp, and using samples of about 167bp for quality control will not reveal this problem.

[0011] Secondly, the mechanism of ctDNA production has not yet been fully and clearly verified. The mutation information stored in fragments of different lengths will also be different. Current ctDNA detection methods include methods based on different principles such as NGS, and the comparability of the detection results remains to be evaluated. Therefore, standard substances with the same length as clinical samples are needed to be correctly used for comparison of methods based on different principles.

[0012] Therefore, it is necessary to optimize and control the nucleosome enzyme cleavage system to obtain samples that can produce a size consistent with ctDNA as standard material candidates for the next step of calibration research.

[0013] (3) Using the nucleosome digestion method, the existing technology may also produce fragments of about 145bp, but the sample size obtained after digestion generally exists in a gradient, that is, 100-200bp, 200-300bp, or even longer fragments. The development of standard substances needs to meet the requirements of purity and uniformity, so the prepared fragmented DNA needs to be purified to obtain samples of specific fragment lengths. Therefore, after digestion, the sample needs to be further purified to remove large fragments of DNA and retain the required characteristic fragments. The commonly used DNA purification method is column purification, which uses the adsorption column and collection tube provided by the kit to transfer the mixed sample to the adsorption column for DNA purification. This method can remove impurities such as proteins and salts, but cannot remove impurity DNA fragments.

[0014] In summary, for ctDNA detection, the field urgently needs to develop a new standard substance and its determination method, which can not only meet the technical requirements of standard substances for purity and accuracy, but also be consistent with the fragment length of clinical samples, and be applied to the calibration, evaluation, quality control and metrological traceability of ctDNA clinical detection methods for large multi-gene panels. Summary of the invention

[0015] The purpose of the present invention is to provide a circulating tumor DNA standard substance and a preparation method and a determination method thereof.

[0016] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0017] A method for preparing a circulating tumor DNA standard substance comprises the following steps:

[0018] (1) Cell culture

[0019] Tumor cell lines and normal cell lines containing hotspot mutations were used as materials, and all cells were cultured in a CO2 incubator;

[0020] (2) Cell nucleus extraction;

[0021] (3) Nucleolytic cleavage;

[0022] (4) Purification of fragmented DNA

[0023] ①The first round of magnetic bead purification

[0024] Add magnetic beads at a ratio of sample volume: magnetic bead volume = 1:0.9, blow and stir, leave at room temperature, place on a magnetic stand for adsorption, and retain the supernatant in a new EP tube;

[0025] ② Ethanol precipitation method to remove impurities

[0026] 1) Add 1 / 10 volume of sodium acetate to the DNA solution and mix thoroughly to make the final concentration 0.3 mol / L;

[0027] 2) Add 2 volumes of ice-cold anhydrous ethanol, mix thoroughly, and place at -20°C;

[0028] 3) Centrifuge in a 4°C centrifuge, carefully remove the supernatant, and remove all droplets on the tube wall;

[0029] 4) Add 1 / 2 volume of 70% ethanol to the centrifuge tube, centrifuge, remove the supernatant, and remove all droplets on the tube wall;

[0030] 5) Leave the opened EP tube at room temperature to allow the remaining liquid to evaporate to dryness;

[0031] 6) Add ddH2O to dissolve the DNA precipitate.

[0032] ③Second round of magnetic bead purification

[0033] 1) Vortex to mix the magnetic beads thoroughly, add 1.8 times the volume of the sample, pipette and stir, leave at room temperature, place on a magnetic stand for adsorption, and discard the supernatant;

[0034] 2) Add ethanol, place at room temperature, adsorb on a magnetic rack, discard the supernatant, and repeat the wash twice;

[0035] 3) Add TE, pipette, place at room temperature, adsorb on a magnetic rack, and the supernatant is ctDNA;

[0036] (5) Take samples and use nanodrop to measure the samples for quantification;

[0037] (6) Take samples to verify the fragment length;

[0038] (7) According to the final theoretical mutation abundance, different ctDNAs are mixed to prepare standard substances.

[0039] Furthermore, in step (1), the tumor cell line containing hotspot mutations includes at least one of the following:

[0040]

[0041] The normal cell line is PLCL6.

[0042] Furthermore, in the step (1), when the cells are cultured to cover 80-90% of the bottom of the flask, the original culture medium is discarded, PBS is added to rinse the bottom of the culture flask, and then the cells are digested with trypsin containing EDTA, and complete culture medium is added to terminate the digestion, and the cell suspension is collected into a centrifuge tube; the collected cells are counted, the cells are divided into centrifuge tubes, and the cell precipitates are collected after centrifugation and stored at -80°C for further use.

[0043] Furthermore, the step (2) specifically includes adding PBS to the collected cell precipitate to wash the cells; centrifuging and discarding the supernatant; adding pre-cooled NP-40 to resuspend the cells, and incubating on ice to lyse the cells; and using a cell nuclear extraction kit to extract the cell nuclear precipitate into a centrifuge tube.

[0044] Furthermore, the step (3) specifically comprises: enzymatically hydrolyzing the nuclear DNA with micrococcal nuclease, adding ultrapure water, micrococcal nuclease buffer, protease inhibitors, recombinant albumin, and micrococcal nuclease to the nuclear precipitate, incubating in a metal bath, and then inactivating the enzyme; adding ultrapure water, proteinase K solution, and SDS solution to the digested nuclei, and incubating at 60°C.

[0045] Furthermore, in step (5), the DNA purity is evaluated by measuring A260 / A280 and A260 / A230. If the DNA satisfies A260 / A280 of 1.8 to 2.0 and A260 / A230 is greater than 2, then the purity requirement of the DNA standard substance is met.

[0046] Furthermore, in step (7), different ctDNAs are mixed to prepare three standard substances according to the final theoretical mutation abundance of 10%, 5%, and 1%.

[0047] The method for determining the value of the circulating tumor DNA standard substance of the present invention is specifically as follows: digital PCR amplification is performed on the sample by using a double-labeled probe (FAM channel labels mutant genes, and VIC channel labels wild probes; or FAM channel labels target genes, and VIC channel labels reference genes) to obtain the copy number concentrations of mutant genes and wild genes (or target genes and reference genes), and the gene mutation frequency and amplification multiple are calculated by a formula;

[0048]

[0049] Compared with the prior art, the outstanding effects of the present invention are:

[0050] (1) The present invention utilizes micrococcal nuclease to enzymatically hydrolyze nuclear DNA, and combines two rounds of magnetic bead purification and ethanol precipitation to efficiently, stably, and reproducibly prepare standard substances that can be used for ctDNA detection.

[0051] (2) The present invention obtains fragmented DNA with a length similar to that of ctDNA through overall optimization and adjustment of the enzyme cleavage system and the enzyme cleavage method, and can correctly perform quality control, calibration and quantity traceability on ctDNA detection methods based on different principles and different detection products.

[0052] (3) The present invention can remove 330 bp and large fragments through two rounds of purification by magnetic bead method, and can eliminate the interference of impurities in the solution on subsequent analysis through ethanol precipitation method to obtain a single fragment standard substance. The purification method is simple and efficient, and meets the requirements of purity and uniformity for the development of standard substances.

[0053] (4) The method of the present invention can be applied to different cell lines to obtain ctDNA containing different mutation sites, which can be mixed to prepare standard materials containing multiple gene mutation sites. The application and verification in 10 different cell types demonstrated that the method of the present invention is universal and reproducible.

[0054] (5) The present invention uses a digital PCR method to accurately determine the value of standard substances, thereby obtaining standard substances with accurate mutation frequencies (amplification mutation multiples), which are applied to the calibration, evaluation, quality control and metrological traceability of large-panel detection methods and detection products of multiple gene loci.

[0055] The circulating tumor DNA standard substance and its preparation method and determination method described in the present invention are further described below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1-Figure 10 Verification results of ctDNA fragment length and purity obtained from different cells.

[0057] Figure 11-13 These are the purity verification results of the three standard substances.

[0058] Figure 14-15 This is the ctDNA purification effect (SNU-5).

[0059] Figure 16-Figure 24 This is the two-dimensional amplification diagram of ctDNA digital PCR of standard substance three. DETAILED DESCRIPTION

[0060] 1. Preparation of Standard Substances

[0061] 1. Cell culture

[0062] Tumor cell lines and normal cell lines containing hotspot mutations were used as materials for ctDNA preparation. The following protocol uses a mixture of 10 cell lines as an example for preparation. The sources of samples in the preparation of standard substances are shown in Table 1 below. All cells were cultured in a CO2 incubator with a CO2 concentration of 5% and a temperature of 37°C.

[0063] Table 1 Correspondence between mutations and source cell lines

[0064]

[0065] Among them, SK-BR-3, MIA-PaCa-2, PLC / PRF / 5, HCC38, BT20 (EGFR and PIK3CA), 293T-AKT, SNU-5, SNU-16 and PLCL6 were used to prepare standard substance 1. SK-BR-3, MIA-PaCa-2, PLC / PRF / 5, HCC38, C3A, BT20 (PIK3CA), 293T-AKT, SNU-5, SNU-16 and PLCL6 were used to prepare standard substances 2 and 3.

[0066] When the cells are cultured to 80-90% of the bottom of the bottle, discard the original culture medium, add PBS to rinse the bottom of the culture bottle, and then digest the cells with trypsin containing 0.25% EDTA, and add complete culture medium to stop digestion, collect the cell suspension into a centrifuge tube. Count the collected cells, centrifuge the cells at 1500g / min for 5 minutes, collect the cell pellet, and store it at -80℃ for further use.

[0067] 2. Cell nuclear extraction

[0068] Add 1 mL of PBS to the collected cell pellet and wash the cells twice. Centrifuge at 4°C, 800 g for 10 min in a refrigerated centrifuge and discard the supernatant. Add 1 mL of pre-cooled NP-40 to resuspend the cells and incubate on ice for 10 min to lyse the cells. Use a nuclear extraction kit to extract the nuclear pellet into a centrifuge tube.

[0069] 3. Nucleolytic enzyme digestion

[0070] Micrococcal nuclease was used to enzymatically digest the nuclear DNA. 100 μL ultrapure water, 10 μL 10× micrococcal nuclease buffer, 10 μL 10× protease inhibitor, 0.5 μL 20 mg / mL recombinant albumin, 0.5 μL 2×10 6 gel units / mL micrococcal nuclease, incubate at 37°C for 10 min in a metal bath, and inactivate the enzyme at 65°C for 10 min. Add 120 μL ultrapure water, 3 μL 20 g / l proteinase K solution, and 10 μL 20% SDS solution to the digested nuclei and incubate at 60°C for 20 min.

[0071] 4. Purification of fragmented DNA

[0072] (1) First round of magnetic bead purification

[0073] Add magnetic beads at a ratio of sample volume: magnetic bead volume = 1:0.9, pipette and beat 10 times, leave at room temperature for 5 minutes, place on a magnetic stand for adsorption for 2 minutes, and retain the supernatant in a new EP tube.

[0074] (2) Ethanol precipitation method to remove impurities

[0075] 1) Add 1 / 10 volume of sodium acetate (3 mol / L, pH=5.2) to the DNA solution and mix thoroughly to make the final concentration 0.3 mol / L;

[0076] 2) Add 2 volumes of ice-cold anhydrous ethanol, mix thoroughly, and place at -20°C for 25 minutes;

[0077] 3) Centrifuge at 12000g for 10 minutes at 4°C, carefully remove the supernatant, and remove all droplets on the tube wall;

[0078] 4) Add 1 / 2 volume of 70% ethanol to the centrifuge tube, centrifuge at 12000g for 2 minutes, carefully remove the supernatant, and remove all droplets on the tube wall;

[0079] 5) Leave the opened EP tube at room temperature to allow the remaining liquid to evaporate to dryness;

[0080] 6) Add 200 μL of ddH2O to dissolve the DNA precipitate.

[0081] (3) Second round of magnetic bead purification

[0082] 1) Vortex to mix the magnetic beads thoroughly, add 1.8 times the volume of the sample, pipette 10 times, let stand at room temperature for 5 minutes, place on a magnetic stand for 2 minutes, and discard the supernatant.

[0083] 2) Add 200 μL of 70% ethanol, place at room temperature for at least 30 seconds, adsorb on a magnetic rack for 2 minutes, discard the supernatant, and repeat the wash twice.

[0084] 3) Add 100 μL of TE and pipette 20 times, place at room temperature for 5 minutes, and adsorb on a magnetic rack for 2 minutes. The supernatant is ctDNA.

[0085] 5. Take 2 μL of sample and use nanodrop to measure and quantify the sample. DNA purity is evaluated by measuring A260 / A280 and A260 / A230. If DNA meets the A260 / A280 range of 1.8 to 2.0 and A260 / A230 is greater than 2, it meets the purity requirements of DNA standard material. The results are shown in Table 2.

[0086] Table 2 ctDNA preparation yield and quality

[0087]

[0088] 6. Take 1 μL of sample and use Agilent 2100 Bioanalyzer to verify the fragment length. The result is as follows: Figure 1-Figure 10 shown.

[0089] 7. According to the final theoretical mutation abundance of 10%, 5%, and 1%, different ctDNAs were mixed to prepare standard substance 1, standard substance 2, and standard substance 3 respectively. The purity verification results of the three standard substances are as follows Figure 11-13 Taking SNU-5 as an example, the ctDNA purification effect is as follows Figure 14-15 shown.

[0090] 2. Determination of the value of reference materials

[0091] The digital PCR method is used to determine the value of the standard substance, and the sample is amplified by digital PCR using a double-labeled probe (FAM channel labels the mutant gene, and the VIC channel labels the wild-type probe; or FAM channel labels the target gene, and the VIC channel labels the reference gene) to obtain the copy number concentration of the mutant gene and the wild-type gene (or the target gene and the reference gene), and the gene mutation frequency and amplification multiple are calculated using the formula.

[0092]

[0093] The sequences of the primer probes used are shown in Table 3 (shown in SEQ ID NOs: 1-40), the concentrations of the primer probes are shown in Table 4, and the reaction program is set in a PCR instrument according to Table 5 for PCR amplification.

[0094] Table 3 Primer probe sequence information

[0095]

[0096]

[0097]

[0098] Table 4 Primer probe concentration and annealing temperature information

[0099] Gene mutation site Final primer concentration Final probe concentration Annealing temperature TP53-R175H 500nM 250nM 56℃ TP53-R248W 500nM 500nM 56℃ TP53-R249S 500nM 250nM 56℃ TP53-R273L 500nM 125nM 56℃ NRAS-Q61L 500nM 250nM 56℃ EGFR 900nM 250nM 56℃ PIK3CA-H1047R 900nM 350nM 56℃ AKT1-E17K 500nM 250nM 56℃ MET 500nM 125nM 56℃ MYC 500nM 250nM 56℃ Reference gene 250nM 100nM 56℃

[0100] Table 5 PCR instrument reaction program

[0101]

[0102] The results of the determination are shown in Table 6. Taking standard substance 3 as an example, its amplification diagram is shown in Figure 16-Figure 24 shown.

[0103] Table 6 Digital PCR determination results of standard substances

[0104]

[0105] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a circulating tumor DNA standard substance, characterized in that: The following steps are involved: (1) Cell culture Tumor cell lines and normal cell lines containing hotspot mutations were used as materials, and all cells were cultured in a CO2 incubator; (2) Cell nucleus extraction; (3) Nucleolytic cleavage; (4) Purification of fragmented DNA ①The first round of magnetic bead purification Add magnetic beads at a ratio of sample volume: magnetic bead volume = 1:0.9, blow and stir, leave at room temperature, place on a magnetic stand for adsorption, and retain the supernatant in a new EP tube; ② Ethanol precipitation method to remove impurities ③Second round of magnetic bead purification 1) Vortex to mix the magnetic beads thoroughly, add 1.8 times the volume of the sample, pipette and stir, leave at room temperature, place on a magnetic stand for adsorption, and discard the supernatant; 2) Add ethanol, place at room temperature, adsorb on a magnetic rack, discard the supernatant, and repeat the wash twice; 3) Add TE, pipette, place at room temperature, adsorb on a magnetic rack, and the supernatant is ctDNA; (5) Take samples and use nanodrop to measure the samples for quantification; (6) Take samples to verify the fragment length; (7) According to the final theoretical mutation abundance, different ctDNAs are mixed to prepare standard substances.

2. The method for preparing circulating tumor DNA standard material according to claim 1, characterized in that: In step (1), the tumor cell line containing hotspot mutations includes at least one of the following: The normal cell line is PLCL6.

3. The method for preparing circulating tumor DNA standard material according to claim 2, characterized in that: In the step (1), when the cells are cultured to 80-90% of the bottom of the flask, the original culture medium is discarded, PBS is added to rinse the bottom of the culture flask, and then the cells are digested with trypsin containing EDTA, and complete culture medium is added to terminate the digestion, and the cell suspension is collected into a centrifuge tube; The collected cells were counted, the cells were dispensed into centrifuge tubes, the cell pellets were collected after centrifugation, and stored at -80°C for further use.

4. The method for preparing circulating tumor DNA standard material according to claim 3, characterized in that: The step (2) specifically comprises adding PBS to the collected cell precipitate to wash the cells; centrifuging and discarding the supernatant; adding pre-cooled NP-40 to resuspend the cells, incubating on ice to lyse the cells; and using a cell nuclear extraction kit to extract the cell nuclear precipitate into a centrifuge tube.

5. The method for preparing circulating tumor DNA standard material according to claim 4, characterized in that: The step (3) specifically comprises: enzymatically hydrolyzing the nuclear DNA with micrococcal nuclease, adding ultrapure water, micrococcal nuclease buffer, protease inhibitors, recombinant albumin, and micrococcal nuclease to the nuclear precipitate, incubating in a metal bath, and then inactivating the enzyme; adding ultrapure water, proteinase K solution, and SDS solution to the digested nucleus, and incubating at 60°C.

6. The method for preparing circulating tumor DNA standard material according to claim 5, characterized in that: In the step (4), the specific method of removing impurities by ethanol precipitation is: 1) Add 1 / 10 volume of sodium acetate to the DNA solution and mix thoroughly to make the final concentration 0.3 mol / L; 2) Add 2 volumes of ice-cold anhydrous ethanol, mix thoroughly, and place at -20°C; 3) Centrifuge in a 4°C centrifuge, carefully remove the supernatant, and remove all droplets on the tube wall; 4) Add 1 / 2 volume of 70% ethanol to the centrifuge tube, centrifuge, remove the supernatant, and remove all droplets on the tube wall; 5) Leave the opened EP tube at room temperature to allow the remaining liquid to evaporate to dryness; 6) Add ddH2O to dissolve the DNA precipitate.

7. The method for preparing circulating tumor DNA standard material according to claim 6, characterized in that: In the step (5), the purity of the DNA is evaluated by measuring A260 / A280 and A260 / A230. If the DNA satisfies A260 / A280 of 1.8 to 2.0 and A260 / A230 is greater than 2, then the purity requirement of the DNA standard substance is met.

8. The method for preparing circulating tumor DNA standard material according to claim 7, characterized in that: In the step (7), different ctDNAs are mixed to prepare three standard substances according to the final theoretical mutation abundance of 10%, 5%, and 1%.

9. A circulating tumor DNA standard substance prepared by the preparation method according to any one of claims 1 to 8.

10. The method for determining the value of circulating tumor DNA standard substance according to claim 9, characterized in that: The samples are digitally amplified by double-labeled probes to obtain the copy number concentration of mutant genes and wild-type genes, or the copy number concentration of target genes and reference genes, and the gene mutation frequency and amplification multiple are calculated by formula;

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