Tumor circulating free DNA reference and preparation method thereof

Abstract

The invention provides a tumor circulating free DNA reference and a preparation method thereof, and the preparation method comprises the following steps: S1, preparation of fragmented DNA: breaking mixed wild gDNA and mutant gDNA by using ultrasound to prepare the fragmented DNA; or respectively breaking the wild type gDNA and the mutant type gDNA by utilizing ultrasound, and then mixing to prepare the fragmented DNA; s2, preparing a ctDNA reference product: performing magnetic bead purification on the fragmented DNA to obtain the ctDNA reference product; wherein the wild type gDNA is genome DNA which is not mutated, and the mutant type gDNA is genome DNA which is mutated at a preset site. According to the method disclosed by the invention, the proportion of the sizes of DNA fragments of 100bp-200bp is increased, and the distribution of the sizes of the fragments is closer to that of natural ctDNA.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and particularly relates to a circulating free DNA reference product for tumors and a preparation method thereof. Background Art

[0002] According to the latest global cancer data released by the International Agency for Research on Cancer (IARC) of the World Health Organization, in 2020, the number of newly diagnosed cases of breast cancer in women was approximately 2.26 million, exceeding that of lung cancer (2.2 million) for the first time to become the "world's largest cancer". In 2020, both the number of newly diagnosed cases of breast cancer and the number of breast cancer deaths in China ranked first in the world. The growth rate of breast cancer incidence in China ranked first in the world, being twice the global average growth rate. The prevention and treatment situation of breast cancer in China faces huge challenges. At the same time, the five-year survival rate of breast cancer in China exceeds 80%, and the number of existing patients is large. However, existing patients still have the risk of recurrence or metastasis, and there is a great demand for effectively monitoring the patient's condition and timely detecting the postoperative recovery and recurrence and metastasis status.

[0003] Among breast cancer patients, more than 70% are positive for estrogen receptor α (ERα). Endocrine therapy is currently the standard treatment for ER-positive patients. However, almost all endocrine therapies develop drug resistance after several years, leading to recurrence and metastasis and ultimately death, which is a huge challenge in clinical practice. The main drug resistance mechanisms of endocrine therapy include acquired mutations in the estrogen receptor α gene (ESR1), activation of the PI3K / AKT / mTOR signaling pathway, etc. ESR1 point mutation is an important cause of endocrine resistance in ER-positive breast cancer, and its high-frequency mutation sites are mainly concentrated in the ligand-binding domain. The PI3K pathway includes genes such as PIK3CA, AKT1, and PTEN. Mutations in these genes are highly correlated with pathway overactivation and endocrine therapy resistance. The occurrence of mutations is associated with an increased risk of disease progression and is an indicator of poor prognosis. Analyzing the above gene mutation status is of great significance for guiding the clinical precision treatment of breast cancer, early detection of recurrence, and monitoring of treatment response.

[0004] At present, the main source of obtaining tumor gene-related information is still the tumor tissue samples of patients. The methods of surgical resection or diagnostic tissue biopsy are limited by factors such as the tumor growth site, the size of the tissue sample taken, and tumor heterogeneity, and cannot completely describe the genetic changes of the whole tumor. The conclusion that overgeneralizes may even mislead the treatment plan. For advanced patients and those not suitable for surgery, it is impossible to obtain tumor tissue samples, and it is even less suitable for dynamic monitoring. Liquid biopsy involves collecting and testing the patient's peripheral blood. The plasma DNA sample contains circulating tumor DNA (ctDNA), and its detection has the advantages of sensitivity, homogeneity, real-time nature, etc. It can avoid invasive tissue biopsy and is also convenient for repeated sample collection. Moreover, research shows that ctDNA is more sensitive than protein indicators and imaging, and can detect the spread and recurrence of advanced breast cancer tumors 3 to 6 months in advance, which is of great significance in tumor recurrence prediction, drug resistance monitoring, and medication guidance. Therefore, the ctDNA gene detection of plasma samples has been recommended in the clinical consensus of certain cancers.

[0005] The mutation information of tumor-related genes can be obtained using ctDNA in plasma samples. However, due to the extremely low absolute and relative content of ctDNA in plasma, the detection of ctDNA usually requires detecting extremely low (0.1% or even lower) gene mutation frequencies, which poses extremely high requirements for the sensitivity of ctDNA detection. Although the technical sensitivity for detecting tumor-related mutant genes in ctDNA has been greatly improved at present, the detection of rare gene mutations and ultra-low frequency gene mutations still poses great challenges. Therefore, in the relevant detection process, it is necessary to strictly and fully evaluate or verify various performances to ensure that lower frequency mutation information can be detected stably, reliably, and reproducibly. This also requires establishing a set of simulated plasma ctDNA that can be stably prepared, has stable performance, can be stored for a relatively long time, and has a high mutation coverage rate as a detection reference product.

[0006] To meet the main clinical requirements of clinicians for sufficient technical verification data, high product precision, and comprehensive detectable indicators of ctDNA detection products, to make the detection results of diagnostic reagents stable and reproducible, and to ensure the reliability of clinical applications, more stringent requirements are put forward for parameters such as the type (mutation coverage), stability, and minimum detection limit of reference products during the R & D process. It is necessary to reasonably set reference products and conduct quality control on the various performances of reference products to ensure the quality of the developed diagnostic reagents. At the same time, reference products are closely related to R & D costs and R & D cycles, and reference products are required in many links during the R & D process of diagnostic reagents. Therefore, the configuration of reference products / reference product plates can not only reduce R & D costs, but also reduce the number of detection reactions, thereby reducing clinical use costs; at the same time, it can also speed up the R & D progress, shorten the R & D cycle, accelerate the clinical application of diagnostic reagents, and further reduce the medical expense burden on patients and society.

[0007] At present, many companies have launched tumor mutation ctDNA detection kits and corresponding gene mutation reference products. On the one hand, these products usually detect tumor mutation genes that appear in many common cancers, and there is no product for detecting gene mutations related to endocrine therapy resistance in advanced breast cancer. On the other hand, the gene types and mutation sites covered by the above products are few and scattered, especially the detection of ESR1 hotspot mutations is even less. Therefore, the current reference products cannot meet the needs of this specific detection and product research and development, and there is an urgent need to develop them. It is expected that with a small number of reference products, as many genes and / or multiple mutation sites related to endocrine therapy resistance in breast cancer can be covered simultaneously as possible.

[0008] CtDNA in normal human body is mainly small and uniform DNA fragments released into plasma through apoptosis. The sizes of ctDNA fragments vary, with more than 70% of plasma ctDNA fragments being less than 300bp in length, ranging from about 50bp to 300bp, and the average length being 167bp. Currently, the main methods for preparing simulated ctDNA are artificial synthesis of plasmids or PCR products, non-specific enzymatic digestion, and physical and mechanical fragmentation. The artificial synthesis of plasmids or PCR products is being gradually phased out because their sequence complexity is much lower than that of natural ctDNA in its normal biological state and they cannot simulate the characteristics of natural ctDNA. By using non-specific endonucleases to digest and fragment gDNA, it usually starts from culturing cells and extracting cell nuclei, which has a long cycle and high cost; moreover, the degree of DNA fragmentation has high requirements for sample quality, and a large number of exploratory experiments need to be carried out on the enzymatic digestion reaction conditions. It is impossible to accurately control the time for molecular-level reactions, and the simulated ctDNA obtained has a large deviation from natural ctDNA; in addition, enzymatic digestion fragmentation also has a drawback: most nucleases have a cleavage preference for regions rich in AU or AT at base sites. The method of enzymatic digestion fragmentation has unstable fragmentation, large differences between batches, and low repeatability, and is not the best choice for industrialized and large-scale production. Mechanical fragmentation, especially ultrasonic fragmentation, is currently the main method for obtaining fragmented DNA or simulated ctDNA. By using mechanical vibration or acoustic vibration to break relatively intact genomic DNA into fragments. Different from the method of forming simulated ctDNA by enzymatic digestion fragmentation, mechanical fragmentation, especially ultrasonic fragmentation, is relatively more controllable in experimental methods and repeatability, and can also avoid costly operations such as culturing cells, extracting cell nuclei, and exploring reaction conditions, making it more suitable for automated and industrialized production. However, the size distribution of simulated ctDNA fragments obtained by ultrasonic fragmentation is different from that of natural ctDNA. Most simulated ctDNA fragments obtained by ultrasonic fragmentation methods are smaller, with a narrower distribution range, usually 25bp - 500bp or even lower to below 300bp. In natural ctDNA, there are some large fragments of specific sizes due to the intracellular cleavage mechanism, such as obvious 300 - 400bp and 400 - 500bp. And the main peak of the simulated DNA fragments generated by ultrasonic fragmentation is smaller, with about 50% or even lower proportion around 170bp, far lower than the proportion of about 85% of the main peak of natural ctDNA fragment distribution at 167bp, and the sizes of natural ctDNA fragments are normally distributed between 150bp - 180bp. The recommended size of ctDNA amplicons is about 80 - 100bp. Too many small DNA fragments will inhibit PCR amplification, thus affecting the detection effect of ctDNA. Therefore, the size of ctDNA fragments directly affects the PCR amplification effect, resulting in problems such as low detection limit and high background noise in subsequent experiments, increasing the R & D cost and cycle and also affecting the detection performance.

[0009] Patent CN 110894524 A describes a method for rapidly preparing a gene mutation reference product. This method uses a plasmid containing a target gene mutation sequence and PCR to obtain a PCR product containing the target gene mutation sequence, which is mixed with the genomic DNA wild type of a wild-type cell line (293T) to prepare BRAF and KRAS gene mutation reference products, which can be used for subsequent ultrasonic fragmentation, but does not further describe the relevant verification and application of ultrasonic fragmentation. Patent CN 109810973 A describes a method for using ultrasonic fragmentation to simulate cfDNA. This method uses gradient ultrasonic fragmentation of gDNA to improve the fragmentation effect, but the size distribution of the DNA generated by fragmentation is very different from that of natural cfDNA. Patent CN 114717314 A describes a reference product and reference disk for plasma-free DNA mutation detection, which are obtained by two methods of enzymatic digestion and fragmentation, but do not present the verification data of the size distribution characteristics of the prepared simulated cfDNA. Patent CN 113106149 B describes a preparation method of a fusion gene detection reference product and the application of the fusion gene detection reference product in NGS library construction and sequencing analysis. In this method, an in vitro artificial synthesis of DNA is used to construct a DNA fusion positive fragment, which is mixed with negative genomic DNA and purified after ultrasonic fragmentation to obtain a reference product before the initial library preparation. Since the ctDNA detection sample is fragmented human genomic DNA, and the preparation method in this patent uses synthetic DNA, it cannot truly reflect the complexity of the human tumor ctDNA sample and is not an ideal sample. In addition, the average size of the main peak of the simulated ctDNA fragment obtained by fragmentation is about 192bp, which is still quite different from the main peak of natural ctDNA, which is 167bp. In the implementation schemes of some reference product manufacturers, the length of the simulated ctDNA fragment obtained by ultrasonic fragmentation is between 35bp and 1000bp; more preferably, it is between 35bp and 500bp.

[0010] CN111321136A discloses a cfDNA wild-type reference product and its preparation method. This method limits the size range of the target DNA fragment: most of the product fragments in this method are distributed in the range <300bp, and the proportion of the 300 - 400bp part is extremely low or even absent; the SPRIselect kit (purchased from Beckman-Coulter, B23318) is used in the purification process, and the cost is relatively high; and the purification process uses a two-step magnetic bead purification method, which further increases the usage amount of the SPRIselect reagent, increases the operation complexity and time cost; at the same time, the DNA loss is large, the yield is low, the DNA input amount is increased, and the time and economic costs for preparing the reference product are increased; at the same time, the wild-type reference product prepared by this method is designed for library construction and sequencing methods, and the sensitivity for fluorescence PCR methods needs to be optimized and verified.

[0011] In summary, there is an urgent need for a method to stably prepare a simulated ctDNA reference product with a source of human genomic DNA, a fragment size distribution closer to natural ctDNA, high sensitivity, and low cost. Summary of the Invention

[0012] Aiming at the deficiencies existing in the prior art, the present invention provides a tumor circulating free DNA reference product and a preparation method thereof. Specifically, the present invention provides a reference product and a preparation method thereof that can be used for tumor-related mutant gene detection technologies based on ARMS and Taqman qPCR. Specifically, it is a reference product and a reference product plate for detecting gene mutations related to endocrine therapy resistance in breast cancer using plasma free DNA.

[0013] On the one hand, the present invention optimizes the source of the sample for preparing the simulated ctDNA reference product, extracts the genomic DNA of an edited cell line with a specific mutation site, and mixes it with negative genomic DNA in a specific proportion. Compared with other artificial synthetic plasmids, DNA fragments, or PCR products, the similarity with natural ctDNA in terms of sample characteristics, structure, and sequence complexity is improved; on the other hand, by optimizing the parameters of ultrasonic fragmentation, the absolute proportion of DNA fragments in the range of 150 - 180 bp is increased, the fragmentation efficiency of genomic DNA is improved, and at the same time, some fragments of 300 - 400 bp are retained, making it more in line with the fragment size ratio of natural ctDNA and reducing the cost of preparing simulated ctDNA; finally, combined with secondary magnetic bead sorting and purification, by optimizing the magnetic bead ratio, products with single bases, multiple bases, and ultra-short genomic DNA fragments below 50 bp that are inevitably generated during the ultrasonic process are further removed, thereby increasing the proportion of DNA fragment sizes of 100 bp - 200 bp and being closer to natural ctDNA in terms of fragment size distribution.

[0014] In the first aspect of the present invention, a method for preparing a tumor circulating free DNA reference product is provided, including the following steps:

[0015] S1 Prepare fragmented DNA: Use ultrasound to fragment the mixed wild-type gDNA and mutant gDNA to obtain fragmented DNA; or use ultrasound to fragment the wild-type gDNA and mutant gDNA separately; then mix to obtain fragmented DNA;

[0016] S2 Prepare the ctDNA reference product: Purify the fragmented DNA with magnetic beads to obtain the ctDNA reference product;

[0017] Among them, the wild-type gDNA is genomic DNA that has not mutated, and the mutant gDNA is genomic DNA that has mutated at a preset site.

[0018] In one embodiment of the present invention, the wild-type gDNA is NA12878.

[0019] In one embodiment of the present invention, the mutant gDNA is from the genomic DNA of an edited cell line with specific mutation sites.

[0020] In one embodiment of the present invention, the amino acid sequence encoded by the mutant gDNA includes one or more of the following mutation sites and does not contain any amino acid mutations in other groups: the amino acid mutation p.E17K of AKT1, the amino acid mutation p.E380Q of ESR1, the amino acid mutation p.Y537S of ESR1, the amino acid mutation p.Y537N of ESR1, the amino acid mutation p.D538G of ESR1, the amino acid mutation p.E542K of PIK3CA, the amino acid mutation p.E545K of PIK3CA, the amino acid mutation p.H1047R of PIK3CA, the amino acid mutation p.H1047L of PIK3CA.

[0021] In one embodiment of the present invention, the nucleotide sequence of the mutant gDNA includes one or more of the following mutation sites and does not contain any base mutations in other groups: the base mutation corresponding to p.E17K is c.49G>A, the base mutation corresponding to p.E380Q is c.1138G>C, the base mutation corresponding to p.Y537S is c.1610A>C, the base mutation corresponding to p.Y537N is c.1609T>A, the base mutation corresponding to p.D538G is c.1613A>G, the base mutation corresponding to p.E542K is c.1624G>A, the base mutation corresponding to p.E545K is c.1633G>A, the base mutation corresponding to p.H1047R is c.3140A>G, the base mutation corresponding to p.H1047L is c.3140A>T.

[0022] In one embodiment of the present invention, the mutant gDNA is the amino acid mutation p.Y537N of ESR1.

[0023] In one embodiment of the present invention, the mutant gDNA is ESR1 p.Y537N Reference Standard Plus - 1% Mutation.

[0024] In one embodiment of the present invention, the mass ratio of the mutant gDNA to the wild-type gDNA makes the base mutation frequency of the reference product one or more of the following: VAF>10%, 1%<VAF<10%, VAF<1%.

[0025] In one embodiment of the present invention, the mass ratio of the mutant gDNA to the wild-type gDNA is such that the base mutation frequency of the reference product is any one of 50%, 25%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0.3%, and 0.1%.

[0026] In one embodiment of the present invention, the mass ratio of the mutant gDNA to the wild-type gDNA is 1:1 - 9. In one embodiment of the present invention, the mass ratio of the mutant gDNA to the wild-type gDNA is 1:1. In one embodiment of the present invention, the mass ratio of the mutant gDNA to the wild-type gDNA is 1:9.

[0027] In one embodiment of the present invention, the operating conditions of the ultrasound include: peak power of 175 - 210 W, load ratio of 10%, number of single-pulse cycles of 200, and processing time of 300 - 580 S.

[0028] In a preferred embodiment of the present invention, the peak power of the ultrasound is 200 - 210 W, the load ratio is 10%, the number of single-pulse cycles is 200, and the processing time is 400 - 450 S.

[0029] In a preferred embodiment of the present invention, the peak power of the ultrasound is 210 W, the load ratio is 10%, the number of single-pulse cycles is 200, and the processing time is 430 S.

[0030] In one embodiment of the present invention, the input amount of the gDNA sample in the ultrasound is 0.5 μg / tube.

[0031] In one embodiment of the present invention, the steps of purifying the fragmented DNA with magnetic beads include:

[0032] 1) Adding magnetic beads to the fragmented DNA solution so that the fragmented DNA with the first length binds to the magnetic beads, while the fragmented DNA with the second length remains in the solution;

[0033] 2) Separating the magnetic beads bound to the fragmented DNA with the first length from the solution containing the fragmented DNA with the second length;

[0034] 3) Separating the fragmented DNA with the first length from the magnetic beads; obtaining the ctDNA reference product;

[0035] In one embodiment of the present invention, the size of the first length is 100 - 400 bp, preferably 100 - 220 bp, 300 - 400 bp, and more preferably 160 bp ± 10%; the size of the second length is 35 - 100 bp.

[0036] In one embodiment of the present invention, the volume ratio of the fragmented DNA to the magnetic beads is 1:0.6 - 1:2.8, preferably 1:2 - 1:2.5.

[0037] In one embodiment of the present invention, the concentration of the solution of the fragmented DNA is 0.005 ng / μL - 120 ng / μL; preferably 0.1 ng - 120 ng / μL.

[0038] In a specific embodiment of the present invention, the specific steps for purifying the fragmented DNA with magnetic beads include:

[0039] (1) Take the fragmented DNA and dissolve it in TE buffer to form a fragmented DNA solution, where the concentration of the fragmented DNA in the fragmented DNA solution is 0.005 ng / μL - 120 ng / μL, preferably 0.1 ng - 120 ng;

[0040] (2) Add magnetic beads to the fragmented DNA solution, mix evenly, and incubate at room temperature to allow the fragmented DNA with the first length to bind to the magnetic beads, while the fragmented DNA with the second length remains in the solution. The size of the first length is 100 - 400 bp, preferably 100 - 220 bp, 300 - 400 bp, more preferably 160 bp ± 10%; the size of the second length is 35 - 100 bp; preferably the magnetic beads are AMPure XP magnetic beads;

[0041] (3) Use a magnetic stand to adsorb the magnetic beads. After the solution becomes clear, remove the solution;

[0042] (4) Wash the magnetic beads, incubate at room temperature, and remove the supernatant; preferably wash with ethanol;

[0043] (5) Repeat step (4);

[0044] (5) After drying the magnetic beads, add an eluent for elution and incubate at room temperature to obtain an elution mixture; preferably the eluent is TE buffer;

[0045] (6) Use a magnetic stand to adsorb the magnetic beads to make the elution mixture obtained in step (5) clear, and then separate the clear liquid to complete fragment sorting and obtain a ctDNA reference product.

[0046] In one embodiment of the present invention, the magnetic beads are AMPure XP magnetic beads.

[0047] In one embodiment of the present invention, it further includes the step of detecting the sensitivity of the ctDNA reference product.

[0048] In one embodiment of the present invention, the sensitivity of the ctDNA reference sample detection is as follows: the initial sample concentration is 0.005 ng / μL - 120 ng / μL; the quantification range is 0.1 ng - 120 ng.

[0049] In one embodiment of the present invention, it further includes the step of quality inspection of the ctDNA reference sample.

[0050] In one embodiment of the present invention, the quality inspection includes detecting the fragment distribution of the reference sample, the DNA concentration of the detection sample, and the allelic gene mutation frequency / genotype frequency.

[0051] In one embodiment of the present invention, the fragment distribution of the reference sample is 160 bp ± 10%.

[0052] In one embodiment of the present invention, the DNA concentration of the detection sample is 5 ng / ul ± 25%.

[0053] In one embodiment of the present invention, the reference sample is used to detect the allelic gene mutation frequency / genotype frequency;

[0054] Among them, the acceptance criteria for the reference sample include: the acceptance criteria for the 50% VAF reference sample are ±20%; the acceptance criteria for the 1% VAF reference sample are ±40%.

[0055] In the second aspect of the present invention, there is provided a tumor circulating free DNA reference sample prepared by any of the above methods.

[0056] In the third aspect of the present invention, there is provided a reference sample plate including the above reference sample as a positive reference sample.

[0057] In one embodiment of the present invention, the reference sample plate includes 10 positive reference samples, and the 10 positive reference samples cover a total of gene mutation sites related to endocrine therapy resistance in breast cancer; each positive reference sample contains a group of mutations with different mutation frequencies: a strong positive reference sample of 50% VAF, a medium positive reference sample of 10% VAF, a weak positive reference sample of 1%, and the lowest detection limit reference samples of 0.1%, 0.2%, 0.3%, 0.5% VAF. In addition, it also includes a mixed reference sample such as Y537N - 33% VAF & H1047L - 15% VAF & E17K - 33% VAF).

[0058] In the fourth aspect of the present invention, there is provided a kit including the above reference sample plate.

[0059] In one embodiment of the present invention, the kit further includes a negative reference sample.

[0060] In the fifth aspect of the present invention, there is provided the use of the above reference product, reference product plate, and kit in the preparation of a reagent for detecting endocrine therapy resistance in breast cancer or a reagent for detecting the allele mutation frequency / genotype frequency in ctDNA.

[0061] In the sixth aspect of the present invention, a method for detecting the allele mutation frequency / genotype frequency in ctDNA for non-diagnostic purposes, which uses the above reference product, the above reference product plate, and the above kit to detect ctDNA.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) By optimizing the ultrasonic fragmentation parameters in the first step, the present invention reduces the proportion of large DNA fragments, increases the absolute proportion of DNA fragments in the range of 150-180 bp, improves the fragmentation efficiency of genomic DNA, and at the same time retains some fragments of 300-400 bp, making it more in line with the fragment size ratio of natural ctDNA, laying a foundation for omitting large fragment magnetic bead purification later and reducing the cost of preparing simulated ctDNA.

[0064] (2) By comparing different brands of magnetic beads and optimizing the magnetic bead purification methods, ratios, and parameters of the two-step method and the one-step method, the present method realizes the removal of single-base and multi-base products and ultra-short genomic DNA fragments below 50 bp inevitably generated in the ultrasonic fragmented genomic DNA by one-step magnetic bead purification, thereby increasing the proportion of DNA fragments with a size of 100 bp - 200 bp, being closer to natural ctDNA in terms of fragment size distribution, while reducing the cost and simplifying the operation.

[0065] (3) The reference product prepared by the method of the present invention has high complexity and high sensitivity, and is similar to natural plasma ctDNA.

[0066] (4) By optimizing the reference product combination method and the preparation process, the present invention improves the performance and significantly reduces the cost. This method can be flexibly customized according to the dynamic needs in the enterprise R & D process, prepare a large number of simulated plasma ctDNAs with stable performance and controllable mutation frequency, reduce the enterprise R & D cost and shorten the R & D cycle. And the preparation method is simple and the cost is low, which is suitable for industrial production.

[0067] (5) The preparation method of the reference product of the present invention is applicable to plasma-free DNA (including cfDNA and ctDNA) samples. The reference product of the present invention includes 10 independently usable positive reference products, and each positive reference product contains a set of mutations with different mutation frequencies. The reference product plate of the present invention contains positive reference products, including 3 independently usable positive reference products, and each positive reference product contains a set of mutations. The 10 positive reference products cover the gene mutation sites related to endocrine therapy resistance in breast cancer, can meet the needs in the R & D process; the detection needs of multiple genes and multiple mutations, and has excellent quality control performance. Description of the Drawings

[0068] Figure 1 It is a distribution result diagram of fragmented gDNA in experimental group 1;

[0069] Figure 2 It is a distribution result diagram of fragmented gDNA in experimental group 2;

[0070] Figure 3 It is a final yield result diagram of fragmented gDNA with different single-tube loading amounts;

[0071] Figure 4 It is a distribution result diagram of fragmented gDNA in experimental groups 3 and 4, sample 2-175-500 is experimental group 3, and sample 3-175-580 is experimental group 4;

[0072] Figure 5 It is a distribution result diagram of fragmented gDNA in experimental groups 5-8, sample 4-200-300 is experimental group 5, sample5-200-430 is experimental group 6; sample 6-200-500 is experimental group 7, and sample 7-200-580 is experimental group 8;

[0073] Figure 6 It is a distribution result diagram of fragmented gDNA in experimental groups 9-11, sample 8-210-300 is experimental group 9, sample9-210-350 is experimental group 10; sample 10-210-430 is experimental group 11;

[0074] Figure 7 It is an electrophoresis diagram of gDNA fragments in experimental groups 2-11;

[0075] Figure 8a It is a size distribution diagram of DNA fragments prepared after ultrasonic fragmentation (without magnetic bead purification) in the method of the present invention; 8b and 8c are size distribution diagrams of DNA fragments of two different brands one and two purchased.

[0076] Figure 9Electrophoresis diagram of gDNA fragments in experimental groups 1'-3'

[0077] Figure 10 Diagram of the distribution of fragmented gDNA in experimental groups 1'-3'. D1-2 are large fragments in experimental group 1', X1-2 are small fragments in experimental group 1', D3-4 are large fragments in experimental group 2', X3-4 are small fragments in experimental group 2', D5 is the large fragment in experimental group 3', and X5-6 are small fragments in experimental group 3'.

[0078] Figure 11 is a diagram of the size distribution of simulated ctDNA fragments using the method of the present invention (ultrasonic fragmentation + purification and ultrasonic fragmentation alone).

[0079] Figure 12 is a diagram of the size distribution of the simulated ctDNA reference product using the method of the present invention (ultrasonic fragmentation + purification) (12a is the blue line; 12b is the red line), two commercially available simulated ctDNAs of different brands (brand one in 12a is the green & bright blue line, brand two in 12b is the dark blue, light blue & green line), and natural ctDNA (12a is the red line, extracted by the QIAamp Circulating Nucleic Acid Kit column method).

[0080] Figure 13 Diagram of the distribution of fragmented gDNA in Comparative Example 1; 0.8X-YS-D-4-6.29 is the product of the first-step magnetic bead large fragment screening; 0.7X-YS-X-1-6.29 is the product of the second-step magnetic bead small fragment screening.

[0081] Figure 14 Diagram of the distribution of fragmented gDNA in Comparative Example 2; WD-D-1 is the product of the first-step magnetic bead large fragment screening; WD-X-1 is the product of the second-step magnetic bead small fragment screening.

[0082] Figure 15 Cp values for PIK3CA H1047R qPCR mutation detection using the simulated ctDNA reference product prepared by the method of the present invention and the purchased brand one reference product, with VAF being 1%.

[0083] Figure 16 Flowcharts for the preparation of 10% reference product and 50% reference product. Detailed implementation manners

[0084] The technical solutions of the present invention will be further described in detail below with specific embodiments. It should be understood that the following embodiments are only for illustrative and explanatory purposes of the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope intended to be protected by the present invention.

[0085] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0086] Technical terms:

[0087] VAF is the abbreviation of Variant Allel Frequency, usually referred to as variant allele frequency, which refers to the ratio of the number of variant alleles to wild-type alleles.

[0088] cfDNA (cell free DNA) refers to cell-free DNA, which is released into the blood through apoptosis, necrosis and secretion. cfDNA is usually a double-stranded fragment with a length of about 150-200 base pairs.

[0089] ctDNA (circluting-tumor DNA) refers to circulating tumor DNA, whose molecular genetic and epigenetic information can reflect the genome or epigenome of the origin cell.

[0090] The mutant gene types and mutation sites used in the present invention are shown in Table 1. The reference species and mutation frequencies used in the present invention are shown in Table 2. The reference species and mutation frequencies used in the present invention are shown in Table 3.

[0091] Table 1 Mutation gene types and mutation sites

[0092]

[0093] Table 2 Reference species and mutation frequencies

[0094]

[0095] Table 3 Reference disk types and mutation frequencies

[0096]

[0097] The preparation method of the reference product of the present invention: According to the characteristics of the accuracy of the reference product mutation frequency determination, in order to achieve a more accurate target mutation frequency, two methods (mixing first and then ultrasonic interruption or ultrasonic interruption first and then mixing) are adopted to prepare the reference product:

[0098] 1. Reference samples with VAF > 10%: First, mix wild-type gDNA with gDNA with a specific VAF at the mutation site, then perform ultrasonic shearing, and then perform magnetic bead purification. The wild-type gDNA used is NA12878, purchased from Coriell; the 100% mutant site gDNA used is purchased from Nanjing Kebai. The concentrations of the wild-type and mutant mother liquors are measured using Qubit (a nucleic acid and protein quantifier), and by calculating the mass ratio, samples with 50% and 10% mutation frequencies are mixed proportionally. Then, DNA fragments with a length of 50bp - 300bp are prepared using the ultrasonic shearing method, with the main peak of the fragments concentrated at 160bp - 170bp. Subsequently, secondary purification is performed using the magnetic bead method, and after passing the quality inspection, it is used as a reference sample.

[0099] 2. Reference samples with 1% < VAF < 10%: Mix the qualified high-frequency sample with the closest target VAF and negative NA12878 in the corresponding proportion, and obtain it according to the method of the reference sample with VAF > 10% in 1 above.

[0100] 3. Reference samples with VAF < 1%: First, perform ultrasonic shearing on wild-type gDNA, then mix it with qualified 1% VAF sheared gDNA, and then perform magnetic bead purification. The wild-type gDNA used is NA12878, purchased from Coriell; the 1% VAF sheared gDNA with different mutations used is purchased from Nanjing Kebai. DNA fragments with a length of 50bp - 300bp are prepared using the ultrasonic shearing method, with the main peak of the fragments concentrated at 160bp - 170bp; the concentrations of the wild-type and mutant mother liquors are measured using Qubit, and by calculating the mass ratio, samples with mutation frequencies such as 0.3% and 0.5% are mixed proportionally. After purification and passing the quality inspection, it is used as a reference sample.

[0101] Example 1 Sample Extraction

[0102] 1. Extract genomic DNA from cells: Extract human blood genomic DNA using the TianAmp Blood DNA Extraction Kit (product number: DP314);

[0103] 2. Use Qubit to detect the nucleic acid content in gDNA (NA12878), sheared and purified CH-NA12878, ESR1p.Y537N Reference Standard Plus - 100% mutation and p.Y537N.

[0104] 3. Mix according to the mass ratios in Table 4 and dilute with TE to a final nucleic acid concentration of 10 ng / μL (CH-NA12878 and p.Y537N-1% are fragmented DNA, no need for fragmentation and magnetic bead purification operations, just directly perform concentration and fragment length quality inspections after mixing). In Table 4, WT is the mass of the non-mutated fragment and MuT is the mass of the mutated fragment.

[0105] Table 4 Sample mass ratio

[0106] Sample Name Mass Ratio (WT:MuT) Mix 1 - 50% 1：1 Mix 2 - 10% 9：1 Mix 3 - 0.5% 1:1 (CH - NA12878:p.Y537N - 1%)

[0107] 4. Use Qubit to detect the nucleic acid content in the mixed sample.

[0108] 5. In the biosafety cabinet, respectively take 130 μL of the prepared Mix 1 - 50% and Mix 2 - 10% samples and add them into the MicroTUBE AFA Fiber Pre-Slit Snap-Cap for standby (need to use immediately, the MicroTUBE AFA Fiber Pre-Slit Snap-Cap cannot store samples). The specific steps are as Figure 16 shown.

[0109] Example 2 Ultrasonic fragmentation

[0110] 1. Use a Covaris S220 non-contact ultrasonic disruptor (purchased from Covaris, USA) to ultrasonically fragment the extracted genomic DNA. The ultrasonic fragmentation conditions are shown in Table 5.

[0111] Since the setting of ultrasonic parameters has a great influence on the fragmentation efficiency of genomic DNA and the size of the products, in order to obtain products closer to the fragment distribution characteristics of ctDNA, the input amount of genomic DNA, reaction system, ultrasonic fragmentation parameters, etc. were optimized. Among them, the ultrasonic fragmentation parameters include Peak Incident Power (peak power), Duty Factor (duty ratio), Cycles per Burst (number of cycles per single pulse), and Treatment Time (treatment time), as shown in Table 5.

[0112] Table 5 Ultrasonic parameter settings

[0113]

[0114] 2. Transfer the fragmented sample into a 1.5 ml centrifuge tube and store it in an ice box.

[0115] 3. Detect the DNA fragment length in the collected sample.

[0116] 4. Use Qubit to detect the nucleic acid concentration in the sample.

[0117] 5. Ultrasonic fragmentation results

[0118] 1) Experimental group 1: The genomic DNA extracted in step 1 was fragmented to prepare fragmented gDNA using Program 1 in Table 5. The results are as Figure 1 shown. From the results, it can be seen that for the fragmented DNA prepared with these ultrasonic parameters, the main peak position is 130 bp, and there are a large number of large fragments > 1000 bp.

[0119] 2) Experimental group 2: 0.5 μg of the genomic DNA extracted in step 1 was dissolved in 130 μL of nuclease-free water; Program 2 in Table 5 was used for fragmentation to prepare fragmented gDNA. The results are as Figure 2 shown. From the results, it can be seen that after changing the fragmentation parameters, the fragmentation efficiency was improved. The proportion of large fragments > 1000 bp was extremely low, the main product was < 600 bp, and the main peak was 140 bp. Control group 1 was set up: the dosage of genomic DNA was 1 μg. Control group 2: the dosage of genomic DNA was 4 μg.

[0120] The final yield is as Figure 3 shown. From the results, it can be seen that the final yield of 0.5 μg single-tube loading was 62%, the final yield of 1 μg single-tube loading was 54%, and the final yield of 4 μg single-tube loading was 46%. Thus, it can be seen that the more the single-tube loading amount of the fragmentation tube, the greater the loss. The highest recovery rate was obtained with a single-tube input of 0.5 μg genomic DNA / 130 uL nuclease-free water.

[0121] 3) Experimental group 3: The same as experimental group 2, except that Program 3 in Table 5 was used for fragmentation, TreatmentTime (processing time) was 500 s, and the final yield was 67%.

[0122] 4) Experimental group 4: The same as experimental group 2, except that Program 4 in Table 5 was used for fragmentation, TreatmentTime (processing time) was 580 s, and the final yield was 63%. The results are as Figure 4 shown.

[0123] From the results, it can be seen that: for experimental group 3 (175 - 500), most of the products were < 700 bp, the main peak position was 141 bp, and there were a small number of fragments > 2000 bp; for experimental group 4 (175 - 580), most of the products were < 600 bp, the main peak position was 130 bp, and there were a small number of fragments > 1000 bp.

[0124] 5) Experimental group 5: The same as experimental group 2, except that Program 5 in Table 5 was used for fragmentation, PeakIncident Power (W): 200, Treatment Time (processing time) 300 s, and the final yield was 65%.

[0125] 6) Experimental Group 6: Similar to Experimental Group 2, except that interruption is carried out using Procedure 6 in Table 5. Peak Incident Power (W): 200, Treatment Time: 430 s, and the final yield is 55%.

[0126] 7) Experimental Group 7: Similar to Experimental Group 2, except that interruption is carried out using Procedure 7 in Table 5. Peak Incident Power (W): 200, Treatment Time: 500 s, and the final yield is 61%.

[0127] 8) Experimental Group 8: Similar to Experimental Group 2, except that interruption is carried out using Procedure 8 in Table 5. Peak Incident Power (W): 200, Treatment Time: 580 s, and the final yield is 60%.

[0128] The results of Experimental Groups 5 - 8 are as Figure 5 shown. From the results, it can be seen that for Experimental Group 5 (200 - 300), most of the products are < 600 bp, the main peak position is 160 bp, and there are a small number of fragments > 5000 bp; for Experimental Group 6 (200 - 430), most of the products are < 600 bp, the main peak position is 130 - 180 bp, and there are a small number of fragments > 3000 bp; for Experimental Group 7 (200 - 500), most of the products are < 500 bp, the main peak position is 120 - 160 bp, and there are a small number of fragments > 3000 bp; for Experimental Group 8 (200 - 580), most of the products are < 500 bp, the main peak position is 140 - 150 bp, and there are a small number of fragments > 2000 bp.

[0129] 9) Experimental Group 9: Similar to Experimental Group 2, except that interruption is carried out using Procedure 9 in Table 5. Peak Incident Power (W): 210, Treatment Time: 300 s, and the final yield is 62%.

[0130] 10) Experimental Group 10: Similar to Experimental Group 2, except that interruption is carried out using Procedure 10 in Table 5. Peak Incident Power (W): 210, Treatment Time: 350 s, and the final yield is 62%.

[0131] 11) Experimental Group 11: Similar to Experimental Group 2, except that interruption is carried out using Procedure 11 in Table 5. Peak Incident Power (W): 210, Treatment Time: 430 s, and the final yield is 59%.

[0132] The results of experimental groups 9 - 11 are as Figure 6 shown. It can be seen from the results that most of the products of experimental group 9 (210 - 300) are < 525 bp, the main peak position is 157 - 174 bp, and there are a small number of fragments > 1000 bp; most of the products of experimental group 10 (210 - 350) are < 500 bp, the main peak position is 152 - 179 bp, and there are a small number of fragments > 6000 bp; most of the products of experimental group 11 (210 - 430) are < 500 bp, the main peak position is 166 bp, and there are a small number of fragments > 2000 bp.

[0133] By comprehensive comparison, it can be known that the main peak position and fragment size distribution in experimental group 11 are more in line with the expectations of simulated ctDNA. The results are as Figure 7 shown.

[0134] 12) The fragment size distributions of the simulated ctDNA fragmented by two different commercial brands were compared with that of experimental group 11, and the results are shown in Figure 8.

[0135] It can be seen from the results that for the DNA fragments after ultrasonic fragmentation in the present invention ( Figure 8a ), the distribution is mainly < 500 bp, and there are small fragments below 50 bp; for the two different commercial brands purchased ( Figure 8b , Figure 8c ), there are also small fragments of 35 bp - 50 bp, and the proportion of fragments < 100 bp is relatively large; the main peak position of the method of the present invention is 153 - 170 bp. For the DNA fragmented by ultrasonic waves of the other two brands, Figure 8b the main peak is 120 bp smaller, Figure 8c and the main peak position in Figure 8b is 191 bp larger. Compared with the method of the present invention, it is closer to the main peak of 167 bp in the natural state; at the same time, the proportion of the fragment size distribution of 100 - 200 bp in the method of the present invention increases significantly; and the proportion of larger fragments of about 400 bp increases. For the DNA fragmented by the other two brands, Figure 8b the fragments are concentrated < 400 bp, Figure 8c and in

[0136] Example 3 Magnetic bead purification and recovery

[0137] 1. Two - step magnetic bead purification was used to remove large fragments and small fragments respectively

[0138] 1) Dilute the fragmented TE obtained from experimental group 11 to 2 ng / μl; restore the AMPure XP magnetic beads to room temperature, mix well and set aside.

[0139] 2) The AMPure XP magnetic beads can purify fragments > 100 bp. However, the effect of removing small fragments has a very large relationship with the ratio of magnetic beads. At the same time, the range of fragments > 100 bp is very broad, and the ratio of magnetic beads used must also be optimized to obtain DNA fragments that meet our expectations and requirements. Therefore, it is necessary to further optimize the magnetic bead purification method. The optimization of magnetic bead purification also includes the purification method and the initial DNA concentration to minimize DNA loss during the purification process. Recover according to the two-step method in Table 6.

[0140] Table 6 DNA fragmentation recovery system

[0141]

[0142] 3) After vortexing and mixing, let it stand at room temperature for 5 min.

[0143] 4) Place the sample on the magnetic stand. After adsorption and the solution becomes clear, carefully remove the supernatant.

[0144] 5) Add 500 μL of freshly prepared 70% ethanol to wash the magnetic beads, incubate at room temperature for 30 s, and carefully remove the supernatant; (the volume of ethanol ≥ the sum of the sample and magnetic bead volumes).

[0145] 6) Repeat step 5.

[0146] 7) Keep the centrifuge tube in the magnetic stand all the time. Open the lid and dry the magnetic beads until just cracking appears (about 5 min).

[0147] 8) Take out the centrifuge tube from the magnetic stand, add 50 μL of TE (≥ 40 μL), vortex or gently pipette to mix well, and incubate at room temperature for 2 min.

[0148] 9) Briefly centrifuge the centrifuge tube and place it in the magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 1 min), carefully aspirate the supernatant into a clean tube to complete the fragment sorting.

[0149] 10) Use Qubit to detect the nucleic acid content in the sample. The detected sensitivity is: the initial sample concentration is 0.005 ng / μL - 120 ng / μL; the quantification range is: 0.1 ng - 120 ng.

[0150] 11) Detect the length of the DNA fragments in the collected samples. Operate twice using the same method for detection respectively.

[0151] The results are as Figure 9 and Figure 10As shown, in the products after the first-step magnetic bead screening of large fragments in experimental groups 1'-3', large fragments were not detected using either the highly sensitive Qubit or the Agilent High Sensitivity DNA Chip (Bioanalyzer). This result indicates that the optimization of the ultrasonic fragmentation operation significantly reduced the proportion of large fragments. After the second-step magnetic bead screening to remove small fragments, the <100bp fragments in the product were significantly reduced. Among them, experimental group 1' corresponds to D1-2&X1-2, experimental group 2' corresponds to D3-4&X3-4, and experimental group 3' corresponds to D5&X5-6.

[0152] 2. Use one-step magnetic bead purification to remove <100bp small fragments.

[0153] Similar to step 1, the difference is that one-step magnetic bead purification method is adopted. Among them, the volume of magnetic beads: the volume of fragmented DNA sample = 2.2:1. The final average recovery efficiency is 68%. The results are shown in Figure 11. Among them, 11a is the fragment distribution diagram before and after the purification of PIK3CA H1047R 50% VAF (refer to the preparation method of the reference product of the present invention above), red is ultrasonic fragmentation + purification, and blue is ultrasonic fragmentation alone (control); 11b is the fragment distribution diagram before and after the purification of ESR1 Y537S 10% VAF (refer to the preparation method of the reference product of the present invention above), red and green are ultrasonic fragmentation + purification, and blue is ultrasonic fragmentation alone (control); 11c is the fragment distribution diagram (ultrasonic fragmentation + purification) of ESR1 Y537S 1% VAF (refer to the preparation method of the reference product of the present invention above). And it is compared with the fragment size distributions of commercialized simulated ctDNA of two different brands. The results are shown in Figure 12.

[0154] As can be seen from Figure 11, after purification, the <50bp small fragments were significantly reduced, almost none, the proportion of 100-220bp fragments increased, and the proportion of the main peak at 170bp increased. At the same time, the proportion of 300-400bp fragments was increased to make it more simulate natural ctDNA, so as to make the downstream mutation detection and verification more real.

[0155] As can be seen from Figure 12: The proportion of DNA fragments below 50bp after fragmentation by the present invention was significantly reduced, almost none; while for the two different brands purchased ( Figure 12a , Figure 12b ) the proportion of 35bp-100bp was relatively large; the main peak position of the method of the present invention is 170-180bp, Figure 12a in which the main peak position is relatively large, 150-200bp, Figure 12bThe main peak is relatively small, concentrated at 120 - 140 bp. Compared with the other two brands, the method of the present invention is closer to the main peak of 167 bp in the natural state; at the same time, the proportion of the fragment size distribution of 100 - 200 bp increases significantly; and the proportion of larger fragments around 300 - 400 bp is increased.

[0156] At the same time, Schmidt B and Vermeulen C respectively published research in 2005 and 2017 and found that when using Qiagen columns to separate and purify cell-free plasma ctDNA, some DNA fragments less than 150 bp will be lost. Therefore, the method of the present invention is also significantly superior to the effect of using Qiagen columns to separate and purify ctDNA.

[0157] Comparative Example 1 uses Yeasen magnetic beads Hieff DNA Selection Beads DNA sorting magnetic beads (12601ES08) to remove large fragments and small fragments in two steps respectively. The dosages of Yeasen magnetic beads are 0.55 - 0.8 times the volume of the fragmented sample and 0.7 - 0.8 times the volume of the sample after large fragment removal respectively.

[0158] The results are as follows Figure 13 As shown, the product of the first-step magnetic bead large fragment screening (YS-D) is concentrated at 3000 - 7000 bp, and small fragments of 300 bp - 500 bp can also be adsorbed and removed. The adsorption of large fragments is not specific, resulting in the loss of small fragments; the second-step magnetic bead small fragment screening (YS-X): the main peak of small fragments in the recovered product is concentrated at 163 - 177 bp, and at the same time, some large fragments (>400 bp) are captured. The expected purification purpose cannot be achieved.

[0159] Comparative Example 2 uses Weidu magnetic beads to screen the left and right side fragments in the ultrasonically fragmented DNA in two steps. The dosage of Weidu magnetic beads is 0.8 times the volume of the fragmented sample and 0.2 times the volume of the sample after large fragment removal.

[0160] The results are as follows Figure 14 As shown, in the first-step magnetic bead large fragment screening (WD-D-1): the large fragments in the product are concentrated at 4000 - 6000 bp, and a small number of 400 - 600 bp fragments can be seen; the adsorption of large fragments corresponding to this magnetic bead dosage is not specific; in the second-step magnetic bead <500 bp fragment screening (WD-X): the product contains large fragments of 2000 - 9000 bp; the main peak of small fragments is 240 bp, and DNA fragments <200 bp are not detected. The results indicate that Weidu magnetic beads fail to adsorb <200 bp DNA fragments and cannot meet the expected requirements.

[0161] 3. Quality inspection

[0162] 1) Fragment distribution: Use a bioanalyzer to detect the distribution of DNA fragments of different lengths in the reference: 160bp ± 10%;

[0163] 2) Concentration: Use 2.0 Fluorometer to detect sample DNA concentration: concentration 5ng / ul±25%;

[0164] 3) Allele mutation frequency / genotype frequency: Droplet digital PCR determination:

[0165] The acceptance criteria for 50% VAF reference products are: ±20%;

[0166] The acceptance criteria for 1% VAF reference products are: ±40%;

[0167] <1%VAF reference acceptance criteria: ±50%.

[0168] Example 4: qPCR detection of weak positive mutation sites

[0169] 1) Reaction system configuration, as shown in Table 7:

[0170] Table 7 Reaction system

[0171] Component Name Volume (μL) 5×buffer (probe) 4 Forward Primer (10uM) 0.3 Reverse Primer (10uM) 0.3 Probe (10uM) 0.2 Enzyme (5U / μL) 0.4 Template 3 Nuclease - Free - Water To 20 Total Volume 20

[0172] 2) Reaction conditions are set as shown in Table 8:

[0173] Table 8 Reaction conditions

[0174]

[0175]

[0176] The simulated ctDNA reference prepared in Example 3 and the purchased brand 1 reference were subjected to PIK3CA H1047R1%VAF weak positive mutation qPCR detection. The detection method was the same as that in Example 4. The results are shown in Figure 15 shown.

[0177] Figure 15 The results showed that there was no significant difference in the Cp value of the reference gene amplification prepared in step 2 of Example 3 (<0.5), but the Cp value of the target gene mutation site was significantly reduced (1.31), and the ΔCp was significantly reduced (0.8), which improved the amplification performance of the mutation site and reduced the Cp value of the mutation site, indicating that a lower LoD (minimum detection limit) can be achieved. Therefore, the method of the present invention is more conducive to the performance of ctDNA mutation detection.

[0178] It can be seen that by optimizing the source of the sample for preparing the simulated ctDNA reference product, the ultrasonic fragmentation parameters, introducing a small fragment removal method and optimizing the proportion of purification magnetic beads, the size and distribution characteristics of the prepared simulated ctDNA are closer to those of natural plasma-free DNA, contain more mutation sites, and can be stably prepared and stored for a long time, and the amplification performance is close to that of natural ctDNA.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a reference product of circulating free DNA in tumors, comprising the following steps: S1 Prepare fragmented DNA: Use ultrasound to break the mixed wild-type gDNA and mutant gDNA to obtain fragmented DNA; Or use ultrasound to break wild-type gDNA and mutant gDNA separately, and then mix them to obtain fragmented DNA; S2 Prepare a ctDNA reference product: Purify the fragmented DNA with magnetic beads to obtain the ctDNA reference product; Wherein, the wild-type gDNA is genomic DNA that has not undergone mutation, and the mutant gDNA is genomic DNA that has undergone mutation at a preset site.

2. The preparation method of a tumor circulating free DNA reference product according to claim 1, characterized in that: The amino acid sequence encoded by the mutant gDNA includes one or more of the following mutation sites: the amino acid mutation p.E17K of AKT1, the amino acid mutation p.E380Q of ESR1, the amino acid mutation p.Y537S of ESR1, the amino acid mutation p.Y537N of ESR1, the amino acid mutation p.D538G of ESR1, the amino acid mutation p.E542K of PIK3CA, the amino acid mutation p.E545K of PIK3CA, the amino acid mutation p.H1047R of PIK3CA, the amino acid mutation p.H1047L of PIK3CA; Preferably, the nucleotide sequence of the mutant gDNA includes one or more of the following mutation sites: the base mutation corresponding to p.E17K is c.49G>A, the base mutation corresponding to p.E380Q is c.1138G>C, the base mutation corresponding to p.Y537S is c.1610A>C, the base mutation corresponding to p.Y537N is c.1609T>A, the base mutation corresponding to p.D538G is c.1613A>G, the base mutation corresponding to p.E542K is c.1624G>A, the base mutation corresponding to p.E545K is c.1633G>A, the base mutation corresponding to p.H1047R is c.3140A>G, the base mutation corresponding to p.H1047L is c.3140A>T.

3. The preparation method of a tumor circulating free DNA reference product according to claim 1, wherein: By presetting the mass ratio of the mutant gDNA and the wild-type gDNA, the base mutation frequency of the reference product is one or more of the following, VAF>10%, 1%<VAF<10%, VAF<1%; Preferably, by presetting the mass ratio of the mutant gDNA and the wild-type gDNA, the base mutation frequency of the reference product is any one of 50%, 25%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%; Preferably, the mass ratio of the mutant gDNA to the wild-type gDNA is 1:1-9.

4. The preparation method of a tumor circulating free DNA reference product according to claim 1, characterized in that: The operating conditions of the ultrasound include: peak power of 175 - 210 W, duty cycle of 10%, number of single - pulse cycles of 200, and treatment time of 300 - 580 s; preferably, the peak power of the ultrasound is 200 - 210 W, duty cycle of 10%, number of single - pulse cycles of 200, and treatment time of 400 - 450 s; more preferably, the peak power of the ultrasound is 210 W, duty cycle of 10%, number of single - pulse cycles of 200, and treatment time of 430 s; Preferably, the input amount of the gDNA sample in the ultrasound is 0.5 μg / tube.

5. The preparation method of a tumor circulating free DNA reference product according to claim 1, characterized in that: The steps of purifying the fragmented DNA with magnetic beads include: 1) Adding magnetic beads to the fragmented DNA solution, so that the fragmented DNA with the first length binds to the magnetic beads, while the fragmented DNA with the second length remains in the solution; 2) Separating the magnetic beads bound with the fragmented DNA of the first length from the solution containing the fragmented DNA of the second length; 3) Separating the fragmented DNA of the first length from the magnetic beads; obtaining the ctDNA reference sample; Among them, the size of the first length is 100 - 400 bp, preferably 100 - 220 bp, 300 - 400 bp, and more preferably 160 bp ± 10%; the size of the second length is 35 - 100 bp; Preferably, the volume ratio of the fragmented DNA to the magnetic beads is 1:2.

2. Preferably, the concentration of the fragmented DNA solution is 0.005 ng / μL - 120 ng / μL; preferably 0.1 ng - 120 ng / μL; Preferably, the magnetic beads are AMPure XP magnetic beads.

6. A tumor - circulating free DNA reference sample, prepared by the preparation method according to any one of claims 1 - 5; Preferably, the sensitivity of the ctDNA reference sample detection is: initial sample concentration of 0.005 ng / μL - 120 ng / μL; quantification range: 0.1 ng - 120 ng.

7. A reference sample plate, including the reference sample according to claim 6 as a positive reference sample.

8. A kit, including the reference sample plate according to claim 7, and preferably further including a negative reference sample.

9. Use of the reference sample according to claim 6, the reference sample plate according to claim 7, and the kit according to claim 8 in the preparation of reagents for detecting endocrine - therapy resistance of breast cancer, or in the preparation of reagents for detecting the allele mutation frequency / genotype frequency in ctDNA.

10. A method for detecting the allele mutation frequency / genotype frequency of ctDNA for non-diagnostic purposes, characterized in that: Detecting ctDNA using the reference sample according to claim 6, the reference sample plate according to claim 7, and the kit according to claim 8.

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