DNA / RNA extraction kit based on magnetic bead method without enzyme digestion

Abstract

The application discloses a DNA / RNA reagent kit based on a magnetic bead method and free of enzyme digestion, and belongs to the technical field of nucleic acid extraction and purification. The reagent kit comprises a lysis solution AL, protease K, a specific binding buffer CLB, amino magnetic bead suspension, silicon hydroxyl magnetic bead suspension and other components. The application solves the problems of nucleic acid degradation, incomplete removal of cross contamination and complicated process caused by enzyme digestion in the prior art, is suitable for automatic high-throughput processing, and has a wide application prospect in scientific research and clinical application.

Description

Technical Field

[0001] This invention relates to the field of reagent kit technology, and more specifically to a reagent kit for enzyme-free digestion and extraction of DNA / RNA based on magnetic beads. Background Technology

[0002] Nucleic acid extraction is a fundamental step in molecular biology experiments. After total nucleic acid extraction, downstream applications (such as gene amplification and sequencing analysis) often require high-purity nucleic acids of a single type (RNA or DNA). Bacterial samples, due to their complex cell wall structure and active endogenous nucleases, present a typical challenge for the extraction of high-integrity and high-purity nucleic acids, especially in the simultaneous acquisition of RNA and DNA while avoiding cross-contamination, where current technologies still have significant limitations.

[0003] Currently, the mainstream methods for obtaining high-purity RNA from bacteria mainly involve magnetic bead or centrifugal column methods combined with enzymatic digestion. The core workflow of typical commercial kits (such as the Qiagen RNeasy series and Thermo Fisher Scientific's MagMAX™ Microbial RNA Isolation Kit) is as follows: ① Lysis and binding: Cells are lysed using a lysis buffer containing dissociative salts and surfactants to inactivate nucleases, and total nucleic acids (RNA + DNA) are adsorbed onto silica-based magnetic beads or silica membranes; ② Washing: Impurities are removed using a washing buffer containing ethanol; ③ Enzymatic digestion: The magnetic beads are resuspended in DNase I buffer and incubated for 15-30 minutes to degrade DNA contaminants; ④ Second wash: DNase enzymes and digestion products are removed; ⑤ Elution: Purified RNA is obtained. Similarly, RNase A digestion is used to remove RNA contamination when extracting DNA.

[0004] However, existing technologies have significant drawbacks: 1. Risks of cross-contamination and degradation coexist: The digestion efficiency of DNase I on the surface of a solid support is limited by spatial accessibility, which can easily lead to incomplete DNA removal and affect the accuracy of downstream experiments; at the same time, commercial DNase I may leave trace amounts of RNase, or long-term incubation (37°C) may lead to RNA degradation, reducing yield and integrity. 2. The process is cumbersome and costly: The additional enzyme digestion steps require the preparation of buffer solutions, control of incubation conditions, and the addition of washing steps, which extends the operation time by 15-45 minutes, increasing labor and reagent costs. 3. Low sample utilization: Traditional methods can only obtain one type of nucleic acid, while the other type of nucleic acid is degraded and discarded during digestion, which cannot meet the needs of parallel DNA / RNA analysis of precious samples; 4. Poor automation adaptability: The requirements for incubation and buffer preparation in enzyme digestion steps increase the complexity of automation integration, which is not conducive to high-throughput sample processing. 5. Insufficient nucleic acid adsorption efficiency: Traditional magnetic bead systems have limited specific adsorption capacity for nucleic acids, especially in low-concentration samples, where nucleic acid loss is likely to occur, leading to a decrease in extraction recovery rate and failing to meet the extraction needs of trace samples.

[0005] Therefore, developing an extraction kit that requires no enzyme digestion, has a simple process, can simultaneously obtain high-purity DNA and RNA, and can improve nucleic acid adsorption efficiency, in order to solve the core pain points of existing technologies, has become an urgent need in the field of molecular biology. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a DNA / RNA extraction kit based on magnetic bead-based enzyme-free digestion, thereby resolving the problems mentioned in the background section.

[0007] The present invention solves the technical problem by adopting the following technical solution: This invention provides a DNA / RNA extraction kit based on enzyme-free digestion using magnetic beads, comprising lysis buffer AL, proteinase K, specific binding buffer CLB, amino magnetic bead suspension, silanol magnetic bead suspension, washing buffer DW1, washing buffer DW2, elution buffer, enzyme-free water, and nano-silica modifier. The specific composition and function of each component are as follows: (1) Lysis buffer AL: It consists of ionizing salt, surfactant and RNase inhibitor. The concentrations of each component are: guanidine hydrochloride 5-7 mol / L, sodium dodecyl sulfate (SDS) 1-3% (v / v), EDTA 10-20 mmol / L, β-mercaptoethanol 5-10 mmol / L, pH 5.5-6.5. Its function is to break the bacterial cell wall, release nucleic acid (RNA+DNA), and at the same time inactivate endogenous nucleases to protect the integrity of nucleic acid.

[0008] (2) Proteinase K: concentration 20 mg / mL. Its function is to degrade proteins in the sample, promote the release of nucleic acids, and avoid interference of proteins with nucleic acid binding.

[0009] (3) Specific binding buffer CLB: composed of Tris-HCl, sodium chloride and ethanol, with the following concentrations: Tris-HCl 20-40 mmol / L, sodium chloride 1.0-1.5 mol / L, ethanol 40-60% (v / v), pH 7.0-8.0; its function is to create a specific ionic strength and pH environment to promote the specific binding of RNA to amino magnetic beads, while inhibiting the interaction between DNA and amino magnetic beads.

[0010] (4) Amino magnetic bead suspension: The concentration of amino functionalized magnetic beads is 10-20 mg / mL, the particle size of magnetic beads is 500-1000 nm, the surface is modified with amino (-NH2) groups, and the zeta potential is +20~+30 mV; its function is to specifically capture RNA by preferentially binding RNA through electrostatic interaction and hydrogen bonds under the binding buffer CLB conditions.

[0011] (5) Silyl hydroxyl magnetic bead suspension (MBVB): The concentration of silyl hydroxyl functionalized magnetic beads is 10-20 mg / mL, the particle size of the magnetic beads is 500-1000 nm, and the surface is rich in silyl hydroxyl (-SiOH) groups; its function is to specifically bind DNA in the remaining solution system to achieve DNA capture and separation.

[0012] (6) Washing solution DW1: composed of Tris-HCl, sodium chloride and ethanol, with the following concentrations: Tris-HCl 10-20 mmol / L, sodium chloride 0.5-1.0 mol / L, ethanol 70-80% (v / v), pH 7.0-8.0; its function is to remove impurities such as proteins and salts adsorbed on the surface of amino magnetic beads / silicone magnetic beads without affecting the binding of nucleic acids to magnetic beads.

[0013] (7) Washing solution DW2: is a 75-85% (v / v) ethanol solution; its function is to further remove residual salts and organic impurities and improve the purity of nucleic acids.

[0014] (8) Elution buffer: composed of Tris-HCl and EDTA, with the following concentrations: Tris-HCl 5-10 mmol / L, EDTA 0.1-0.5 mmol / L, pH 8.0-8.5; its function is to gently elute the nucleic acids bound to the magnetic beads and protect the integrity of the nucleic acids.

[0015] (9) Enzyme-free water: DEPC treated, free from RNase and DNase contamination; used to dilute reagents or replace elution buffers, adaptable to different downstream experimental needs.

[0016] (10) Nano-silica modifier: The concentration is 5-10 mg / mL. Its function is to synergistically enhance the adsorption efficiency of nucleic acid with bifunctional magnetic beads, strengthen the specific binding ability of magnetic beads to nucleic acid, reduce the loss of nucleic acid in low-concentration samples, significantly improve the nucleic acid extraction recovery rate and purity, and at the same time help stabilize the nucleic acid structure, further reducing the risk of nucleic acid degradation. Its specific preparation method is as follows: ① Pretreatment: Place nano-silica in a sufficient amount of sulfuric acid solution with a mass fraction of 5-8%, stir at room temperature for 2-3 hours until it is mixed evenly, then wash repeatedly with deionized water until the washing liquid is neutral, filter to collect the solid, and dry in an oven at 60-80℃ for 4-6 hours to obtain pretreated nano-silica. ② Preparation of montmorillonite solution: Take 3-5 parts of montmorillonite and 2-5 parts of carbon nanotubes, add them to 5-8 parts of sodium dodecylbenzenesulfonate solution with a mass fraction of 4-5%, and ultrasonically disperse for 30-60 minutes to obtain a uniformly dispersed montmorillonite solution (all parts are by mass). ③ Blending and ball milling: Take 3-5 parts of the dried nano-silica from step ①, 2-5 parts of nano-cellulose and 5-8 parts of the montmorillonite liquid prepared in step ②, put them into a ball mill, adjust the ball milling speed to 1000-1500 r / min, and continue ball milling for 2 hours; ④ Post-processing: After ball milling, the mixture is filtered, the solid product is collected, and dried in an oven at 60-80℃ for 4-6 hours. After cooling to room temperature, it is pulverized and passed through an 80-mesh sieve to obtain nano-silica modifier.

[0017] 2. Preferred Reagent Kit Scheme Furthermore, the optimal composition of each component of the kit is as follows: (1) Lysis buffer AL: guanidine hydrochloride 6 mol / L, SDS 2% (v / v), EDTA 15 mmol / L, β-mercaptoethanol 8 mmol / L, pH=6.0; (2) Specific binding buffer CLB: Tris-HCl 30 mmol / L, sodium chloride 1.2 mol / L, ethanol 50% (v / v), pH=7.5; (3) Amino magnetic bead suspension: concentration 15 mg / mL, magnetic bead particle size 800 nm, Zeta potential +25 mV; (4) Silyl hydroxyl magnetic bead suspension: concentration 15 mg / mL, magnetic bead particle size 800 nm; (5) Washing solution DW1: Tris-HCl 15mmol / L, sodium chloride 0.8mol / L, ethanol 75% (v / v), pH=7.5; (6) Washing solution DW2: 80% (v / v) ethanol solution; (7) Eluent: Tris-HCl 8 mmol / L, EDTA 0.3 mmol / L, pH=8.3; (8) Nano silica modifier: concentration 8 mg / mL, its optimal preparation method is as follows: Pretreatment: Nano-silica was placed in a 6% sulfuric acid solution and stirred at room temperature for 2.5 hours, washed with water until neutral, filtered, and dried at 70°C for 5 hours; Preparation of montmorillonite solution: 4 parts montmorillonite, 3 parts carbon nanotubes, 6 parts sodium dodecylbenzenesulfonate solution (4.5% by mass) were added and ultrasonically dispersed for 45 minutes; Blending and ball milling: 4 parts pretreated nano silica, 3 parts nano cellulose, 6 parts montmorillonite liquid, ball milling speed 1200 r / min, ball milling for 2 hours; Post-processing: After filtration, dry at 70℃ for 5 hours, pulverize and pass through an 80-mesh sieve to obtain nano-silica modifier.

[0018] 3. Instructions for using the reagent kit The specific steps for enzyme-free digestion and extraction of DNA / RNA from bacterial samples using this kit are as follows: S1. Sample pretreatment: Take 1 mL of sample with a concentration of 10... 7 For bacterial samples of CFU / mL, centrifuge at 4000×g for 5 minutes, discard the supernatant, and retain the bacterial precipitate; S2. Cell lysis: Add 500 μL of lysis buffer AL and 20 μL of proteinase K to the bacterial pellet, vortex to mix, and incubate at 56°C for 15-20 minutes, vortexing every 5 minutes to ensure complete cell lysis and release of nucleic acids (RNA+DNA). S3. Add nano silica modifier: Add 30 μL of nano silica modifier to the lysis buffer, vortex to mix, and let stand at room temperature for 3-5 minutes to allow the modifier to be fully dispersed and initially bound to the nucleic acid; S4. RNA-specific binding: Add 300 μL of specific binding buffer CLB to the above system, vortex to mix, then add 50 μL of amino magnetic bead suspension, incubate at room temperature (25±2℃) for 5-8 minutes, gently inverting to mix 3-4 times during the process; use a magnetic rack to adsorb the amino magnetic beads, and aspirate the supernatant (containing DNA) into a new centrifuge tube for subsequent DNA extraction. S5. RNA Washing and Purification: ① Add 800 μL of washing buffer DW1 to the centrifuge tube containing amino-adsorbed magnetic beads, vortex to mix, then use a magnetic rack to adsorb the magnetic beads and discard the supernatant; ② Add 800 μL of washing buffer DW2, vortex to mix, then use a magnetic rack to adsorb the magnetic beads and discard the supernatant; ③ Repeat step ② once to ensure that impurities are completely removed; ④ Open the centrifuge tube cap and let it stand at room temperature for 5-10 minutes to dry any residual ethanol on the surface of the magnetic beads; S6. RNA elution: Add 50-100 μL of elution buffer to the dried magnetic beads, vortex to mix, incubate at 56°C for 5 minutes, use a magnetic rack to adsorb the magnetic beads, collect the supernatant, which is the purified RNA without DNA contamination, and store at -80°C for later use. S7. DNA-specific binding: Add 100 μL of silanol magnetic bead suspension to the supernatant (containing DNA) collected in step S4, incubate at room temperature for 5-8 minutes, gently inverting and mixing 3-4 times during the process; use a magnetic rack to adsorb the silanol magnetic beads and discard the supernatant. S8. DNA Washing and Purification: ① Add 800 μL of washing buffer DW1, vortex to mix, then use a magnetic rack to adsorb the magnetic beads and discard the supernatant; ② Add 800 μL of washing buffer DW2, vortex to mix, then use a magnetic rack to adsorb the magnetic beads and discard the supernatant; ③ Repeat step ② once; ④ Open the centrifuge tube cap and let it stand at room temperature for 5-10 minutes to dry any residual ethanol on the surface of the magnetic beads; S9. DNA elution: Add 50-100 μL of elution buffer to the dried magnetic beads, vortex to mix, incubate at 56°C for 5 minutes, use a magnetic rack to adsorb the magnetic beads, collect the supernatant, which is the purified DNA free of RNA contamination, and store at -20°C for later use.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention features an enzyme-free design, avoiding enzyme-related risks: It eliminates the traditional DNase / RNase digestion step, achieving nucleic acid separation through physicochemical selective binding, thus preventing nucleic acid degradation caused by enzyme residue at the source, while ensuring thorough removal of cross-contamination (DNA / RNA contamination rate ≤0.5%), improving nucleic acid purity and integrity; it achieves dual DNA / RNA recovery: simultaneously obtaining high-purity RNA and DNA from the same sample, increasing sample utilization by 100%, meeting the needs of genome and transcriptome association analysis, ensuring data consistency, and is especially suitable for precious samples: the synergistic effect of bifunctional magnetic beads and specific buffers... This invention ensures a stable and controllable extraction process with small intra- and inter-batch coefficients of variation, stable nucleic acid recovery rate and purity, and guarantees the reproducibility of downstream experiments. The newly added nano-silica modifier in this invention achieves synergistic modification of nano-silica, nano-cellulose and montmorillonite through a specific preparation process, which can significantly improve the adsorption efficiency of magnetic beads for nucleic acids, reduce nucleic acid loss, especially improve the extraction effect of low concentration samples, and at the same time help stabilize the nucleic acid structure, further reduce the risk of degradation, and increase the nucleic acid recovery rate by 10-15%, solving the pain point of insufficient adsorption efficiency of traditional reagent kits. Moreover, the preparation process of this modifier is simple, low-cost and easy to scale up. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 A DNA / RNA extraction kit based on magnetic beads using enzyme-free digestion, with the following components and concentrations: (1) Lysis buffer AL: guanidine hydrochloride 6 mol / L, SDS 2% (v / v), EDTA 15 mmol / L, β-mercaptoethanol 8 mmol / L, pH=6.0; (2) Proteinase K: 20 mg / mL; (3) Specific binding buffer CLB: Tris-HCl 30 mmol / L, sodium chloride 1.2 mol / L, ethanol 50% (v / v), pH=7.5; (4) Amino magnetic bead suspension: 15 mg / mL, particle size 800 nm, Zeta potential +25 mV; (5) Silyl hydroxyl magnetic bead suspension: 15 mg / mL, particle size 800 nm; (6) Washing solution DW1: Tris-HCl 15mmol / L, sodium chloride 0.8mol / L, ethanol 75% (v / v), pH=7.5; (7) Washing solution DW2: 80% (v / v) ethanol solution; (8) Eluent: Tris-HCl 8 mmol / L, EDTA 0.3 mmol / L, pH=8.3; (9) Enzyme-free water: DEPC treated; (10) Nano silica modifier: concentration 8 mg / mL, preparation method as follows: ① Pretreatment: Nano-silica was placed in a 6% sulfuric acid solution and stirred at room temperature for 2.5 hours, washed with water until neutral, filtered, and dried at 70°C for 5 hours; ② Preparation of montmorillonite solution: 4 parts montmorillonite, 3 parts carbon nanotubes, add 6 parts sodium dodecylbenzenesulfonate solution with a mass fraction of 4.5%, and ultrasonically disperse for 45 minutes; ③ Blending and ball milling: 4 parts pretreated nano silica, 3 parts nano cellulose, 6 parts montmorillonite liquid, ball milling speed 1200 r / min, ball milling for 2 hours; ④ Post-processing: After filtration, dry at 70℃ for 5 hours, pulverize and pass through an 80-mesh sieve to obtain nano-silica modifier.

[0022] How to use: S1. Take 1 mL of E. coli sample (10 7 Centrifuge at 4000×g for 5 minutes (CFU / mL), discard the supernatant; S2. Add 500 μL of lysis buffer AL and 20 μL of proteinase K, and incubate at 56 °C for 18 minutes, mixing once every 5 minutes during the incubation period. S3. Add 30μL of nano silica modifier, let stand at room temperature for 4 minutes, and vortex mix well. S4. Add 300 μL of CLB buffer and 50 μL of amino magnetic beads, incubate at room temperature for 6 minutes, use a magnetic rack to adsorb the magnetic beads, and collect the supernatant for later use. S5. RNA washing: Wash with 800μL DW1 → Wash twice with 800μL DW2 → Air dry at room temperature for 5 minutes; S6. Add 80 μL of elution buffer, incubate at 56°C for 5 minutes, and collect the RNA solution; S7. Add 100 μL of silanol magnetic beads to the supernatant of step S4 and incubate at room temperature for 6 minutes to allow the magnetic beads to be adsorbed by the magnetic rack. S8. DNA washing: Wash with 800μL DW1 → Wash twice with 800μL DW2 → Air dry at room temperature for 5 minutes; S9. Add 80 μL of elution buffer, incubate at 56°C for 5 minutes, and collect the DNA solution.

[0023] Example 2 A DNA / RNA extraction kit based on magnetic beads using enzyme-free digestion differs from Example 1 in that: (1) Lysis buffer AL: guanidine hydrochloride 5 mol / L, SDS 1% (v / v), EDTA 10 mmol / L, β-mercaptoethanol 5 mmol / L, pH=5.5; (2) Specific binding buffer CLB: Tris-HCl 20 mmol / L, sodium chloride 1.0 mol / L, ethanol 40% (v / v), pH=7.0; (3) The concentration of amino magnetic beads is 10 mg / mL, and the concentration of silanol magnetic beads is 10 mg / mL; (4) Nano silica modifier: concentration 5 mg / mL, preparation method is as follows: ① Pretreatment: Nano-silica was placed in a 5% sulfuric acid solution and stirred at room temperature for 2 hours, washed with water until neutral, filtered, and dried at 60°C for 4 hours; ② Preparation of montmorillonite solution: 3 parts montmorillonite, 2 parts carbon nanotubes, add 5 parts sodium dodecylbenzenesulfonate solution with a mass fraction of 4%, and ultrasonically disperse for 30 minutes; ③ Blending and ball milling: 3 parts pretreated nano silica, 2 parts nano cellulose, 5 parts montmorillonite liquid, ball milling speed 1000 r / min, ball milling for 2 hours; ④ Post-processing: After filtration, dry at 60℃ for 4 hours, then pulverize and pass through an 80-mesh sieve.

[0024] The method of use is the same as in Example 1, except that the incubation time is adjusted to 8 minutes and the standing time of the nano silica modifier is adjusted to 3 minutes.

[0025] Example 3 A DNA / RNA extraction kit based on magnetic beads using enzyme-free digestion differs from Example 1 in that: (1) Lysis buffer AL: guanidine hydrochloride 7 mol / L, SDS 3% (v / v), EDTA 20 mmol / L, β-mercaptoethanol 10 mmol / L, pH=6.5; (2) Specific binding buffer CLB: Tris-HCl 40 mmol / L, sodium chloride 1.5 mol / L, ethanol 60% (v / v), pH=8.0; (3) The concentration of amino magnetic beads is 20 mg / mL, and the concentration of silanol magnetic beads is 20 mg / mL; (4) Nano silica modifier: concentration 10 mg / mL, preparation method is as follows: ① Pretreatment: Nano-silica was placed in an 8% sulfuric acid solution and stirred at room temperature for 3 hours, washed with water until neutral, filtered, and dried at 80°C for 6 hours; ② Preparation of montmorillonite solution: 5 parts montmorillonite, 5 parts carbon nanotubes, add 8 parts 5% sodium dodecylbenzenesulfonate solution, and ultrasonically disperse for 60 minutes; ③ Blending and ball milling: 5 parts pretreated nano silica, 5 parts nano cellulose, 8 parts montmorillonite liquid, ball milling speed 1500 r / min, ball milling for 2 hours; ④ Post-processing: After filtration, dry at 80℃ for 6 hours, then pulverize and pass through an 80-mesh sieve.

[0026] The method of use is the same as in Example 1, except that the incubation time is adjusted to 5 minutes and the standing time of the nano silica modifier is adjusted to 5 minutes.

[0027] Comparative Example 1 The Qiagen RNeasy Bacterial RNA Extraction Kit and the Qiagen DNeasy Bacterial DNA Extraction Kit were used to extract RNA and DNA from the same bacterial sample, respectively, following the instructions of the kits (including the DNase I / RNase A enzyme digestion steps), as a prior art control.

[0028] Performance testing standards The performance tests of Examples 1-3 and the comparative examples of this invention were all performed in accordance with the following standards to ensure the accuracy, repeatability and comparability of the test data. All tests were conducted in an environment with a room temperature of 25±2℃ and a relative humidity of 50±5%. Three parallel samples were set for each test group, and the average value was taken as the final test result.

[0029] 1. Nucleic acid concentration and purity testing standards The concentrations (ng / μL), A260 / A280 ratio, and A260 / A230 ratio of RNA and DNA were detected using a Nanodrop nucleic acid detection instrument. The detection steps were as follows: ① Blank calibration was performed using enzyme-free water; ② 2 μL of nucleic acid solution was used for measurement; ③ Data were recorded, and the purity was determined to meet the requirements (RNA: A260 / A280 1.8-2.0, A260 / A230 ≥ 1.5; DNA: A260 / A280 1.8-1.9, A260 / A230 ≥ 1.5).

[0030] 2. Nucleic Acid Integrity Testing Standards (1) RNA integrity: RNA integrity (RIN value) was detected using an Agilent 2100 bioanalyzer. A RIN ≥ 8.0 was considered acceptable. (2) DNA integrity: DNA fragment length was detected by agarose gel electrophoresis (0.8% agarose gel, 120V electrophoresis for 30 minutes). Fragment length ≥20kb is acceptable.

[0031] 3. Cross-contamination rate detection standards (1) DNA contamination rate in RNA: RNA-specific qPCR primers (targeting bacterial 16S rRNA gene) were used for amplification. No Ct value or Ct value > 35 indicates no obvious DNA contamination (contamination rate ≤ 0.5%). (2) RNA contamination rate in DNA: DNA samples were directly amplified by qPCR (without reverse transcription). No Ct value or Ct value > 35 indicates no obvious RNA contamination (contamination rate ≤ 0.5%).

[0032] 4. Extraction efficiency testing standards Using the same amount of bacterial sample (1 mL, 10) 7 The nucleic acid concentration (CFU / mL) was used as the detection target. Extraction was performed using the kit of this invention and a commercially available kit (Comparative Example 1). The nucleic acid concentration was detected by Nanodrop, and the extraction recovery rate was calculated as (extraction concentration of this invention / extraction concentration of the comparative example × 100%). The recovery rate was required to be ≥85%.

[0033] 5. Precision testing standards (1) Intra-batch imprecision: Take the same batch of kits, extract the same bacterial sample 10 times, detect the RNA and DNA concentrations, calculate the coefficient of variation (CV), and require CV ≤ 10.0%; (2) Inter-batch imprecision: Take 3 different batches of kits, extract the same bacterial sample 3 times, detect the concentration of RNA and DNA, calculate the coefficient of variation (CV), and require CV≤15.0%.

[0034] Performance summary of Examples 1-3 To clarify the overall performance of the reagent kit of the present invention, the products prepared in Examples 1-3 were comprehensively tested, and the specific data are shown in the table below:

[0035] To verify the technical advantages of the reagent kit of the present invention, the specific settings are as follows: Comparative Example 1: Existing technology, containing enzyme digestion, without nano-silica modifier; Comparative Example 2: The formulation was the same as in Example 1, but only amino magnetic beads were used (without adding silanol magnetic beads), and DNA / RNA contamination was removed by traditional enzymatic digestion. Comparative Example 3: The formulation was the same as in Example 1, except that the ethanol content in the specific binding buffer CLB was 30% (v / v). Comparative Example 4: The formulation is the same as that of Example 1, except that the preparation method of the nano silica modifier is different (no sulfuric acid pretreatment step, nano silica, nano cellulose and montmorillonite liquid are directly mixed and ball-milled). Comparative Example 5: The formulation is the same as that of Example 1, except that the preparation method of the nano silica modifier is different (ball milling speed 800 r / min, ball milling time 1 hour, and no carbon nanotubes in the montmorillonite solution). Comparative Example 6: The formulation is the same as in Example 1, but without the nano-silica modifier.

[0036] The above 7 groups of samples were subjected to performance testing. The testing items were the same as in Example 1. The specific performance data are shown in the table below:

[0037] Example 1 shows that the enzyme-free digestion design resulted in an RNA RIN value (8.5) that was significantly higher than that of Comparative Example 1 (7.2) and Comparative Example 2 (7.5), while the cross-contamination rate (<0.5%) was lower than that of Comparative Example 1 (1.2%) and Comparative Example 2 (0.8%). This demonstrates that the synergistic effect of bifunctional magnetic beads and specific buffer can replace enzyme digestion to achieve efficient decontamination while avoiding nucleic acid degradation and improving integrity. The low ethanol content (30%) in the buffer solution of Comparative Example 3 led to an increased cross-contamination rate (1.0%) and a decreased recovery rate (82%). In contrast, the ethanol content of Example 1 (50%) allowed for precise regulation of the specific binding of RNA to amino magnetic beads, demonstrating that the ratio of buffer components is key to achieving selective separation of nucleic acids. Example 1 has a total operation time of 55 minutes, which is more than 35% shorter than Comparative Example 1 (85 minutes) and Comparative Example 2 (80 minutes), and does not require expensive nucleases, thus significantly reducing time and economic costs; Example 1 can simultaneously obtain high-purity RNA and DNA with a recovery rate of 92%, while Comparative Example 1 requires two extractions of the same sample, resulting in low sample utilization and the enzyme digestion step is not conducive to automated integration. This demonstrates the significant advantages of the kit of the present invention in terms of sample utilization and automation adaptability. In addition, the addition of nano silica modifier and the preparation process show a trend of improving the performance of the product.

[0038] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0039] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A kit for enzyme-free digestion and extraction of DNA / RNA based on magnetic beads, characterized in that: The kit includes lysis buffer AL, proteinase K, specific binding buffer CLB, amino magnetic bead suspension, silanol magnetic bead suspension, washing buffer DW1, washing buffer DW2, elution buffer, enzyme-free water, and nano-silica modifier. The kit does not rely on DNase or RNase digestion steps. Through the synergistic effect of bifunctional magnetic beads and specific binding buffer, it achieves the separation of RNA and DNA without cross-contamination from a single lysis buffer.

2. The DNA / RNA extraction kit based on magnetic bead-based enzyme-free digestion according to claim 1, characterized in that: The specific composition of each component is as follows: (1) Lysis buffer AL: guanidine hydrochloride 5-7 mol / L, sodium dodecyl sulfate 1-3% (v / v), EDTA 10-20 mmol / L, β-mercaptoethanol 5-10 mmol / L, pH=5.5-6.5; (2) Proteinase K: concentration 20 mg / mL; (3) Specific binding buffer CLB: Tris-HCl 20-40 mmol / L, sodium chloride 1.0-1.5 mol / L, ethanol 40-60% (v / v), pH=7.0-8.0; (4) Amino magnetic bead suspension: concentration 10-20 mg / mL, particle size 500-1000 nm, Zeta potential +20~+30 mV; (5) Silyl hydroxyl magnetic bead suspension: concentration 10-20 mg / mL, particle size 500-1000 nm; (6) Washing solution DW1: Tris-HCl 10-20 mmol / L, sodium chloride 0.5-1.0 mol / L, ethanol 70-80% (v / v), pH=7.0-8.0; (7) Washing solution DW2: 75-85% (v / v) ethanol solution; (8) Eluent: Tris-HCl 5-10 mmol / L, EDTA 0.1-0.5 mmol / L, pH=8.0-8.

5.

3. The DNA / RNA extraction kit based on magnetic bead-based enzyme-free digestion according to claim 1 or 2, characterized in that: The optimal composition of each component is as follows: (1) Lysis buffer AL: guanidine hydrochloride 6 mol / L, sodium dodecyl sulfate 2% (v / v), EDTA 15 mmol / L, β-mercaptoethanol 8 mmol / L, pH=6.0; (2) Specific binding buffer CLB: Tris-HCl 30 mmol / L, sodium chloride 1.2 mol / L, ethanol 50% (v / v), pH=7.5; (3) Amino magnetic bead suspension: concentration 15 mg / mL, particle size 800 nm, Zeta potential +25 mV; (4) Silyl hydroxyl magnetic bead suspension: concentration 15 mg / mL, particle size 800 nm; (5) Washing solution DW1: Tris-HCl 15mmol / L, sodium chloride 0.8mol / L, ethanol 75% (v / v), pH=7.5; (6) Washing solution DW2: 80% (v / v) ethanol solution; (7) Eluent: Tris-HCl 8 mmol / L, EDTA 0.3 mmol / L, pH=8.

3.

4. The DNA / RNA extraction kit based on magnetic bead-based enzyme-free digestion according to claim 1, characterized in that: The concentration of the nano-silica modifier is 5-10 mg / mL; its specific preparation method is as follows: Pretreatment: Place nano-silica in a sufficient amount of sulfuric acid solution with a mass fraction of 5-8%, stir at room temperature for 2-3 hours until the mixture is uniform, then wash repeatedly with deionized water until the washing solution is neutral, filter to collect the solid, and dry in an oven at 60-80℃ for 4-6 hours to obtain pretreated nano-silica. Preparation of montmorillonite solution: Take 3-5 parts of montmorillonite and 2-5 parts of carbon nanotubes, add them to 5-8 parts of sodium dodecylbenzenesulfonate solution with a mass fraction of 4-5%, and ultrasonically disperse for 30-60 minutes to obtain a uniformly dispersed montmorillonite solution (all parts are by mass). Blending and ball milling: Take 3-5 parts of the dried nano-silica from step ①, 2-5 parts of nano-cellulose and 5-8 parts of the montmorillonite liquid prepared in step ②, put them into a ball mill, adjust the ball milling speed to 1000-1500 r / min, and continue ball milling for 2 hours; Post-processing: After ball milling, the mixture is filtered, the solid product is collected, dried in an oven at 60-80℃ for 4-6 hours, cooled to room temperature, and then pulverized through an 80-mesh sieve to obtain nano-silica modifier.

5. A method for extracting DNA / RNA using the kit described in any one of claims 1-4, characterized in that: Includes the following steps: S1. Sample pretreatment: Centrifuge bacterial samples to collect the precipitate; S2, Cell lysis: Add lysis buffer AL and proteinase K, and incubate at 56°C for 15-20 minutes; S3, RNA-specific binding: Add specific binding buffer CLB and amino magnetic beads, incubate at room temperature for 5-8 minutes, magnetic rack adsorbs magnetic beads, collect supernatant for later use; Add nano silica modifier: Add 30 μL of nano silica modifier to lysis buffer, vortex mix, let stand at room temperature for 3-5 minutes to allow the modifier to be fully dispersed and initially bind to nucleic acid; S4. RNA washing and purification: Wash the magnetic beads sequentially with washing buffer DW1 and washing buffer DW2, and then air dry. S5. RNA elution: Add elution buffer, incubate at 56°C for 5 minutes, and collect the RNA solution; S6, DNA-specific binding: Add silanol magnetic beads to the supernatant and incubate at room temperature for 5-8 minutes to allow the magnetic beads to be adsorbed by the magnetic rack. S7. DNA washing and purification: Wash the magnetic beads sequentially with washing buffer DW1 and washing buffer DW2, and then air dry. S8. DNA elution: Add elution buffer, incubate at 56°C for 5 minutes, and collect the DNA solution.

6. The extraction method according to claim 5, characterized in that: In step S3, the amount of amino magnetic beads added is 50 μL, and the amount of specific binding buffer CLB added is 300 μL; in step S6, the amount of silanol magnetic beads added is 100 μL.

7. The DNA / RNA extraction kit based on magnetic bead-based enzyme-free digestion according to claim 5, characterized in that: The kit is suitable for bacterial samples and can be adapted to automated liquid handling workstations for downstream experiments such as reverse transcription-quantitative PCR, transcriptome sequencing, or genome sequencing.

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