Composition and method for cell preservation and DNA extraction of NGS detection

Abstract

The invention discloses a composition and method for cell preservation and DNA extraction of NGS detection, and relates to the technical field of biological sample treatment.The composition is prepared from a cell sample preservation solution, a solid phase, a binding solution, a washing solution and an eluent. The preservation solution can avoid cell solidification and nucleic acid and protein cross-linking, reduce DNA degradation and false positive risks, and significantly improve DNA extraction quality, and meanwhile, the preservation solution has a lysis function, a sample can be stably preserved for 3-30 days at 4-30 DEG C without low-temperature conditions, additional lysis steps and protease are not needed during subsequent extraction, the operation can be completed by directly combining with a solid phase, the process is greatly simplified, and the cost is reduced. The efficiency is improved, and a reliable DNA template is provided for high-precision molecular diagnosis such as NGS.

Description

Technical Field

[0001] This invention relates to the field of biological sample processing technology, specifically to a composition and method for cell preservation and DNA extraction using NGS detection. Background Technology

[0002] Cells, as a common biological sample, are widely studied in fields such as molecular diagnostics, disease monitoring, and treatment. For example, in the diagnosis and treatment of tumors, cell DNA analysis is a commonly used and important technique. However, once cells are removed from the body, their endogenous DNA degrades rapidly. If DNA extraction cannot be performed immediately, cell samples usually need to be cryopreserved and transported under low-temperature conditions such as dry ice. This not only increases transportation costs but also significantly increases the operational burden.

[0003] To replace cryopreservation and transportation, cell preservation solutions are currently commonly used to achieve room temperature preservation and transportation of cell samples. However, existing cell preservation and cell DNA extraction methods generally have the following shortcomings:

[0004] Liquid-based cell preservation solutions are used to preserve cell samples. While they can maintain the integrity of cell morphology relatively well, they are mainly used for cell morphology examination. The formulations often contain fixatives such as methanol, ethanol, and formaldehyde. These components can interfere with the subsequent DNA extraction process. On the one hand, alcohol reagents can easily cause cell dehydration and solidification, increasing the difficulty of cell lysis. On the other hand, aldehyde reagents have a strong destructive effect on DNA. For example, formalin can cause nucleic acid and protein cross-linking, forming non-amplifiable fragments. Deamination reactions may also trigger cytosine artifacts, thus producing false positive results in next-generation sequencing (NGS) mutation analysis. In addition, methanol and formaldehyde themselves are also toxic.

[0005] The applicable cell sample types are limited, while the sources of cell samples in clinical and research are complex and diverse, such as cervical exfoliated cells, sputum cells, urine exfoliated cells, cerebrospinal fluid cells, and fine needle aspiration cells. In addition to cells, these samples are often accompanied by a large number of impurities such as fat, protein, mucus, and bacteria. Moreover, the components of impurities accompanied by cell samples from different sources vary significantly. Currently, commercially available cell preservation solutions are often designed for single cell samples, such as cervical cell preservation solutions and sputum cell preservation solutions, and lack compatibility and universality for multiple cell samples.

[0006] Cell preservation and DNA extraction are separate processes. When cell sample preservation solution is mixed with DNA extraction reagent lysis buffer, components such as guanidine salts, buffers, and reducing agents in the preservation solution may affect the normal operation of the lysis buffer, deviating from the optimal cell lysis and DNA binding conditions, thereby reducing the efficiency of DNA extraction.

[0007] Therefore, it is of great significance to develop a technology that can avoid cell solidification and nucleic acid cross-linking, is compatible with multiple cell sample types, and is compatible with the preservation and extraction process. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a composition and method for cell preservation and DNA extraction in NGS detection. The cell sample preservation solution provided is free of interfering components such as methanol and formaldehyde, which can avoid cell solidification and nucleic acid-protein cross-linking, reduce DNA degradation and false positive risks, and significantly improve DNA extraction quality. At the same time, the preservation solution also has a lysis function, and the sample can be stably preserved for 3-30 days at 4-30℃ without the need for low temperature conditions. Subsequent extraction does not require additional lysis steps and proteases; the operation can be completed directly by binding to the solid phase, greatly simplifying the process, improving efficiency, and providing a reliable DNA template for high-precision molecular diagnostics such as NGS.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In one aspect, a composition for cell preservation and DNA extraction using NGS detection, the composition comprising: a cell sample preservation solution, a solid phase, a binding solution, a washing solution, and an elution solution.

[0010] The cell sample preservation solution includes a liquid release agent, a buffer compound, an alkali metal salt, a metal ion chelating agent, a nonionic surfactant, a reducing agent, and an antifoaming agent.

[0011] The solid phase is selected from glass fiber membranes, silanol-coated magnetic beads, and carboxyl-coated magnetic beads;

[0012] The binding liquid is a polyol selected from ethanol, propanol, isopropanol, butanol, and 1,3-butanediol;

[0013] The washing solution is a 60-80% ethanol solution;

[0014] The elution buffer is TE buffer.

[0015] Furthermore, in the cell sample preservation solution, the dissociation agent is a dissociation salt selected from one or a mixture of two or more of guanidine thiocyanate, guanidine isothiocyanate, guanidine hydrochloride, sodium iodide, sodium perchlorate, sodium thiocyanate, and sodium trichloroacetate, and the dissociation agent is present in the cell sample preservation solution at a concentration of 0.1 to 6 M.

[0016] Furthermore, in the cell sample preservation solution, the buffer compound is selected from N-(tris(hydroxymethyl)methyl)glycine (TRICINE), tris(hydroxymethyl)aminomethane (TRIS), di(2-hydroxyethyl)iminotris(hydroxymethyl)methane (BIS-TRIS), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N,N-bis(2-hydroxyethyl)glycine (BICINE), 2-(N-morpholino)ethanesulfonic acid (MES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS) and / or phosphate buffer, and the concentration of the buffer compound is ≤500mM.

[0017] Furthermore, in the cell sample preservation solution, the alkali metal salt is selected from one or a mixture of two or more of sodium chloride, potassium chloride, and lithium chloride, and the concentration of the alkali metal salt is ≤500mM.

[0018] Furthermore, in the cell sample preservation solution, the metal ion chelating agent is selected from EDTA, EGTA, NTA, citric acid, iminodisuccinic acid, isoascorbic acid, triethanolamine, tartaric acid, sodium gluconate, and sodium alginate.

[0019] Furthermore, the nonionic surfactant in the cell sample preservation solution is a combination of TrionX-100 and Tween 20;

[0020] The defoamer is an organosilicone oil emulsion;

[0021] The reducing agent is dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, dithioerythritol, sodium thiosulfate, β-mercaptoethanol, or a mixture thereof.

[0022] On the other hand, a method for cell preservation and DNA extraction using NGS detection, the specific steps of which are as follows:

[0023] S100: Contact the cell sample with the cell sample preservation solution;

[0024] S200, Add the binding liquid to the mixture from step S100;

[0025] S300: Bind the DNA contained in the mixture of step S200 to the solid phase;

[0026] S400. Wash the solid phase containing bound DNA with washing solution;

[0027] S500, use elution buffer to elute DNA from the solid phase.

[0028] Furthermore, in S100, after the cell sample comes into contact with the cell sample preservation solution, the cell DNA is stabilized for ≥3 days in an environment of 4-30℃.

[0029] In S100 and S200, proteolytic enzymes, proteases, and serine proteases are not used.

[0030] Furthermore, in S200, after adding the binding liquid to the mixture of S100, S300 is carried out directly without pyrolysis incubation, wherein the pyrolysis incubation includes heating, mixing, shaking, and standing.

[0031] Furthermore, in S100, the cell samples are taken from the human respiratory system, digestive system, urinary system, reproductive system, musculoskeletal system, endocrine system, nervous system, lymphohematopoietic system, mammary glands and regional lymph nodes, skin and soft tissue, bones and joints, serous cavities, and eyes. The sampling methods include scraping, wiping, brushing, natural excretion, puncture and aspiration, irrigation, flushing, and drainage.

[0032] Compared with existing technologies, this composition and method for cell preservation and DNA extraction using NGS detection has the following advantages:

[0033] I. The cell sample preservation solution of this invention does not contain interfering components such as methanol and formaldehyde, which can avoid cell solidification and nucleic acid-protein cross-linking, reduce DNA degradation and false positive risks, and significantly improve DNA extraction quality. At the same time, the preservation solution also has a lysis function, and the sample can be stably preserved for 3-30 days at 4-30℃ without the need for low temperature conditions. Subsequent extraction does not require additional lysis steps and proteases; the operation can be completed directly by binding to the solid phase, which greatly simplifies the process, improves efficiency, and provides a reliable DNA template for high-precision molecular diagnostics such as NGS.

[0034] Second, the composition of the present invention can effectively handle multiple sample types containing impurities such as fat, protein, and mucus. It can not only further extend the preservation time of cellular DNA, but also significantly simplify the subsequent DNA extraction process. Since the DNA has been fully released from the cells into the preservation solution during room temperature storage, there is no need to use a lysis buffer separately in the subsequent nucleic acid extraction process. This design effectively simplifies the reagent system and operation process required for DNA extraction. At the same time, the integration of the functions of the preservation solution and the lysis buffer avoids component interference between different systems, thereby improving extraction efficiency and consistency of results.

[0035] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0037] Figure 1 This is a flowchart of a method for cell preservation and DNA extraction using NGS detection. Detailed Implementation

[0038] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0039] Example 1

[0040] This embodiment verifies the preservation effect of the cell sample preservation solution of the present invention on exfoliated cells in human saliva through comparative experiments. Saliva samples were collected and divided into two groups, which were stored at room temperature and frozen at -20℃ respectively using the cell sample preservation solution of the present invention. After 3 days, DNA was extracted and the concentration was compared. The results showed that the DNA concentration of the sample stored at 30℃ for 3 days with the sample frozen at -20℃ was not significantly different, proving that it can effectively achieve stable preservation of cell samples at room temperature, and the operation is simple and does not require low temperature conditions.

[0041] (1) Preparation of experimental materials

[0042] Sample collection tools: 20mL sterile saline, 50mL sterile centrifuge tubes, 1.5mL sterile EP tubes, pipettes (100-1000μL) and matching sterile pipette tips.

[0043] Cell sample preservation solution:

[0044] 3M guanidine hydrochloride: As a dissociative agent, it can disrupt cell structure, denature proteins, release nucleic acids, and inhibit nuclease activity;

[0045] 50mM Tris-HCl (pH 8.0): Maintains stable solution pH and creates a suitable alkaline environment for nucleic acid preservation;

[0046] 100mM NaCl and 100mM KCl: provide suitable ionic strength and promote nucleic acid stability;

[0047] 5mM EDTA: chelates divalent metal ions and inhibits metal ion-dependent nuclease activity;

[0048] 10% Triton X-100 and 10% Tween 20 (volume ratio 1:1): a combination of nonionic surfactants that enhance cell membrane permeability and aid in cell lysis;

[0049] 100mM dithiothreitol: a reducing agent that breaks disulfide bonds in proteins and promotes protein denaturation;

[0050] 1% silicone oil (by volume): an antifoaming agent to reduce the interference of air bubbles generated during sample processing.

[0051] Prepared with ultrapure water, filtered through a 0.22μm filter membrane for sterilization, and stored at 4℃ away from light;

[0052] Binding solution: anhydrous ethanol (analytical grade), used to adjust the solution polarity and promote the binding of DNA to solid-phase materials;

[0053] Solid phase: Nucleic acid adsorption column (including glass fiber filter membrane), with high adsorption capacity and specificity;

[0054] Washing solution: 75% ethanol (prepared with DEPC-treated water), used to remove impurities without affecting DNA binding;

[0055] Elution buffer: TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0) provides a suitable pH environment to promote DNA elution from the solid phase;

[0056] (2) Sample collection process

[0057] Each volunteer rinsed their mouth with 20 mL of sterile saline for 1 minute, discarded the mouthwash, and sat quietly for 5 minutes. Then, a 50 mL sterile centrifuge tube was placed below the lower lip to collect approximately 6 mL of naturally secreted saliva. The saliva sample was immediately inverted and mixed 10 times to ensure homogeneity. Each saliva sample was then divided into 6 equal portions, 1 mL each, and transferred to 1.5 mL sterile EP tubes. All EP tubes were centrifuged at 2000 × g for 3 minutes at room temperature. The supernatant was carefully discarded, and the bottom cell pellet (approximately 50-100 μL) was retained.

[0058] (3) Sample preservation and processing

[0059] Experimental group (processing of cell sample preservation solution of the present invention): 450 μL of cell sample preservation solution pre-equilibrated to room temperature was added to one sample from each of the three volunteers (a total of three samples). The samples were vortexed for 10 seconds (3000 rpm) to completely suspend the cell pellet. After labeling the sample information, the samples were stored in a 30°C constant temperature water bath in the dark for 3 days to observe the sample status.

[0060] Control group (low-temperature cryopreservation): The remaining 3 samples from 3 volunteers were not added with any preservation solution. After the sample information was marked, they were directly placed in a -20°C freezer for cryopreservation, and repeated freeze-thaw cycles were avoided during the cryopreservation process.

[0061] (4) DNA extraction

[0062] Sample preparation: After 3 days of storage, all samples were removed. Control group samples were taken out of the -20℃ freezer and thawed at room temperature for 10 minutes. Then, 450μL of cell sample preservation solution was added and the samples were vortexed for 10 seconds. Experimental group samples were taken directly from the 30℃ water bath without any additional treatment.

[0063] Binding steps: Accurately add 250 μL of binding buffer (anhydrous ethanol) to all sample tubes and mix thoroughly by pipetting 10 times (the total volume of the solution at this point is approximately 750 μL). Transfer the entire mixture to the nucleic acid adsorption column (approximately 600 μL each time, done in two separate transfers).

[0064] Centrifuge at 8000×g for 30 seconds at room temperature to allow DNA to fully bind to the glass fiber filter membrane. Discard the filtrate in the collection tube and reassemble the adsorption column into the collection tube.

[0065] Washing procedure: Add 500 μL of washing buffer (75% ethanol) to the adsorption column, centrifuge at 8000×g for 30 seconds at room temperature to remove unbound proteins, salts and other impurities, discard the filtrate, reassemble the adsorption column into the collection tube, repeat the washing once, centrifuge the empty column at 14,000×g for 2 minutes to completely remove residual ethanol to prevent it from affecting subsequent experiments.

[0066] Elution procedure: Transfer the adsorption column to a new 1.5 mL sterile centrifuge tube, add 50 μL of TE buffer (preheated to 65 °C) to the center of the filter membrane of the adsorption column, let stand at room temperature for 2 minutes to allow the DNA to dissolve completely, centrifuge at 14,000 × g for 1 minute, collect the eluent (containing purified DNA), immediately place the extracted DNA on ice and store it for concentration detection within 2 hours.

[0067] (5) DNA concentration detection methods

[0068] DNA concentration was detected using a Qubit4 fluorometer. The results showed that salivary cells preserved with the cell sample preservation solution described in this invention did not exhibit significant degradation compared to those preserved at low temperatures. Specific experimental data are shown in the table below:

[0069]

[0070] In summary, this embodiment, through controlled comparative experiments, verified the preservation effect of the cell sample preservation solution of the present invention on saliva-exfoliated cells. The results showed that the DNA extraction concentration of samples preserved at 30°C for 3 days using the preservation solution containing 3M guanidine hydrochloride, a complex buffer system, and a surfactant was not statistically different from that of samples frozen at -20°C (P>0.05). Furthermore, the samples exhibited good stability and no significant degradation. The components worked synergistically: the exfoliant disrupted cell structure and inhibited nucleases; the buffer maintained pH stability; the metal ion chelating agent further protected DNA; and the surfactant enhanced the lysis effect. This method requires no cryogenic equipment, is easy to operate, and provides an effective solution for the room-temperature preservation and transportation of saliva and other bodily fluid samples.

[0071] Example 2

[0072] This embodiment evaluated the treatment effect of the cell sample preservation solution of the present invention on bronchoalveolar lavage fluid samples containing mucus. Five clinically collected bronchoalveolar lavage fluid samples containing mucus were selected and treated with liquid-based cell preservation solution and cell sample preservation solution of Example 1, respectively. The dispersion state of the mucus was observed. The results showed that the cell sample preservation solution of Example 1 could completely disperse the mucus in all samples within 10 minutes, while the liquid-based cell preservation solution still left obvious mucus residue and shrinkage. This proves that the preservation solution of the present invention has excellent treatment ability for samples containing mucus and can effectively solve the problem of mucus interference.

[0073] (1) Preparation of experimental materials

[0074] Sample source: 5 clinical bronchoalveolar perfusion fluid samples, all of which showed obvious mucus components to the naked eye;

[0075] Control reagent: Liquid-based cell preservation solution (mainly composed of methanol and buffer solution);

[0076] Experimental reagents: Same as the cell sample preservation solution in Example 1;

[0077] Experimental equipment: 1.5mL sterile EP tubes, pipettes (100-1000μL), vortex mixer;

[0078] (2) Sample processing

[0079] From each bronchoalveolar lavage fluid sample, two 200 μL aliquots of lavage fluid were pipetted into labeled 1.5 mL EP tubes using the same pipette.

[0080] Control group: Add 1 mL of liquid-based cell preservation solution to one of the samples and gently invert to mix 10 times;

[0081] Experimental group: Add 1 mL of the cell sample preservation solution from Example 1 to another sample, and gently invert and mix 10 times as well;

[0082] All samples were placed in a room temperature (25℃) environment and left to stand. The condition of the samples was observed at 0 minutes, 5 minutes and 10 minutes.

[0083] (3) Observation and evaluation

[0084] Observation: Record the macroscopic state of the mucus in the samples at each time point, including whether mucus clumps are visible and changes in the size of the mucus clumps. The specific results are shown in the table below:

[0085]

[0086] In summary, this embodiment verified the ability of the preservation solution of the present invention to process samples containing mucus through clinical samples. The results showed that the liquid-based preservation solution, due to the presence of methanol and other components, caused the mucus protein to coagulate and shrink, resulting in obvious mucus residue. In contrast, the preservation solution of the present invention, through the breaking of the hydrogen bond structure of the mucus protein by the dissociation agent (guanidine hydrochloride) and the synergistic effect of the composite surfactants (Triton X-100 and Tween 20), effectively dispersed the mucus components, making the mucus in all samples completely dispersed, thus removing obstacles for subsequent DNA extraction.

[0087] Example 3

[0088] This embodiment verifies the preservation effect of the cell sample preservation solution of the present invention on exfoliated human cervical epithelial cells and its compatibility with HPV detection. Sixteen HPV-positive cervical samples were selected and preserved at room temperature for 10 days using liquid-based preservation solution and the preservation solution of the present invention, respectively. After DNA extraction, HPV fluorescent PCR detection was performed. The results showed that the samples treated with the preservation solution of the present invention had a higher HPV positive detection rate and a lower average Ct value, indicating that the DNA was preserved more completely and the amplification efficiency was higher, making it suitable for molecular diagnostic applications such as clinical HPV detection.

[0089] (1) Preparation of experimental materials

[0090] Sample source: 16 clinically confirmed HPV-positive cervical epithelial cell samples were collected using a dedicated cervical sampling brush;

[0091] Experimental reagents:

[0092] Control group: Liquid-based cell preservation solution;

[0093] Experimental group: Cell sample preservation solution from Example 1;

[0094] Human papillomavirus (HPV) nucleic acid detection kit (PCR fluorescence method);

[0095] The composition and operating procedures are the same as in Example 1;

[0096] (2) Sample processing and preservation

[0097] Each patient was sampled simultaneously using two cervical sampling brushes to ensure sample consistency.

[0098] After sampling, one of the sampling brushes was immediately placed in a special preservation tube containing 5 mL of liquid-based cell preservation solution (control group);

[0099] Another sampling brush was placed in a preservation tube containing 5 mL of the cell sample preservation solution of the present invention (experimental group);

[0100] Gently rotate the sampler to ensure that the cells are fully eluted into the preservation solution, then squeeze the sampling brush against the tube wall and remove it.

[0101] After all samples were labeled, they were stored at 25°C in the dark, and the condition of the samples was observed during the storage period.

[0102] (3) DNA extraction

[0103] After 10 days of storage, 200 μL of the preservation solution was taken from each group of samples for DNA extraction.

[0104] The extraction steps are the same as in Example 1, including adding binding solution, column chromatography, washing, and elution;

[0105] The elution volume is 50 μL, and the extracted DNA is immediately used for HPV detection or stored at -20℃ for a short period of time.

[0106] (4) HPV fluorescent PCR detection

[0107] The extracted DNA was analyzed, and the Ct values ​​of each sample in the FAM, VIC, and CY5 fluorescence channels were monitored. The results showed that the cell sample preservation solution in Example 1 had a higher positive detection rate and a lower Ct value than the liquid-based cell preservation solution. The specific experimental results are shown in the table below:

[0108]

[0109] In summary, this embodiment verified the practicality of the preservation solution of the present invention using clinical HPV positive samples. The results showed that, compared with liquid-based preservation solutions, the preservation solution of the present invention can still maintain the integrity and amplifiability of DNA after 10 days of storage at room temperature. This is due to the fact that the preservation solution of the present invention avoids nucleic acid cross-linking and degradation caused by formaldehyde / methanol, and the synergistic effect of the dissociation agent and the buffer system effectively protects the integrity of DNA. These results confirm that the preservation solution of the present invention is suitable for long-term preservation of clinical cervical cell samples and can meet the requirements of molecular diagnostic technologies such as HPV detection.

[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A composition for cell preservation and DNA extraction using NGS detection, characterized in that, The composition comprises: cell sample preservation solution, solid phase, binding solution, washing solution, and elution solution. The cell sample preservation solution includes a liquid release agent, a buffer compound, an alkali metal salt, a metal ion chelating agent, a nonionic surfactant, a reducing agent, and an antifoaming agent. The solid phase is selected from glass fiber membranes, silanol-coated magnetic beads, and carboxyl-coated magnetic beads; The binding liquid is a polyol selected from ethanol, propanol, isopropanol, butanol, and 1,3-butanediol; The washing solution is a 60-80% ethanol solution; The elution buffer is TE buffer.

2. The composition for cell preservation and DNA extraction using NGS detection according to claim 1, characterized in that, In the cell sample preservation solution, the dissociation agent is a dissociation salt selected from one or more of guanidine thiocyanate, guanidine isothiocyanate, guanidine hydrochloride, sodium iodide, sodium perchlorate, sodium thiocyanate, and sodium trichloroacetate. The dissociation agent is present in the cell sample preservation solution at a concentration of 0.1 to 6 M.

3. The composition for cell preservation and DNA extraction using NGS detection according to claim 1, characterized in that, The cell sample preservation solution contains a buffer compound selected from N-(tris(hydroxymethyl)methyl)glycine (TRICINE), tris(hydroxymethyl)aminomethane (TRIS), di(2-hydroxyethyl)iminotris(hydroxymethyl)methane (BIS-TRIS), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N,N-bis(2-hydroxyethyl)glycine (BICINE), 2-(N-morpholino)ethanesulfonic acid (MES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS), and / or phosphate buffers, with a concentration ≤500 mM.

4. The composition for cell preservation and DNA extraction using NGS detection according to claim 1, characterized in that, In the cell sample preservation solution, the alkali metal salt is selected from one or two or more mixtures of sodium chloride, potassium chloride, and lithium chloride, and the concentration of the alkali metal salt is ≤500mM.

5. The composition for cell preservation and DNA extraction using NGS detection according to claim 1, characterized in that, The metal ion chelating agent in the cell sample preservation solution is selected from EDTA, EGTA, NTA, citric acid, iminodisuccinic acid, isoascorbic acid, triethanolamine, tartaric acid, sodium gluconate, and sodium alginate.

6. The composition for cell preservation and DNA extraction using NGS detection according to claim 1, characterized in that, The cell sample preservation solution contains a combination of TrionX-100 and Tween 20 as a nonionic surfactant. The defoamer is an organosilicone oil emulsion; The reducing agent is dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, dithioerythritol, sodium thiosulfate, β-mercaptoethanol, or a mixture thereof.

7. A method for cell preservation and DNA extraction using NGS detection, applicable to the composition for cell preservation and DNA extraction using NGS detection as described in any one of claims 1-6, characterized in that, The specific steps of this method are as follows: S100: Contact the cell sample with the cell sample preservation solution; S200, Add the binding liquid to the mixture from step S100; S300: Bind the DNA contained in the mixture of step S200 to the solid phase; S400. Wash the solid phase containing bound DNA with washing solution; S500, use elution buffer to elute DNA from the solid phase.

8. The method for cell preservation and DNA extraction by NGS detection according to claim 7, characterized in that, In S100, after the cell sample comes into contact with the cell sample preservation solution, the cell DNA is stabilized for ≥3 days in an environment of 4-30℃. In S100 and S200, proteolytic enzymes, proteases, and serine proteases are not used.

9. The method for cell preservation and DNA extraction by NGS detection according to claim 7, characterized in that, In step S200, after adding the binding liquid to the mixture of S100, S300 is carried out directly without pyrolysis incubation. The pyrolysis incubation includes heating, mixing, shaking, and standing.

10. The method for cell preservation and DNA extraction by NGS detection according to claim 7, characterized in that, In S100, the cell samples are taken from the human respiratory system, digestive system, urinary system, reproductive system, musculoskeletal system, endocrine system, nervous system, lymphohematopoietic system, mammary glands and regional lymph nodes, skin and soft tissue, bones and joints, serous cavities, and eyes. The sampling methods include scraping, wiping, brushing, natural excretion, puncture and aspiration, irrigation, flushing, and drainage.

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