CfDNA detection standard substance and preparation method thereof

Abstract

The invention discloses a cfDNA detection standard substance and a preparation method thereof. The preparation method comprises the following steps: carrying out multiplication culture on cells; performing cell apoptosis treatment on the amplified cells; after cell apoptosis treatment is finished, incubating for a period of time, and collecting supernate; adding the supernate into plasma to prepare a plasma standard substance for cfDNA detection; or extracting cfDNA in the supernate, and preparing a DNA standard substance for cfDNA detection; according to the preparation method, the supernatant of a culture cell line is adopted as a source of a cfDNA detection standard substance, rapid death of cells is promoted through a method of nutrient depletion or cytotoxic reagent addition, the content of mononuclear bodies in the supernatant of the culture medium is increased, the concentration of cfDNA is further increased, and the preparation method is easy to operate, high in yield and low in cost.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, specifically to a cfDNA detection standard and its preparation method. Background Technology

[0002] Liquid biopsies are a non-invasive diagnostic method that involves collecting a patient's blood or other bodily fluid samples and analyzing biomarkers such as cell-free DNA (cfDNA), cell-free RNA (cfRNA), exosomes, and circulating tumor cells (CTCs) to assess the patient's health status or disease progression. cfDNA, or cell-free DNA, is the most widely used analyte in liquid biopsies because it can be detected in blood and carries information on various disease-related variants. Currently, cfDNA testing has significant clinical applications in precision oncology diagnosis and recurrence monitoring, fetal genomic abnormality detection, non-invasive paternity testing, pathogen identification and drug resistance assessment, acute rejection assessment after organ transplantation, and the diagnosis and monitoring of autoimmune diseases.

[0003] cfDNA is a double-stranded DNA fragment found in bodily fluids, including blood, urine, and cerebrospinal fluid. cfDNA primarily originates from cell death processes, including apoptosis, necrosis, and inflammatory cell death (NETosis) caused by neutrophils. In healthy individuals, blood cfDNA mainly originates from leukocytes. Specific physiological and pathological conditions, such as pregnancy, tumors, inflammation, infection, autoimmune diseases, acute rejection after organ transplantation, and strenuous exercise, can lead to elevated cfDNA concentrations.

[0004] The widespread application of cfDNA in clinical diagnosis is based on its close association with various physiological and pathological states, as well as the diverse molecular information it carries related to the original cell characteristics. This molecular information can be divided into three omics fields: (1) genomics, covering single nucleotide variants (SNVs), insertion / deletion variants (Indels), copy number variants (CNVs), and fusion gene variants; (2) epigenomics, mainly involving DNA methylation; and (3) fragment omics, including molecular characteristics such as cfDNA terminal sequence distribution, nucleosome imprinting, and fragment size. The rich molecular characteristics of cfDNA provide a multi-dimensional and multi-target information source for scientific research and clinical applications.

[0005] plasma cfDNA contains nuclear DNA, mitochondrial DNA, and microbial DNA. Nuclear cfDNA typically exists as a nucleosome complex, where 143 bp of DNA binds to the nucleosome core histone via electrostatic interactions, and 23 bp binds to the H1 histone to stabilize the nucleosome structure. The linker DNA (20-50 bp) between adjacent nucleosomes may be digested and degraded to varying degrees by nucleases during cfDNA formation. cfDNA bound to the nucleosome is protected by histones, thus mitigating the degradation by nucleases. Therefore, the main peak size of nuclear cfDNA is approximately 167 bp.

[0006] Because cfDNA fragments are small and often carry low mutation frequencies, commonly used techniques for cfDNA detection include fluorescence PCR (qPCR), digital PCR (dPCR), and high-throughput sequencing. cfDNA standards are essential for standardizing cfDNA detection techniques and kits. The applications of cfDNA standards include research and development of detection methods and kits, performance validation, quality control, data standardization, disease diagnosis, and surveillance.

[0007] The most prominent characteristic of cfDNA standards is that the main peak of the fragment is 167bp. Furthermore, there are three main methods for preparing existing cfDNA standards:

[0008] 1) First, long fragments of genomic gDNA are extracted. The DNA is then fragmented using mechanical breaking. Fragments are then selected using different concentrations of Ampure XP magnetic beads, selectively retaining fragments between 150-200 bp [Application No. 202211411050.X][Application No. 201811526763.4][Application No. 201810734800.4]. The cfDNA fragments prepared by this method have a wide size range, more severe DNA damage, and the fragmentation pattern differs significantly from that of cfDNA in real samples.

[0009] 2) During nucleic acid extraction, a weaker protein denaturant is first used to break the cell membrane and nuclear membrane to release the chromosome. Then, micrococcal nuclease is added to degrade the connecting DNA between two nucleosomes, producing unconnected nucleosomes. Finally, a stronger protein denaturant is used to destroy the histones in the nucleosomes. After purification, cfDNA standard is obtained [Paper Clin Chem. 2017 Sep; 63(9):1465-1475]. Although the fragment size of the cfDNA obtained by this method is highly similar to that of cfDNA, the operation steps are cumbersome and the yield is low.

[0010] 3) By highly expressing DFFB (DNA Fragmentation Factor subunit Beta) and / or DNASE1L3 (deoxyribonuclease 1-like 3) in cell lines, cfDNA is present in the cell culture supernatant. DNA is then extracted from the cell culture supernatant to obtain cfDNA standards [Application No. 202411217531.6]. Although the cfDNA fragments obtained by this method closely mimic the size of cfDNA and the extraction is relatively conventional, if a series of cell lines carrying different mutations need to be used as raw materials for the preparation of cfDNA standards, high expression of the above two enzymes in all cell lines is a rather cumbersome step. Therefore, this method is costly and not easy to promote.

[0011] The above-mentioned methods for preparing cfDNA standards have drawbacks such as complicated operation steps, low yield, high cost, or significant differences in the size of the standard fragments compared to cfDNA. Summary of the Invention

[0012] The purpose of this invention is to provide a cfDNA detection standard and its preparation method. The preparation method of this invention uses the supernatant of cultured cell lines as the source of cfDNA detection standard, and promotes rapid cell death by nutrient depletion or the addition of cytotoxic reagents to increase the content of single nucleosomes in the culture medium supernatant, thereby further increasing the concentration of cfDNA. The preparation method of this invention is simple to operate, has a high yield, and is low in cost.

[0013] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0014] The first aspect of this invention provides a method for preparing a cfDNA detection standard, the method comprising the following steps:

[0015] (a) Expanding and culturing cells;

[0016] (b) Inducing apoptosis in the expanded cells;

[0017] (c) After the apoptosis treatment is completed, incubate for a period of time and collect the supernatant;

[0018] (d) Add the supernatant to the plasma to prepare a plasma standard for cfDNA detection; or extract cfDNA from the supernatant to prepare a DNA standard for cfDNA detection.

[0019] Preferably, the preparation method further includes mixing plasma standards or DNA standards from different cell sources in a certain proportion to prepare a combined standard.

[0020] Preferably, in step (b), the apoptosis treatment involves not renewing the culture medium, causing the cells to die due to nutrient depletion; or adding a cytotoxic agent to the cell culture medium to induce apoptosis.

[0021] Preferably, the cytotoxic agent is selected from at least one of the following: American pokeweed antimitotic protein, daunorubicin, rapamycin, paclitaxel, ferulic acid esterase inhibitor, etoposide, anthrone, colchicine, sodium hydroxide, and rotenone.

[0022] Preferably, in step (c), the incubation time is 4 to 24 hours and the incubation temperature is 37°C.

[0023] Preferably, in step (a), the cell amplification culture includes culturing the cells in a culture dish until the cells cover the entire culture dish.

[0024] Preferably, the medium is changed at intervals during the amplification culture process.

[0025] Preferably, the interval is 20 to 28 hours.

[0026] A second aspect of the present invention provides a cfDNA detection standard prepared by the above preparation method.

[0027] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0028] This invention uses the supernatant of cultured cell lines as the source of cfDNA detection standards. By nutrient depletion or the addition of cytotoxic reagents, the cells in the culture dish are rapidly killed, increasing the content of single nucleosomes in the culture supernatant and further increasing the concentration of cfDNA. Furthermore, by mixing the cell culture supernatant with plasma or synthetic plasma, plasma standards with specific variant characteristics and cfDNA concentrations can be obtained. Alternatively, nucleic acids can be extracted from the cell culture supernatant to prepare DNA standards. These standards play an important role in the performance verification and quality control of plasma nucleic acid extraction. In addition, when preparing multivariate cfDNA standards, cell suspensions carrying different variants (with added cytotoxic reagents) or cell culture supernatants can be mixed in a certain proportion to obtain cfDNA standards with nucleosome structure protection.

[0029] The fragment size distribution of the cfDNA prepared by this invention is highly consistent with that of plasma cfDNA under natural conditions, both exhibiting a main peak at 170 bp and a smaller peak at 300-400 bp. The preparation method of this invention is simple, using cell culture supernatant, eliminating the need for high expression of specific proteins in cell lines or adjustments to nucleic acid extraction steps, thus reducing operational steps, lowering costs, and improving the stability of cfDNA standards. Furthermore, the preparation method of this invention can prepare both DNA standards and plasma standards protected by nucleosome structures, which is of great significance for the development and performance validation of plasma nucleic acid extraction kits and the evaluation of full-process testing laboratories. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0031] Figure 1 This is the size distribution of cfDNA fragments in Example 1 of the present invention;

[0032] Figure 2 This is the size distribution of cfDNA fragments in Example 2 of the present invention. Detailed Implementation

[0033] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0034] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by those skilled in the art to which this invention pertains.

[0035] This invention provides a method for preparing a cfDNA detection standard, the method comprising the following steps:

[0036] (a) Expanding and culturing cells;

[0037] (b) Inducing apoptosis in the expanded cells;

[0038] (c) After the apoptosis treatment is completed, incubate for a period of time and collect the supernatant;

[0039] (d) Add the supernatant to the plasma to prepare a plasma standard for cfDNA detection; or extract cfDNA from the supernatant to prepare a DNA standard for cfDNA detection.

[0040] The fragment size distribution of the cfDNA prepared by this invention is highly consistent with that of plasma cfDNA under natural conditions, both exhibiting a main peak at 170 bp and a smaller peak at 300-400 bp. The preparation method of this invention is simple, using cell culture supernatant, eliminating the need for high expression of specific proteins in cell lines or adjustments to nucleic acid extraction steps, thus reducing operational steps, lowering costs, and improving the stability of cfDNA standards. Furthermore, the preparation method of this invention can prepare both DNA standards and plasma standards protected by nucleosome structures, which is of great significance for the development and performance validation of plasma nucleic acid extraction kits and the evaluation of full-process testing laboratories.

[0041] In one embodiment, the preparation method further includes mixing plasma standards or DNA standards from different cell sources in a certain proportion to prepare a combined standard.

[0042] In one embodiment, in step (b), the apoptosis treatment is to not renew the culture medium, causing the cells to die due to nutrient depletion; or to add a cytotoxic agent to the cell culture medium to induce apoptosis.

[0043] In one embodiment, the cytotoxic agent is selected from at least one of the following: pokeweed antimitotic protein, daunorubicin, rapamycin, paclitaxel, ferulic acid esterase inhibitor, etoposide, anthrone, colchicine, sodium hydroxide, and rotenone.

[0044] In one embodiment, in step (c), the incubation time can be any time from 4 to 24 hours, and the incubation temperature is 37°C.

[0045] In one embodiment, step (a), expanding the cell culture, includes culturing the cells in a culture dish until they cover the entire culture dish.

[0046] In one embodiment, the medium is changed at intervals during the amplification culture process.

[0047] In one embodiment, the interval is 20 to 28 hours.

[0048] Another embodiment of the present invention provides a cfDNA detection standard prepared by the above preparation method.

[0049] This invention uses the supernatant of cultured cell lines as the source of cfDNA detection standards. By nutrient depletion or the addition of cytotoxic reagents, the cells in the culture dish are rapidly killed, increasing the content of single nucleosomes in the culture supernatant and further increasing the concentration of cfDNA. Furthermore, by mixing the cell culture supernatant with plasma or synthetic plasma, plasma standards with specific variant characteristics and cfDNA concentrations can be obtained. Alternatively, nucleic acids can be extracted from the cell culture supernatant to prepare DNA standards. These standards play an important role in the performance verification and quality control of plasma nucleic acid extraction. In addition, when preparing multivariate cfDNA standards, cell suspensions carrying different variants (with added cytotoxic reagents) or cell culture supernatants can be mixed in a certain proportion to obtain cfDNA standards with nucleosome structure protection.

[0050] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0051] Example 1

[0052] This embodiment describes a method for preparing a cfDNA detection standard, which includes the following steps:

[0053] HeLa cell lines derived from cervical cancer and carrying HPV18 virus were cultured in three 10cm diameter cell culture dishes. 10mL of culture medium was added for each dish, and the medium was changed every 24 hours until the cells completely covered the dish. Medium changes were then stopped, and incubation continued. Due to insufficient nutrients, the cells gradually underwent apoptosis, exhibiting a loss of adhesion. Broken cell fragments were suspended in the culture supernatant. When more than 90% of the cells no longer adhered, incubation continued at 37°C. Supernatant was collected from one dish at 6, 12, and 18 hours. Nucleic acid extraction was performed using a plasma cfDNA extraction kit from Shenzhen Nanke Zhengtu, with a final DNA elution volume of 500μL TE buffer.

[0054] Concentration was detected using Qubit, and fragment size analysis was performed using an Agilent 2100 bioanalyzer. The results are shown in Table 1 below. The cfDNA fragment size distribution is as follows: Figure 1 As shown, Figure 1 Figures A, B, and C show the fragment distribution of cfDNA in the culture medium supernatant after 6, 12, and 18 hours of incubation, respectively.

[0055] Table 1

[0056]

[0057]

[0058] From Table 1 and Appendix Figure 1 It can be known that:

[0059] Appendix Figure 1 The fragment size distribution showed a clear DNA distribution around 170 bp, and two DNA fragments connected by nucleosomes existed between 300-400 bp, indicating that the DNA fragment distribution obtained in this invention highly matches that of cfDNA in its natural state. Furthermore, statistical analysis of the main peak area around 170 bp and calculation of the cfDNA main peak concentration, along with the Qubit concentration, revealed that the cfDNA concentration gradually decreased with longer incubation times. This is likely due to severe DNA degradation caused by prolonged incubation. Therefore, further optimization of the 37°C incubation time can improve the DNA yield.

[0060] Example 2

[0061] This embodiment describes a method for preparing a cfDNA detection standard, which includes the following steps:

[0062] Lung cancer is the most common cancer worldwide, and mutation sites in the EGFR gene have become targets for many targeted drugs. The NCI-H1975 cell line carries a variety of EGFR mutations (such as L858R and T790M), making it very suitable as a standard material for cfDNA detection.

[0063] Four NCI-H1975 cell lines were cultured in parallel using 10cm cell culture dishes. Once the cells had filled the dishes, specific concentrations of cytotoxic reagents—colchicine, paclitaxel, rotenone, and sodium hydroxide—were added to induce rapid cell death. The cells were incubated at 37°C for 6 hours. The supernatant was then collected and divided into two equal portions. One portion was used for direct nucleic acid extraction using a plasma extraction kit, with a DNA elution volume of 500 μL. The other portion was added to an equal volume of nucleic acid-free synthetic plasma to form a plasma standard for cfDNA detection. Nucleic acid was extracted from the other portion using a plasma extraction kit, with a DNA elution volume of 500 μL. Qubit quantification and fragment distribution analysis were performed on the eight DNA samples. The results are shown in Table 2 below. Figure 2 As shown, Figure 2 In the diagrams, A, B, C, and D represent cfDNA that caused cell death by the addition of reagents such as colchicine, paclitaxel, rotenone, and sodium hydroxide, respectively.

[0064] Table 2

[0065]

[0066]

[0067] From Table 2 and Figure 2 It can be known that:

[0068] The approximate concentration of cfDNA extracted from simulated plasma and raw culture medium supernatant indicates that this invention can be applied to the preparation of plasma standards for cfDNA detection. The varying concentrations and fragment sizes of cfDNA produced by different reagents suggest that different mechanisms inducing cell death affect the preparation efficiency of cfDNA standards, potentially due to factors such as influences on nucleosome structure and intracellular nuclease activity. In summary, this invention utilizes cytotoxic reagents to induce concentrated cell death, facilitating the preparation of standards for cfDNA detection. Furthermore, when mixed with synthetic plasma, these standards can be used as plasma standards for quality control and performance verification of the entire detection process, including nucleic acid extraction.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing a cfDNA detection standard, characterized in that, The preparation method includes the following steps: (a) Expanding and culturing cells; (b) Inducing apoptosis in the expanded cells; (c) After the apoptosis treatment is completed, incubate for a period of time and collect the supernatant; (d) Add the supernatant to the plasma to prepare a plasma standard for cfDNA detection; or extract cfDNA from the supernatant to prepare a DNA standard for cfDNA detection.

2. The preparation method according to claim 1, characterized in that, The preparation method also includes mixing plasma standards or DNA standards from different cell sources in a certain proportion to prepare a combined standard.

3. The preparation method according to claim 1 or 2, characterized in that, In step (b), the apoptosis treatment involves either not renewing the culture medium, causing the cells to die due to nutrient depletion, or adding a cytotoxic agent to the cell culture medium to induce apoptosis.

4. The preparation method according to claim 3, characterized in that, The cytotoxic reagent is selected from at least one of the following: American pokeweed antimitotic protein, daunorubicin, rapamycin, paclitaxel, ferulic acid esterase inhibitor, etoposide, anthrone, colchicine, sodium hydroxide, and rotenone.

5. The preparation method according to claim 1, characterized in that, In step (c), the incubation time is 4 to 24 hours and the incubation temperature is 37°C.

6. The preparation method according to claim 1, characterized in that, In step (a), the cell amplification culture includes culturing the cells in a culture dish until the cells cover the entire culture dish.

7. The preparation method according to claim 1 or 6, characterized in that, The medium is changed at intervals during the amplification culture process.

8. The preparation method according to claim 7, characterized in that, The interval is 20 to 28 hours.

9. The cfDNA detection standard prepared by any one of the preparation methods described in claims 1 to 8.

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