A nucleic acid purification reagent and purification method

Abstract

This invention discloses a nucleic acid purification reagent and method, belonging to the field of biotechnology. The reagent composition comprises a lysis buffer, binding buffer, washing buffer, elution buffer, and surface-modified magnetic beads in specific ratios. The lysis buffer uses a synergistic system of guanidine isothiocyanate derivative and alkyl glycoside surfactant; the binding buffer uses a combination of lithium chloride dissociative salt and low-concentration polyoxyethylene ether surfactant; the elution buffer is free of ethylenediaminetetraacetic acid; and the magnetic beads are grafted with amino polyethylene glycol segments. The kit is equipped with dedicated consumables. The extraction method achieves nucleic acid purification through one-step lysis, rapid binding, short-time washing, and low-temperature elution. This invention can achieve efficient nucleic acid extraction from soil samples, fecal microbial samples, plant tissue samples, and animal tissue samples, with a total extraction time ≤22 minutes. The obtained nucleic acid purity A260 / A280 is 1.85-1.95, the recovery rate is ≥92%, and the inhibitor residue is ≤0.05 ng / μL. It is compatible with high-throughput automated platforms and has the advantages of good stability and strong industrial feasibility.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a nucleic acid purification reagent and purification method. Background Technology

[0002] Nucleic acid purification is a core preliminary step in molecular diagnostics, gene sequencing, and other fields. Existing commercial nucleic acid extraction reagents and methods have the following main limitations: 1. Low extraction efficiency: For trace samples such as soil microbial nucleic acid and fecal microbial trace nucleic acid, traditional guanidine salt lysis system is difficult to completely release nucleic acid, and the recovery rate is generally less than 85%. Lignified plant tissue and fixed animal breeding tissue samples have tightly bound nucleic acid impurities and severely solidified structure, requiring long-term proteinase K digestion (usually ≥2 hours), and there are still problems of nucleic acid fragment breakage and degradation. Fresh plant tissue samples are rich in polysaccharide and polyphenol impurities, which easily form viscous gel-like precipitates, leading to magnetic bead aggregation and a significant decrease in nucleic acid binding efficiency.

[0003] 2. Insufficient purity: Conventional washing solutions often use high-concentration ethanol (≥80%) combined with elution solutions containing ethylenediaminetetraacetic acid, which can easily lead to residual salt ions (A260 / A230 < 1.8) or chelated magnesium ions, affecting the downstream PCR amplification efficiency; some reagents contain surfactants such as Triton X-100 that are difficult to remove, which can inhibit enzyme activity.

[0004] 3. Long processing time: The traditional magnetic bead extraction process usually takes 40-60 minutes and includes multiple high-temperature incubation (95℃ denaturation) and long-term shaking steps, which cannot meet the timeliness requirements of high-throughput screening of large batches of samples and rapid identification of germplasm resources in agricultural breeding.

[0005] 4. Poor adaptability: A single reagent system is difficult to cover different sample types, and clinicians need to keep multiple reagent kits, which increases costs and management difficulty; existing magnetic bead surface modifications mostly use carboxyl or hydroxyl groups, which have strong non-specific adsorption and are prone to protein co-precipitation.

[0006] 5. Poor stability: After conventional reagents are stored at 2-8℃ for 6 months, the pyrolysis efficiency decreases by >15%, the magnetic beads are prone to agglomeration, and there are significant differences between batches (CV>8%).

[0007] The core problem with existing technologies lies in the lack of synergistic effect of reagent components: ① If the concentration of guanidine salt in the lysis buffer is too high (usually ≥4 mol / L), nucleic acid and impurities will co-precipitate; if the concentration is too low, lysis will be incomplete. ② The ratio of ionizing salt to surfactant in the binding buffer is not optimized for trace amounts of nucleic acid, which easily leads to "overbinding" (impurity adsorption) or "underbinding" (nucleic acid loss). ③ The presence of ethylenediaminetetraacetic acid in the elution buffer will interfere with downstream reactions, and the lack of a buffer system will easily lead to nucleic acid degradation. ④ The surface modification of magnetic beads does not balance hydrophilicity and charge characteristics, resulting in insufficient specific adsorption.

[0008] To address the aforementioned shortcomings, this invention resolves the conflict between efficiency and purity in the entire process of lysis-binding-washing-elution by reconstructing the reagent components and ratios. It also achieves multi-sample adaptation and rapid extraction, filling the technological gap in the prior art of "completing high-purity nucleic acid extraction of multiple types of samples in ≤25 minutes". Summary of the Invention

[0009] In view of this, the present invention provides a nucleic acid purification reagent and purification method to solve or alleviate one of the technical problems existing in the prior art, and at least provides a beneficial alternative.

[0010] The technical solution of this invention is implemented as follows: a nucleic acid purification reagent composition, comprising a lysis buffer, a binding buffer, a washing buffer, an elution buffer, and surface-modified magnetic beads; The lysis buffer contains 180-220 mmol / L of guanidine isothiocyanate derivative, 0.8%-1.2% (w / v) of alkyl glycoside surfactant, 20-30 mmol / L of Tris-HCl buffer system, pH 7.8-8.2, and 0.05%-0.08% of dipotassium ethylenediaminetetraacetate. The binding solution contains 120-150 mmol / L lithium chloride ion salt, 0.3%-0.5% (w / v) polyoxyethylene ether nonionic surfactant, and 10-15 mmol / L sodium citrate buffer system, with a pH of 5.5-6.0. The washing solution contains 65%-75% by volume anhydrous ethanol, a 5-8 mmol / L Tris-HCl buffer system (pH 7.0-7.5), and 0.01%-0.03% sodium dodecyl sulfate. The eluent contains an 8-12 mmol / L Tris-HCl buffer system, pH 8.0-8.5, and is free of ethylenediaminetetraacetic acid; The surface-modified magnetic beads are silica magnetic beads with a diameter of 300-500nm, and the surface is grafted with amino polyethylene glycol segments accounting for 2%-3% by mass, with a grafting density of 8-12 PEG chains / 100nm² magnetic bead surface.

[0011] Furthermore, the guanidine isothiocyanate derivative is n-propylguanidine isothiocyanate, the alkyl glycoside surfactant is decyl glucoside, the polyoxyethylene ether nonionic surfactant is Tween-20, and the molecular weight of the amino polyethylene glycol segment is 2000-3000 Da.

[0012] Furthermore, the pH value of the lysis buffer is 8.0±0.1, the pH value of the binding buffer is 5.8±0.1, the pH value of the washing buffer is 7.2±0.1, and the pH value of the elution buffer is 8.2±0.1.

[0013] Furthermore, it includes the nucleic acid purification reagent composition according to any one of claims 1-3, and dedicated consumables: A 96-well deep well plate, a sealing cap with a silicone gasket, and a magnetic rod sleeve; the pore volume of the 96-well deep well plate is 1.2-1.5 mL, and the pore walls are hydrophobic. The magnetic rod sleeve is made of polypropylene, and the gap between the inner diameter and the magnetic rod is 0.1-0.2mm.

[0014] Furthermore, the dosage ratio of each reagent is as follows: The volume ratio of lysis buffer to sample to be processed is 3:1-5:1, the volume ratio of binding buffer to lysed sample is 2:1-3:1, the volume of washing buffer used per wash is 400-600 μL, and the volume of elution buffer used per wash is 50-80 μL.

[0015] Furthermore, this includes the following steps: (1) Sample lysis: Mix the sample to be processed with the lysis buffer at a volume ratio of 1:4, place it in a constant temperature shaker at 35-37℃, shake at 800-1000rpm for 8-10 minutes to obtain the lysis product; (2) Nucleic acid binding: Add binding solution to the lysis product, mix well, add surface-modified magnetic beads, place in a constant temperature shaker at 25-28℃, shake at 600-800 rpm for 5-7 minutes to bind the nucleic acid to the magnetic beads; (3) Magnetic separation and washing: Transfer the reaction system to a 96-well deep well plate, place it on a magnetic rack and let it stand for 2-3 minutes, then discard the supernatant; add washing solution, gently shake and mix, then perform magnetic separation again and discard the supernatant, repeat the washing twice; (4) Drying and elution: Place the washed magnetic beads in a constant temperature drying oven at 45-50℃ for 3-5 minutes, add elution solution, place in a constant temperature shaker at 35-37℃, shake at 800-1000rpm for 5-7 minutes, collect the supernatant after magnetic separation, which is the purified nucleic acid solution.

[0016] Furthermore, in step (2), the amount of surface-modified magnetic beads added is 3-5 μg of magnetic beads per 100 μL of sample to be treated.

[0017] Furthermore, it is applicable to nucleic acid extraction from soil samples, fecal microbial samples, plant tissue samples, and animal tissue samples, and is suitable for various difficult sample extraction scenarios with high impurities and high inhibitors in the field of agricultural breeding. The amount of lysis buffer added can be adjusted for different difficult agricultural samples as follows: the amount added to soil samples is 4 times the sample volume, the amount added to fecal microbial samples is 5 times the sample volume, and the amount added to plant and animal tissue samples is 3 times the sample volume.

[0018] Furthermore, the storage conditions for the reagent kit are as follows: The lysis buffer, binding buffer, washing buffer, elution buffer, and surface-modified magnetic beads were stored at 2-8℃ away from light and had a shelf life of 18 months. After opening, the shelf life of the lysis buffer, binding solution, and washing solution is 3 months, and the shelf life of the surface-modified magnetic beads is 6 months.

[0019] Furthermore, the entire extraction process takes 18-22 minutes, requires no high-temperature denaturation step, and does not require the addition of proteinase K.

[0020] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: 1. High adaptability to multiple samples: The recovery rates of soil microbial nucleic acid, fecal microbial nucleic acid, lignified plant tissue, and animal breeding tissue samples reached 93.5%, 92.1%, and 94.2%, respectively, which are significantly higher than existing agricultural sample extraction reagents (average recovery rate ≤85%). 2. Fast and efficient: Total time is 18-22 minutes, no high-temperature denaturation or long digestion of proteinase K is required, making it suitable for high-throughput automated platforms; 3. Excellent purity: The obtained nucleic acid A260 / A280 is 1.85-1.95, and A260 / A230 is 1.90-2.05. There are no residual inhibitors such as soil humic acid, plant polysaccharides and polyphenols, and fecal organic matter. It can be directly used for downstream experiments such as molecular marker breeding, gene sequencing, and PCR identification. 4. High stability: After storage at 2-8℃ in the dark for 18 months, the performance of each reagent decreases by ≤3%, and the batch-to-batch CV is ≤3%; 5. Low cost: The reagent components are all conventional raw materials with no expensive additives, and the cost of single sample extraction is reduced by more than 40% compared with imported reagents.

[0021] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a flowchart illustrating the overall process of the nucleic acid purification method of the present invention. Figure 2 This is a flowchart of the process for extracting microbial samples from agricultural livestock and poultry manure according to the present invention; Figure 3 This is a flowchart of the nucleic acid extraction process from crop tissue samples according to the present invention; Figure 4 This is a flowchart of the soil sample nucleic acid extraction process of the present invention. Detailed Implementation

[0024] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0025] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0026] Example 1: Nucleic acid extraction from soil samples

[0027] 1. Experimental Materials

[0028] Sample to be processed: Farmland topsoil sample, after removing stones, dead branches and other impurities, air-drying at room temperature, grinding and sieving (sample volume 200 μL); Reagent composition: prepared according to claims 1-3; Kit: prepared according to claims 4-5; Comparative reagent: A commercially available soil nucleic acid extraction kit.

[0029] 2. Experimental Methods

[0030] Extraction is performed according to the method of claims 6-7, with the following specific parameters:

[0031] The control reagent was used according to the instructions, and the total time was 45 minutes.

[0032] 3. Result Detection

[0033] Purity was determined using Nanodrop 2000, concentration was determined using Qubit 4.0, and recovery rate was calculated. Residual inhibitors were detected using qPCR (targeting the 16S rRNA gene of soil microorganisms; a Ct value deviation ≤ 0.5 was considered as no inhibition).

[0034]

[0035] 4. Conclusion

[0036] This embodiment targets farmland soil samples with high humic acid and high impurities. The extraction time is reduced by 55% compared to the control reagent, and the recovery rate is increased by 11.2%. It can effectively remove soil humus and mineral inhibitors, significantly improve nucleic acid purity, and fully meet the needs of soil microbial breeding and detection.

[0037] Example 2: Nucleic acid extraction from plant breeding tissue samples

[0038] 1. Experimental Materials

[0039] Sample to be processed: lignified crop stem tissue, which was prepared into tissue homogenate by low-temperature grinding; reagent composition: same as in Example 1; comparative reagent: a commercially available plant nucleic acid extraction kit (launched in 2024).

[0040] 2. Experimental Methods

[0041] Extraction was performed according to the method of claims 6-8, with the amount of lysis buffer added being 5 times the sample volume, and the specific parameters are as follows:

[0042] 3. Result Detection

[0043] The distribution of nucleic acid fragments was detected using an Agilent 2100 Bioanalyzer, and the proportion of fragments >200bp was calculated. The concentration was detected using a Qubit 4.0, and the recovery rate was calculated.

[0044]

[0045] 4. Conclusion

[0046] This embodiment targets lignified plant tissues rich in polysaccharides and polyphenols, reducing the total processing time by 81.7%, significantly increasing the proportion of nucleic acid fragments >200bp, effectively preserving the complete gene fragments required for breeding, and demonstrating significantly better recovery rate and nucleic acid integrity than traditional reagents.

[0047] Example 3: Verification of reagent storage stability

[0048] 1. Experimental Methods

[0049] The reagent composition and kit of Example 1 were stored at 2-8℃ in the dark. At 0 months, 6 months, 12 months and 18 months, nucleic acid was extracted from soil samples according to the method of Example 1, and the recovery rate, purity and magnetic bead aggregation rate were detected (aggregation rate ≤5% is qualified).

[0050]

[0051] 2. Conclusion

[0052] After 18 months of storage, the performance of the reagent decreased by ≤3%, which meets the stability requirements.

[0053] Example 4: Batch-to-batch repeatability verification

[0054] 1. Experimental Methods

[0055] Three independent batches of reagent composition were prepared, and the same sample was extracted according to the method in Example 1. The coefficient of variation (CV) of the recovery rate was then determined.

[0056]

[0057] 2. Conclusion

[0058] The batch-to-batch CV is ≤3%, indicating good repeatability.

[0059] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A nucleic acid purification reagent composition, characterized in that, It consists of lysis buffer, binding buffer, washing buffer, elution buffer and surface-modified magnetic beads; The lysis buffer contains 180-220 mmol / L of guanidine isothiocyanate derivative, 0.8%-1.2% (w / v) of alkyl glycoside surfactant, 20-30 mmol / L of Tris-HCl buffer system, pH 7.8-8.2, and 0.05%-0.08% of dipotassium ethylenediaminetetraacetate. The binding solution contains 120-150 mmol / L lithium chloride ion salt, 0.3%-0.5% (w / v) polyoxyethylene ether nonionic surfactant, and 10-15 mmol / L sodium citrate buffer system, with a pH of 5.5-6.

0. The washing solution contains 65%-75% by volume anhydrous ethanol, a 5-8 mmol / L Tris-HCl buffer system (pH 7.0-7.5), and 0.01%-0.03% sodium dodecyl sulfate. The eluent contains an 8-12 mmol / L Tris-HCl buffer system, pH 8.0-8.5, and is free of ethylenediaminetetraacetic acid; The surface-modified magnetic beads are silica magnetic beads with a diameter of 300-500nm, and the surface is grafted with amino polyethylene glycol segments accounting for 2%-3% by mass, with a grafting density of 8-12 PEG chains / 100nm² magnetic bead surface.

2. The nucleic acid purification reagent composition according to claim 1, characterized in that, The guanidine isothiocyanate derivative is n-propylguanidine isothiocyanate, the alkyl glycoside surfactant is decyl glucoside, the polyoxyethylene ether nonionic surfactant is Tween-20, and the molecular weight of the amino polyethylene glycol segment is 2000-3000 Da.

3. The nucleic acid purification reagent composition according to claim 1, characterized in that, The pH value of the lysis buffer is 8.0±0.1, the pH value of the binding buffer is 5.8±0.1, the pH value of the washing buffer is 7.2±0.1, and the pH value of the elution buffer is 8.2±0.

1.

4. A nucleic acid purification kit, characterized in that, It comprises the nucleic acid purification reagent composition according to any one of claims 1-3, and dedicated consumables: A 96-well deep well plate, a sealing cap with a silicone gasket, and a magnetic rod sleeve; the pore volume of the 96-well deep well plate is 1.2-1.5 mL, and the pore walls are hydrophobic. The magnetic rod sleeve is made of polypropylene, and the gap between the inner diameter and the magnetic rod is 0.1-0.2mm.

5. The nucleic acid purification kit according to claim 4, characterized in that, The dosage ratio of each reagent is as follows: The volume ratio of lysis buffer to sample to be processed is 3:1-5:1, the volume ratio of binding buffer to lysed sample is 2:1-3:1, the volume of washing buffer used per wash is 400-600 μL, and the volume of elution buffer used per wash is 50-80 μL.

6. The nucleic acid extraction method of the kit according to any one of claims 4-5, characterized in that, Includes the following steps: (1) Sample lysis: Mix the sample to be processed with the lysis buffer at a volume ratio of 1:4, place it in a constant temperature shaker at 35-37℃, shake at 800-1000rpm for 8-10 minutes to obtain the lysis product; (2) Nucleic acid binding: Add binding solution to the lysis product, mix well, add surface-modified magnetic beads, place in a constant temperature shaker at 25-28℃, shake at 600-800 rpm for 5-7 minutes to bind the nucleic acid to the magnetic beads; (3) Magnetic separation and washing: Transfer the reaction system to a 96-well deep well plate, place it on a magnetic rack and let it stand for 2-3 minutes, then discard the supernatant; add washing solution, gently shake and mix, then perform magnetic separation again and discard the supernatant, repeat the washing twice; (4) Drying and elution: Place the washed magnetic beads in a constant temperature drying oven at 45-50℃ for 3-5 minutes, add elution solution, place in a constant temperature shaker at 35-37℃, shake at 800-1000rpm for 5-7 minutes, collect the supernatant after magnetic separation, which is the purified nucleic acid solution.

7. The nucleic acid extraction method of the kit according to claim 6, characterized in that, In step (2), the amount of surface-modified magnetic beads added is 3-5 μg of magnetic beads per 100 μL of sample to be treated.

8. The nucleic acid extraction method of the kit according to claim 6, characterized in that, It is suitable for nucleic acid extraction from soil samples, fecal microbial samples, plant tissue samples, and animal tissue samples. It is adapted to various difficult sample extraction scenarios with high impurities and high inhibitors in the field of agricultural breeding. The amount of lysis buffer added can be adjusted for different difficult agricultural samples as follows: the amount added to soil samples is 4 times the sample volume, the amount added to fecal microbial samples is 5 times the sample volume, and the amount added to plant and animal tissue samples is 3 times the sample volume.

9. A nucleic acid purification kit according to claim 4, characterized in that, The storage conditions for the reagent kit are as follows: The lysis buffer, binding buffer, washing buffer, elution buffer, and surface-modified magnetic beads were stored at 2-8℃ away from light and had a shelf life of 18 months. After opening, the shelf life of the lysis buffer, binding solution, and washing solution is 3 months, and the shelf life of the surface-modified magnetic beads is 6 months.

10. The nucleic acid extraction method of the kit according to claim 6, characterized in that, The entire extraction process takes 18-22 minutes, requires no high-temperature denaturation step, and does not require the addition of proteinase K.

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