Eluent based on nucleic acid extraction and preparation process thereof

Abstract

The invention belongs to the field of biology, and discloses an eluent based on nucleic acid extraction and a preparation process thereof, and the eluent formula comprises sodium bicarbonate (2-30 mM, pH 7.0), Tween 20 (0.1-1.5% v / v), TritonX-100 (0.1-1.5% v / v), EDTA (1-15 mM, chelating metal ions), and dithiothreitol (DTT) (1-15 mM). A composite buffer system is developed, and the elution efficiency and purity are improved through the synergistic effect of multiple components. The ionic strength and the pH value are optimized, and the nucleic acid release and inhibitor removal effects are balanced. The invention provides the nucleic acid extraction eluent which is low in cost, high in elution efficiency (greater than or equal to 95%) and low in residue.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of biotechnology, and in particular relates to an eluent based on nucleic acid extraction and a preparation process thereof. Background Art

[0002] Traditional elution buffers (e.g., TE buffer, pure water) suffer from low nucleic acid elution efficiency (≤80%) and the accumulation of residual inhibitors (e.g., proteins, polysaccharides). Single buffer systems (e.g., containing only Tris-EDTA) are poorly compatible with complex samples, leading to decreased sensitivity in downstream assays (e.g., PCR, sequencing). Commercial elution buffers are expensive and rely on imports (e.g., Qiagen brand elution buffers).

[0003] Prior art CN102229925B (published on June 25, 2014) proposes an "enhanced magnetic bead-based nucleic acid extraction method" that integrates lysis and magnetic bead binding in the same reaction chamber, and adds isopropyl alcohol to the lysis and two wash steps to enhance the magnetic bead's adsorption of DNA / RNA. ([patents.google.com][1]) This process only retains the four steps of "lysis-binding, wash 1, wash 2, and elution," and claims that the reagents for each step can be pre-filled in an automatic extraction instrument, achieving "one-click" purification of multiple samples within 40 minutes.

[0004] However, a high proportion of isopropanol will form a residual film on the surface of the magnetic beads, requiring an additional 5-10 minutes of drying or strong centrifugation (such as the CN110951725A method, which also emphasizes air drying for 5 minutes before low-TE elution) to avoid alcohol from mixing into the eluent, thereby slowing down the process and bringing the risk of solvent contamination. In addition, the above-mentioned technology still uses traditional low-salt Tris / low-TE as elution buffer, lacks DTT and chelating agents, and cannot continuously inactivate RNase and remove divalent metal ions during the elution phase, resulting in easy degradation of trace RNA and insufficient inhibition of downstream PCR. Furthermore, the isopropanol environment will cause the magnetic beads to agglomerate and be sucked away with the liquid, which will increase the error of automated liquid aspiration and colorimetric interference. This is also listed as a problem that needs to be alleviated by adjusting the speed and time in the CN106591297A solution that replaces magnetic frame separation with centrifugation, indicating that the existing technology is still difficult to meet the requirements of high-throughput molecular diagnosis for stability and sensitivity. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides an eluent based on nucleic acid extraction and a preparation process thereof.

[0006] The present invention is achieved by providing an eluent for nucleic acid extraction, characterized in that the eluent for nucleic acid extraction comprises: sodium bicarbonate (2-30 mM, pH 7.0), Tween 20 (0.1-1.5% v / v), Triton X-100 (0.1-1.5% v / v), EDTA (1-15 mM, chelating metal ions), and dithiothreitol DTT (1-15 mM). The eluent is prepared in the following proportions (based on 5 mL), sterilized with a 0.22 μm filter membrane, and stored in aliquots:

[0007] Table 1 Eluent preparation scheme

[0008]

[0009] Furthermore, the eluent based on nucleic acid extraction adopts a composite buffer system to improve elution efficiency and purity through the synergistic effect of multiple components; optimizes ionic strength and pH value, and balances nucleic acid release and inhibitor removal effects.

[0010] Furthermore, the nucleic acid elution process is as follows:

[0011] The eluate was mixed with the magnetic beads that adsorbed nucleic acids, incubated at 80-85°C for 30-60 seconds, vortexed at 1000-1500 rpm for 60-90 seconds, and centrifuged to obtain high-purity nucleic acids.

[0012] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0013] The eluent described in this invention demonstrates exceptional nucleic acid recovery efficiency, achieving a measured recovery rate exceeding 95%, a 15–20% improvement compared to conventional eluents. This high recovery efficiency not only significantly reduces precious sample loss but also yields more high-quality nucleic acids within the same processing time, laying a solid foundation for subsequent molecular experiments.

[0014] In terms of purity, the eluent of the present invention can consistently achieve an A260 / A280 ratio of 1.8–2.0, with residual inhibitors such as proteins as low as ≤0.1 μg / μl. This purity level effectively avoids interference from impurities and significantly improves the accuracy and reliability of downstream PCR, sequencing, and other high-sensitivity assays.

[0015] In terms of cost control, this invention reduces raw material costs by over 50% by using low-cost alternative raw materials and optimizing the preparation process, making it easily scalable to large-scale production. This cost advantage directly translates into a more competitive end-user selling price, providing strong economic support for market promotion and industrial implementation.

[0016] With its combined advantages of high efficiency, high purity, and low cost, the eluent of this invention holds significant commercial potential in applications with high demand for nucleic acid extraction, such as genetic testing, disease diagnosis, and forensic identification. Its technological innovations can shorten the extraction process, reduce impurity interference, and improve experimental accuracy, thereby attracting more partners and end customers. This is expected to generate substantial revenue for the company and promote the rapid development of related biotechnology applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a flow chart of the experimental operation steps of the eluent based on nucleic acid extraction provided by an embodiment of the present invention.

[0018] Figure 2 This is a comparison chart of the RT-qPCR amplification curves of the new coronavirus nucleic acid in sewage.

[0019] Figure 3 Comparison of RT-qPCR amplification curves of Pseudomonas aeruginosa nucleic acid in the air. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] The embodiment of the present invention provides an eluent based on nucleic acid extraction, and the eluent formula is as follows:

[0022] Sodium bicarbonate (2-30 mM, pH 7.0), provides a stable pH environment;

[0023] Tween 20 (0.1-1.5% v / v) and Triton X-100 (0.1-1.5% v / v) reduce surface tension, inhibit nonspecific adsorption, stabilize nucleic acid structure, and reduce physical stress damage;

[0024] EDTA (1-15mM, chelates metal ions), inhibits nuclease activity, prevents nucleic acid degradation, stabilizes nucleic acid structure, and helps maintain pH stability;

[0025] Dithiothreitol DTT (1-15mM) breaks down protein complexes, inhibits nuclease activity, and maintains a reducing environment.

[0026] Prepare the eluent (5 mL) according to the following ratio, sterilize with a 0.22 μm filter membrane, and store in aliquots.

[0027] Table 1 Eluent preparation scheme

[0028]

[0029] The eluent for nucleic acid extraction provided in this embodiment utilizes a composite buffer system. A sodium bicarbonate buffer maintains pH stability, while Tween 20 and Triton X-100 disrupt liquid surface tension, enabling efficient nucleic acid release. EDTA and DTT are combined to reduce nuclease activity and oxidative damage, enhancing nucleic acid integrity.

[0030] The eluent for nucleic acid extraction provided in the embodiment of the present invention is as follows:

[0031] Take 100 μL of nucleic acid-absorbed silica-based magnetic beads and add 300 μL of elution buffer;

[0032] Heat in a metal bath at 80°C for 30 seconds and shake to mix (1000 rpm, 60 seconds);

[0033] The magnetic stand was allowed to adsorb the magnetic beads for 1 minute, and the supernatant was collected to obtain the purified nucleic acid.

[0034] Table 2 Comparison of eluent components

[0035]

[0036] Taking the magnetic bead method as an example, the traditional elution solution experimental operation steps are as follows:

[0037] S1: After completing the washing step, discard the washing solution through magnetic separation and open the lid to dry the magnetic beads for 1-2 minutes;

[0038] S2: Add eluent preheated to 65-70°C (usually 30-100 μL) and gently pipette or vortex to mix thoroughly to suspend the magnetic beads.

[0039] S3: Incubate at 65-70°C for 5-10 minutes;

[0040] S4: Place the centrifuge tube on a magnetic rack and let it stand for 1-2 minutes to allow the magnetic beads to adsorb to the tube wall;

[0041] S5: Carefully transfer the supernatant to a new enzyme-free sterile centrifuge tube.

[0042] like Figure 1 As shown, taking the magnetic bead method as an example, the experimental operation steps of the eluent of the present invention are as follows:

[0043] S1: After completing the washing step, discard the washing solution by magnetic separation, open the lid and dry the magnetic beads for 1 minute or use an automated extractor to dry the magnetic beads for 1 minute;

[0044] S2: Add elution buffer (100 μL) preheated to 80-85°C and mix by pipetting or using an automated extractor for 1 minute to fully suspend the magnetic beads.

[0045] S3: Place the centrifuge tube on a magnetic stand and let it stand for 1 minute to allow the magnetic beads to adsorb to the tube wall or use an automated extraction instrument to allow the magnetic beads to adsorb for 1 minute;

[0046] S4: Carefully transfer the supernatant to a new enzyme-free sterile centrifuge tube.

[0047] The sewage sample was added with a new coronavirus standard sample (diluted 10,000 times, concentration: 2*10 3 After eluting with 500 copies / mL, nucleic acid extraction and elution were performed. The operation steps are as follows:

[0048] (1) Preparation of eluent: 1 mL each of 20 mM sodium bicarbonate, 4 mM EDTA, 5 mM DTT, 1.0% Tween 20, and 0.1% Triton X-100;

[0049] (2) Prepare the nucleic acid eluate at the working concentration according to Table 1, and test 50 units;

[0050] (3) After completing the nucleic acid washing step, discard the washing solution by magnetic separation, open the lid and dry the magnetic beads for 1 minute; add the elution solution (volume 100 μL) preheated to 80-85°C, blow and mix for 1 minute to fully suspend the magnetic beads; place the centrifuge tube on the magnetic rack and let it stand for 1 minute to allow the magnetic beads to adsorb to the tube wall or use an automated extractor to adsorb the magnetic beads for 1 minute; carefully aspirate the supernatant and transfer it to a new enzyme-free sterile centrifuge tube;

[0051] (4) The eluted nucleic acid was tested using the Daan Gene 2019-nCoV Nucleic Acid Detection Kit (fluorescence PCR method), and the mean and standard deviation of the Ct value of each group were calculated.

[0052] The results are recorded in Table 3:

[0053] Table 3 Test results

[0054]

[0055] Figure 2 Comparison of RT-qPCR amplification curves for novel coronavirus nucleic acid in wastewater. The horizontal axis represents the number of amplification cycles (0–32 cycles), and the vertical axis represents the real-time fluorescence intensity (0–2100 RFU). A horizontal threshold line of 100 RFU is included in the figure to determine the Ct value.

[0056] In this figure, the blue curve represents a wastewater sample treated with the eluent of the present invention, while the red curve corresponds to a conventional commercial product. The blue curve crosses the threshold around cycle 23, while the red curve reaches the target after cycle 26. The endpoint fluorescence intensities are approximately 2100 RFU for the blue line and 1100 RFU for the red line, demonstrating that the eluent of the present invention advances the Ct by 2–3 cycles and significantly increases the amplification yield, demonstrating its superior recovery and purity.

[0057] Figure 3 Comparison of RT-qPCR amplification curves for Pseudomonas aeruginosa nucleic acid in air. The horizontal axis shows amplification cycle number ranging from 0 to 42 cycles, and the vertical axis shows real-time fluorescence intensity ranging from 0 to 2400 RFU. A threshold of 100 RFU was also set as the criterion.

[0058] In this figure, the bold bright green line represents the air sample treated with the eluent of the present invention, while the thin brown-red line represents the control using a conventional eluent. The bright green curve crosses the threshold at approximately 29 cycles, while the brown-red curve does so at 35 cycles. The endpoint fluorescence intensity is approximately 2300 RFU for the former and only 300 RFU for the latter. Combining the two figures demonstrates that the eluent of the present invention significantly improves nucleic acid recovery and amplification efficiency in both wastewater and air samples, providing strong data support for its innovative approach.

[0059] Example 1

[0060] Dissolve 10 mM sodium bicarbonate in approximately 4 mL of ultrapure water, stir thoroughly, and adjust to pH 7.0 with a trace amount of HCl / NaOH. Add 0.5% (v / v) Tween 20 and 0.5% (v / v) Triton X-100 to reduce surface tension. Then, add 5 mM EDTA to chelate metal ions and 5 mM DTT to maintain a reducing environment. The volume was adjusted to 5 mL, sterilized through a 0.22 μm PES filter, and aliquoted into 0.5 mL RNase-free centrifuge tubes. Store at -20°C until needed.

[0061] After preheating the eluate to 56°C, the column was used to elute SARS-CoV-2 RNA extracted using a silica gel column. 50 μL of the eluate was added to the loaded column, allowed to stand for 1 minute, and then centrifuged at 10,000 × g for 30 seconds. NanoDrop analysis revealed an A260 / A280 ratio of 1.94, an 18% increase in average recovery compared to the TE buffer control. Real-time RT-qPCR Ct values ​​were also improved by 0.8 to 1.1. The high purity and low background enzyme activity demonstrated the effectiveness of each additive in enhancing nucleic acid integrity and enzyme inhibition.

[0062] Example 2

[0063] Weigh 25 mM sodium bicarbonate into 4 mL of ultrapure water and adjust to pH 7.0. Add 1.2% (v / v) Tween 20 and 0.2% (v / v) Triton X-1000 and mix thoroughly. Add 12 mM EDTA and 2 mM DTT to a volume of 5 mL. Sterilize the solution through a 0.22 μm filter. Aliquot 1 mL into DNase-free EP tubes and store in the dark at 4°C for 6 weeks. For long-term storage, freeze at -80°C.

[0064] In the magnetic bead-based genomic DNA extraction from whole blood, 100 μL of elution buffer was added to the DNA-captured magnetic bead suspension, mixed thoroughly, incubated at 65°C for 5 minutes, and the supernatant was collected by magnetic separation. Qubit quantification revealed a 21% increase in DNA yield compared to the manufacturer's recommended elution buffer, and an A260 / A230 value of 2.10 indicated lower residual protein and salt ions. Subsequent PCR amplification of a 1.5 kb fragment demonstrated improved amplification efficiency and the absence of nonspecific bands, confirming the advantages of a higher Tween 20 content in eluting large genomes.

[0065] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. An eluent for nucleic acid extraction, characterized in that By volume / concentration, it is composed of the following components: Sodium bicarbonate 2-30 mmol / L, pH 7.0; Tween-200.1-1.5vol%; TritonX-1000.1-1.5vol%; Ethylenediaminetetraacetic acid 1-15mmol / L; Dithiothreitol 1-15mmol / L; The remainder is water for injection, which is prepared after being sterilized through a 0.22 μm filter membrane.

2. The eluent according to claim 1, characterized in that The sum of the volume percentages of Tween-20 and TritonX-100 is 0.4-2.0 vol%, and the ratio between the two is in the range of 1:9-9:

1.

3. The eluent according to claim 1, characterized in that After the eluate is sealed and stored at 20-25° C. for 3 months or stored in the dark at 2-8° C. for 6 months, the elution efficiency remains no less than 95% of the initial level.

4. The eluent according to claim 1, wherein The eluent is obtained by dissolving sodium bicarbonate, ethylenediaminetetraacetic acid, and dithiothreitol in sequence, then adding Tween-20 and TritonX-100 in sequence and mixing evenly, finally making up to volume with water for injection, and sterilizing through a 0.22 μm filter membrane.

5. A method for extracting nucleic acid, characterized in that: The following steps are involved: a) preheating the eluent according to claim 1 to 80-85° C.; b) taking a volume of 1-1.5 times the volume of the eluent and adding the magnetic beads-nucleic acid mixture to the preheated eluent, and vortexing and mixing for 60-90 seconds; c) placing the mixture in a magnetic field for 30-60 seconds to allow the magnetic beads to adsorb; d) Remove the supernatant as the nucleic acid elution solution.

6. The method according to claim 5, characterized in that The oscillation mixing in step b) is completed using a vortex mixer at 1000-1500 rpm.

7. The method according to claim 5, characterized in that After step d), steps a) to c) are repeated once for the remaining magnetic beads and the two supernatants are combined to improve the nucleic acid recovery rate.

8. A nucleic acid extraction kit, characterized in that: include: a) the eluent according to claim 1; b) magnetic beads with a hydroxyl-silanized surface treatment; c) nucleic acid binding buffer and wash buffer; d) Instructions.

9. An automated nucleic acid extraction system, characterized in that: The system is provided with a heating module, a mixing module, a magnetic separation module and a program control module, and is equipped with the eluent according to claim 1 for the high-temperature elution step.

10. Use of the eluent according to claim 1 in nucleic acid purification, characterized in that: Used in the high-temperature elution step of the magnetic bead-based nucleic acid extraction process to release DNA, RNA or miRNA bound to the surface of the magnetic beads, while inhibiting the degradation of nucleic acids by metal ions and nucleases.

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