An extraction reagent, extraction method, and kit capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid

Abstract

The present application provides an extraction reagent capable of simultaneously extracting plant pathogenic DNA & RNA and host nucleic acid, which is composed of the following components: sample lysis and combination liquid LS, polysaccharide and polyphenol removal liquid, nucleic acid combination liquid, protein washing liquid WB1, nucleic acid rinsing liquid WB2, and nucleic acid elution liquid EB, wherein each component is stored independently. The extraction reagent can efficiently lyse plant cell walls, effectively remove plant-specific inhibitors, and simultaneously extract plant total DNA with high efficiency and high purity, efficiently enrich pathogenic nucleic acid invading plants, and simultaneously perform nucleic acid extraction by using a method and kit without the need for liquid nitrogen grinding and under the condition of a magnetic bead adsorption method scheme, thereby having important application value.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology, molecular biology, or nucleic acid extraction technology, specifically to an extraction reagent, extraction method, and kit that can simultaneously extract plant pathogen DNA & RNA and host nucleic acid. Background Technology

[0002] Molecular biology research, including the detection of plant pathogenic microorganisms (such as fungi, bacteria, and viruses), identification of transgenic components, gene expression analysis, and high-throughput sequencing, all rely on the efficient and intact extraction of nucleic acids from plant tissues. Plant samples are complex, containing abundant polysaccharides, polyphenols, pigments, secondary metabolites, and tough cell walls. These substances easily co-precipitate with nucleic acids or inhibit subsequent enzymatic reactions during nucleic acid extraction, posing a major challenge to plant nucleic acid extraction.

[0003] Currently, commercially available plant nucleic acid extraction kits are mainly divided into two categories: one is specifically designed for extracting plant genomic DNA, and the other is specifically designed for extracting plant total RNA. Both types of kits target the plant host's own nucleic acids as the primary product, and their lysis systems, buffer compositions, and purification conditions are optimized for the characteristics of plant cell walls, cell membranes, and host nucleic acids.

[0004] However, in practical applications, researchers often need to detect pathogenic microorganisms infecting plants, including fungi, bacteria, and viruses (especially RNA viruses, such as small RNA viruses like Potato Virus Y and Tobacco Mosaic Virus). Because existing kits lack lysis and protection systems for pathogen cell structures, such as fungal thick-walled cells, Gram-positive bacterial cell walls, and nucleic acid types (especially low-abundance, easily degradable RNA virus genomes), directly using host nucleic acid extraction kits to extract pathogen nucleic acids presents the following problems: incomplete disruption of fungal spores, hyphae, and bacterial cell walls leads to incomplete release of pathogen nucleic acids; the low abundance of pathogen nucleic acids in the sample, coupled with the large presence of host nucleic acids, further dilutes the target, and the lack of protection mechanisms against small RNA molecules results in a final yield far below the detection limit; and the inability to simultaneously and efficiently extract plant host DNA, host RNA, and pathogen nucleic acids (including DNA viruses, RNA viruses, bacteria, and fungi) from the same sample limits applications such as multi-target joint detection and pathogen-host interaction analysis.

[0005] Furthermore, existing reagent kits often contain strong liquid salts, high-temperature lysis steps, or lack effective nucleic acid protectants, which can easily lead to the degradation or loss of small RNA virus genomes. Plant samples have rigid cell walls, making conventional methods ineffective for cell lysis. Moreover, nucleic acids are degraded during lysis at room temperature or high temperature, thus requiring liquid nitrogen cryogenic grinding for cell lysis. Nucleic acid extraction from plant samples fundamentally requires this crucial pretreatment step of liquid nitrogen grinding, which is not only complex and poses a risk of liquid nitrogen burns to laboratory personnel, but also lacks access to liquid nitrogen in general or grassroots laboratories, limiting its application in rapid detection of plant pathogens. In addition, plant nucleic acid extraction methodologies either employ adsorption columns with high-speed centrifugation or magnetic bead methods using magnetic racks; currently, no single reagent kit is compatible with both adsorption column and magnetic bead methods, hindering the improvement of plant sample nucleic acid extraction efficiency. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an extraction reagent, extraction method, and kit that can simultaneously extract plant pathogen DNA & RNA and host nucleic acid. This extraction reagent can efficiently lyse plant cell walls, effectively remove plant-specific inhibitors, and simultaneously extract total plant DNA with high efficiency and purity, as well as efficiently enrich pathogen nucleic acids invading plants. Furthermore, it can simultaneously perform nucleic acid extraction using a magnetic bead adsorption method without liquid nitrogen grinding, and has significant application value.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Extraction reagents that can simultaneously extract plant pathogen DNA & RNA and host nucleic acid are provided. They consist of the following components (a) to (f), which are stored separately: Sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer, nucleic acid binding buffer, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB. (a) Sample lysis binding buffer LS, which contains: The first pH buffer component is selected from one or more combinations of Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid, and the concentration of the first pH buffer component is 10 mM to 100 mM, and the pH is 7.5 to 8.8. Nonionic surfactant component: selected from one or more of Triton X-100, NP40, and Tween 20, wherein the volume ratio of the nonionic surfactant component is 0.1% to 10%; Cell wall and cell lysis components: selected from at least one of guanidine isothiocyanate and guanidine hydrochloride, and a combination of one or more of urea, sodium lauroyl sarcosinate and SDS; Polysaccharide and polyphenol scavenging components: containing CTAB 0.5%~3% (w / v), PVP 0.5%~3% (w / v), PVPP 0.1%~2% (w / v), and PEG6000 1%~5% (w / v); Antioxidant components: Contains acetylcysteine ​​5 mM~50 mM, DTT 5 mM~20 mM, TCEP 5 mM~20 mM, and β-mercaptoethanol 0.1%~2% (v / v); Nucleic acid protective agent components: containing trehalose 0.1 M~0.3 M and betaine 0.1 M~1 M; (b) Polysaccharide and polyphenol removal solution PD1, which contains: The second pH buffer component is selected from one or more combinations of Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid, and the concentration of the second pH buffer component is 10 mM to 200 mM, and the pH is 5.5 to 7.0. The liquid salt components are guanidine isothiocyanate and / or guanidine hydrochloride, with a concentration of 1 M to 4 M. Polysaccharide-polyphenol coprecipitation components: containing PVP 0.5%~3% (w / v), PVPP 0.1%~2% (w / v), and aluminum ammonium sulfate 1mM~10 mM; (c) Nucleic acid binding solution LB, which is selected from one or more combinations of anhydrous ethanol, isopropanol, PEG6000, PEG8000, NaCl, KCl, potassium acetate, and sodium acetate; (d) Protein washing solution WB1, which contains Tris-HCl, benzenesulfonic acid and / or sodium citrate and / or citric acid, guanidine hydrochloride and / or guanidine isothiocyanate and / or guanidine thiocyanate and / or urea, Triton 100 and / or Tween 20 and / or NP40, isopropanol and / or anhydrous ethanol. (e) Nucleic acid washing solution WB2, which contains Tris-HCl, NaCl, and anhydrous ethanol; (f) Nucleic acid elution buffer EB, which is nuclease-free water or TE buffer; The mass ratio of sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer PD1, nucleic acid binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB is 0.8~1:0.2~0.6:0.2~0.6:1~4:1~4:0.1~0.3.

[0008] Furthermore, in the sample lysis binding solution LS, the concentration of guanidine isothiocyanate or guanidine hydrochloride in the cell wall and cell lysis components is 1 M to 4 M, the concentration of urea is 2 M to 5 M, the concentration of sodium lauroyl sarcosinate is 1% to 4%, and the concentration of SDS is 0.1% to 2%.

[0009] Furthermore, in the nucleic acid binding solution LB, the concentrations of anhydrous ethanol are 30%–100%, isopropanol is 30%–100%, PEG6000 and PEG8000 are 1%–10%, NaCl is 0.5 M–3 M, KCl is 0.5 M–3 M, potassium acetate is 0.5 M–3 M, and sodium acetate is 0.5 M–3 M.

[0010] Furthermore, in the protein washing solution WB1, the concentration of Tris-HCl is 10mM~100mM; the concentrations of benzenesulfonic acid, sodium citrate, and citric acid are all 10mM~50mM; the concentrations of guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, and urea are all 1M~5M; the concentrations of Triton 100, Tween 20, and NP40 are all 0.5%~5%; and the concentrations of isopropanol and anhydrous ethanol are all 20%~60%. The nucleic acid washing solution WB2 contains Tris-HCl at a concentration of 1 mM to 20 mM, sodium chloride at a concentration of 10 mM to 100 mM, and anhydrous ethanol at a concentration of 50% to 85%.

[0011] Extraction reagents for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid are also provided, including the above-mentioned extraction reagents; nucleic acid adsorption magnetic beads, silicon-based nucleic acid adsorption columns and grinding tubes.

[0012] A method for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid is also provided, using the extraction reagents described above, and including the following steps: Step 1: Sample lysis After the plant tissue sample was broken, it was added to the sample lysis binding buffer LS for lysis, and then the polysaccharide and polyphenol removal buffer PD1 was added. The mixture was vortexed and placed at room temperature or 3~6℃ for 1~3 min to obtain the first mixture. Step 2, Nucleic acid binding: Centrifuge the first mixture, take the supernatant, add 0.5 to 1 times the volume of the supernatant of nucleic acid binding solution LB, mix well, and then use the adsorption column method or magnetic bead method to bind the nucleic acid to the solid support. Step 3: Nucleic acid washing and purification: Wash the nucleic acid-bound solid support obtained in step two with protein washing buffer WB1 1-2 times to remove protein impurities; The solid support is then washed 1-3 times with nucleic acid washing buffer WB2 to remove salt ions and organic reagent residues. Step 4: Nucleic acid elution: Add nucleic acid elution buffer EB to the solid support, incubate at room temperature, then centrifuge or magnetically separate, collect the elution buffer to obtain nucleic acid.

[0013] Furthermore, in step one, if liquid nitrogen is available, the specific steps of lysing the plant tissue sample by breaking it up and adding the sample lysis binding solution LS include: grinding the plant tissue into powder in liquid nitrogen, transferring it to a centrifuge tube under frozen conditions, adding the sample lysis binding solution LS, and then letting it stand at room temperature or 65°C for 10 minutes to complete the lysis. After that, adding the polysaccharide and polyphenol co-precipitant, vortexing and mixing for 1 minute, and then placing it at room temperature for 5-6 minutes or in a 4°C refrigerator for 1-3 minutes. If liquid nitrogen is not available, the specific steps for lysing the plant tissue sample by breaking it up and adding the sample lysis binding solution LS include: cutting the plant tissue into small pieces, taking an appropriate amount and adding it to a grinding tube, adding the sample lysis binding solution LS, and heating at 90~96℃ for 10-20 minutes; after heating, adding a polysaccharide and polyphenol co-precipitant in time, and vortexing for 3~4 minutes on a vortex mixer to completely homogenize the tissue.

[0014] Further, in step two, the adsorption column method or magnetic bead method for binding nucleic acids to the solid-phase support includes: When using the adsorption column method, the first mixture is transferred to the nucleic acid adsorption column; When using the magnetic bead method, add 30 μL to 40 μL of nucleic acid adsorption magnetic beads to the first mixture, mix well, and let stand at room temperature for 5-10 min, mixing once every 2-3 minutes.

[0015] Further, in step three, washing the solid-phase carrier bound with nucleic acid obtained in step two with protein washing solution WB1 1-2 times to remove protein impurities includes: When using the adsorption column method, centrifuge the adsorption column at 12000 rpm to 14000 rpm for 1 to 2 min and discard the filtrate. Add 400 μL to 750 μL of protein washing buffer WB1, centrifuge at 12000 rpm to 14000 rpm for 1 to 2 min, and discard the filtrate; optionally repeat once. Add 400 μL to 750 μL of nucleic acid washing buffer WB2, centrifuge at 12000 rpm to 14000 rpm for 1 to 2 min, discard the filtrate; repeat 1 to 2 times. When using the magnetic bead method Place the solid support on a magnetic rack and let it stand for 1-5 minutes. After the solution becomes clear, remove the supernatant. Add 400 μL to 750 μL of protein washing buffer WB1, resuspend the magnetic beads, place them on a magnetic rack and let stand, then discard the supernatant; repeat once if desired. Add 400 μL~750 μL of nucleic acid washing buffer WB2, resuspend the magnetic beads, place them on a magnetic rack and let stand, then discard the supernatant; repeat 1-2 times. Further, in step four, the addition of nucleic acid elution buffer EB to the solid support, followed by centrifugation or magnetic separation at room temperature, and collection of the elution buffer specifically includes: When using the adsorption column method: Add 30 μL to 200 μL of nucleic acid elution buffer EB to the adsorption column, incubate at room temperature for 3-5 min, centrifuge at 12000 rpm to 14000 rpm for 1 min, and collect the elution buffer; When using the magnetic bead method: Add 30 μL to 200 μL of nucleic acid elution buffer EB to the magnetic beads, incubate at room temperature for 3-5 min, place on a magnetic rack and let stand, then collect the elution buffer.

[0016] The beneficial effects of the extraction reagent of this invention, which can simultaneously extract plant pathogen DNA & RNA and host nucleic acid, are as follows: (1) The nucleic acid co-extraction reagent of the present invention comprises sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer, nucleic acid binding buffer, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB. It effectively targets plant structures, not only removing polysaccharides and polyphenols, but also utilizing a high concentration of guanidine salt to strongly lyse cell walls and cells, releasing their contents. Surfactants such as CTAB, SDS, and sodium lauroyl sarcosinate effectively remove cell walls and cell membranes, complementing the high concentration of guanidine salt and creating a synergistic effect, further thoroughly lysing plant cells and facilitating subsequent nucleic acid extraction. The reagent of the present invention has a dedicated high-temperature lysis protection component, allowing for vortex lysis with grinding beads at high temperatures, making it more convenient.

[0017] CTAB, PVP, and PVPP polysaccharide and polyphenol removers can thoroughly separate polysaccharides and polyphenols from nucleic acids, and then completely remove them through subsequent steps such as centrifugation or magnetic bead separation. In ammonium aluminum sulfate, aluminum ions are 3+ cations. When added to the lysis buffer, due to the alkaline pH of the buffer, aluminum ions will form fine micelle precipitates. This process involves them binding and adhering to negatively charged polysaccharides, polyphenols, humic acids, etc., which are then removed together by centrifugation. Nucleic acids, being large molecules, have higher solubility in the lysis buffer and therefore do not readily bind to aluminum ions. Therefore, ammonium aluminum sulfate works better with CTAB and PVP to achieve superior polysaccharide and polyphenol removal.

[0018] DTT, TCEP, acetylcysteine, and β-mercaptoethanol reducing agents not only protect nucleic acids from oxidative degradation, but also prevent polysaccharides, polyphenols, pigments, and other substances from being oxidized into more difficult-to-clear quinone secondary metabolites when heated at high temperatures, thus providing dual nucleic acid protection.

[0019] The addition of trehalose and betaine ensures that the nucleic acid extraction process, whether it is liquid nitrogen grinding, high-speed vortexing or high-temperature lysis, can maintain the conformational stability of nucleic acids and prevent nucleic acid breakage and degradation.

[0020] (2) Since the present invention uses the above-mentioned nucleic acid co-extraction reagent, nucleic acid extraction can be carried out according to different experimental conditions to cope with different application scenarios. When the laboratory equipment is perfect and the highest nucleic acid purity and yield are pursued, the method of liquid nitrogen grinding and nucleic acid adsorption column purification can be used for nucleic acid co-extraction. When the laboratory conditions are not so perfect, and there is no high-speed centrifuge or liquid nitrogen for grinding, the method of high temperature lysis and magnetic bead separation can be used for nucleic acid extraction, which greatly meets the needs of different laboratories, especially grassroots agricultural epidemic prevention departments.

[0021] (3) The optimized lysis and binding system can simultaneously and efficiently adsorb large molecular weight host DNA, small genome infecting microbial DNA, and has a good simultaneous extraction effect on single and double strand viral RNA. One sample can be extracted to meet different detection applications, saving time and effort and conserving precious samples.

[0022] (4) This invention is applicable to various plant tissue types, including leaves, roots, stems, flowers, fruits, seeds, etc., as well as microorganisms such as bacteria, fungi, and viruses that grow on the plant surface or infect the inside of plant cells. It can meet the needs of different application scenarios such as plant genome research, disease diagnosis, plant-microbe interaction research, and analysis of plant endophytic and epiphytic communities, and has wide applicability. Attached Figure Description

[0023] Figure 1 These are the electrophoretic identification gel images of the four samples in Experiment Example 2.

[0024] Figure 2 These are the electrophoretic gel images of the three samples in Experiment Example 2, obtained under liquid nitrogen grinding, adsorption column conditions, and high-temperature heating and magnetic bead method, respectively.

[0025] Figure 3 This is a diagram showing the results of the identification of four pathogens in Experiment Example 5.

[0026] Figure 4 These are identification images of the seven transgenic F1 samples from Example 7 of this embodiment. Detailed Implementation

[0027] To make the technical problems, technical solutions, and beneficial effects solved by this application clearer, the following will provide a more detailed description of this application in conjunction with the accompanying drawings of the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0028] Molecular biology research, such as plant pathogen detection and transgenic component identification, relies on the efficient extraction of nucleic acids from plant tissues. Plant samples are complex, containing polysaccharides, polyphenols, pigments, and tough cell walls, which can easily co-precipitate nucleic acids or inhibit subsequent enzymatic reactions. Existing technologies suffer from incomplete release of pathogen nucleic acids due to incomplete disruption of fungal spores and bacterial cell walls; a lack of protective mechanisms against small RNA molecules leads to low yields; the inability to simultaneously and efficiently extract both host and pathogen nucleic acids; the use of strong liquid salts or high-temperature lysis easily degrades small RNAs; and the reliance on liquid nitrogen grinding is complex and limited by substrate limitations. Furthermore, existing methods cannot simultaneously integrate adsorption columns and magnetic beads. This application addresses this issue by using a synergistic reagent system to break down the lysis barriers between plants and pathogens, completely removing inhibitors and achieving simultaneous and efficient co-extraction of multiple target nucleic acids in various scenarios.

[0029] Some of the terms or terms that appear in the description of the embodiments of this application shall be interpreted as follows: CTAC: Hexadecyltrimethylammonium chloride.

[0030] The term PNIPAM stands for poly(N-isopropylacrylamide).

[0031] Term Ectoine: Tetrahydropyrimidine carboxylic acid.

[0032] The term LCST stands for Low Critical Dissolution Temperature.

[0033] PEG: Polyethylene glycol. DTT (dithiothreitol)

[0034] The term LAMP: Loop-mediated isothermal amplification

[0035] Term PVP: Polyvinylpyrrolidone

[0036] PVPP: Cross-linked polyvinylpyrrolidone (or polyvinylpyrrolidone)

[0037] Term SDS: Sodium dodecyl sulfate

[0038] The term SPLCV stands for Sweet potato leaf curl virus.

[0039] Term SPFMV: Sweet potato feathery mottle virus

[0040] Terminology TCEP: Tris(2-carboxyethyl)phosphonic hydrochloride

[0041] The term DTT stands for dithiothreitol.

[0042] The nucleic acid extraction kit, microfluidic chip, nucleic acid extraction equipment, and nucleic acid extraction method proposed in this application will be further described below with reference to specific embodiments and accompanying drawings.

[0043] Example 1

[0044] This embodiment discloses an extraction reagent capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid. The extraction reagent includes: (a) Sample lysis binding buffer LS, the pH of which is 7.5, specifically includes: First pH buffer: Tris-HCl 10mM; nonionic surfactant: Triton X-100 0.1% (v / v); cell wall / cell lysis component: guanidine isothiocyanate 1M; polysaccharide and polyphenol scavenging components: CTAB 0.5% (w / v), PVP 0.5% (w / v), PVPP 0.1% (w / v), PEG6000 1% (w / v); antioxidant components: acetylcysteine ​​5mM, DTT 5mM; nucleic acid protectants: trehalose 0.1M, betaine 0.1M. (b) Polysaccharide and polyphenol removal solution PD1 (pH 5.5): second pH buffer sodium acetate-acetic acid 10mM; guanidine hydrochloride 1M; polysaccharide and polyphenol coprecipitation components PVP 0.5% (w / v), PVPP 0.1% (w / v), and ammonium aluminum sulfate 1mM.

[0045] (c) Nucleic acid binding solution LB: 30% (v / v) anhydrous ethanol.

[0046] (d) Protein washing solution WB1: Tris-HCl 10mM, benzenesulfonic acid 10mM, guanidine hydrochloride 1M, Triton 100 0.5% (v / v), isopropanol 20% (v / v).

[0047] (e) Nucleic acid washing solution WB2: Tris-HCl 1mM, NaCl 10mM, anhydrous ethanol 50% (v / v).

[0048] (f) Nucleic acid elution buffer EB: nuclease-free water.

[0049] The mass ratio of sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer PD1, nucleic acid binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB is 0.8:0.2:0.2:1:1:0.1.

[0050] This extraction reagent is used in the extraction method steps: Step 1: Sample lysis After the plant tissue sample was broken, it was added to the sample lysis binding buffer LS for lysis, and then the polysaccharide and polyphenol removal buffer PD1 was added. The mixture was vortexed and placed at room temperature for 1 min to obtain the first mixture. If liquid nitrogen is available, the specific steps for lysing the plant tissue sample by breaking it up and adding the sample lysis binding solution LS include: grinding the plant tissue into powder in liquid nitrogen, transferring it to a centrifuge tube under frozen conditions, adding the sample lysis binding solution LS, and then letting it stand at room temperature for 10 minutes to complete the lysis. After that, adding the polysaccharide and polyphenol co-precipitant, vortexing and mixing for 1 minute, and letting it stand at room temperature for 2 minutes. If liquid nitrogen is not available, the specific steps for lysing the plant tissue sample by breaking it up and adding the sample lysis binding solution LS include: cutting the plant tissue into small pieces, taking an appropriate amount and adding it to a grinding tube, adding the sample lysis binding solution LS, and heating at 90°C for 10 minutes; after heating, adding a polysaccharide and polyphenol co-precipitant in a timely manner, and vortexing for 3 minutes on a vortex mixer to completely homogenize the tissue.

[0051] Step 2, Nucleic acid binding: Centrifuge the first mixture, take the supernatant, add 0.5 times the volume of the supernatant of nucleic acid binding solution LB, mix well, and then use the adsorption column method or magnetic bead method to bind the nucleic acid to the solid-phase carrier. When using the adsorption column method, the first mixture is transferred to the nucleic acid adsorption column; When using the magnetic bead method, add 30 μL of nucleic acid adsorption magnetic beads to the first mixture, mix well, and let stand at room temperature for 5 minutes, mixing once every 2 minutes.

[0052] Step 3: Nucleic acid washing and purification: The solid-phase carrier bound with nucleic acid obtained in step two was washed once with protein washing buffer WB1 to remove protein impurities. The solid support is then washed once with nucleic acid washing buffer WB2 to remove salt ions and organic reagent residues. When using the adsorption column method, the adsorption column is centrifuged at 12,000 rpm for 1 min and the filtrate is discarded. Add 400 μL of protein washing buffer WB1, centrifuge at 12000 rpm for 1 min, and discard the filtrate; Add 400 μL of nucleic acid washing buffer WB2, centrifuge at 12000 rpm for 1 min, and discard the filtrate; When using the magnetic bead method, place the solid support on a magnetic rack and let it stand for 1 minute. After the solution becomes clear, remove the supernatant. Add 400 μL of protein washing buffer WB1, resuspend the magnetic beads, place them on a magnetic rack and let them stand, then discard the supernatant. Add 400 μL of nucleic acid washing buffer WB2, resuspend the magnetic beads, place them on a magnetic rack and let stand, then discard the supernatant.

[0053] Step 4: Nucleic acid elution: Add nucleic acid elution buffer EB to the solid support, incubate at room temperature, centrifuge or magnetically separate, and collect the elution buffer to obtain nucleic acid; When using the adsorption column method: add 30 μL of nucleic acid elution buffer EB to the adsorption column, incubate at room temperature for 3 min, centrifuge at 12000 rpm for 1 min, and collect the elution buffer; When using the magnetic bead method: Add 30 μL of nucleic acid elution buffer EB to the magnetic beads, incubate at room temperature for 3 min, place on a magnetic rack and let stand, then collect the elution buffer.

[0054] Example 2

[0055] This embodiment discloses an extraction reagent capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid. The extraction reagent includes: (a) Sample lysis binding buffer LS (pH 8.0): Tris-HCl 50mM; NP40 5% (v / v); guanidine hydrochloride 2M + urea 3M; CTAB 1.5% (w / v), PVP 1.5% (w / v), PVPP 1.0% (w / v), PEG6000 3% (w / v); acetylcysteine ​​25mM, TCEP 10mM; trehalose 0.2M, betaine 0.5M.

[0056] (b) Polysaccharide and polyphenol removal solution PD1 (pH 6.0): sodium citrate-citric acid 100mM; guanidine isothiocyanate 2.5M; PVP 1.5% (w / v), PVPP 1.0% (w / v), ammonium aluminum sulfate 5mM.

[0057] (c) Nucleic acid binding solution LB: 60% isopropanol (v / v).

[0058] (d) Protein washing buffer WB1: Tris-HCl 50mM, sodium citrate 30mM, guanidine isothiocyanate 3M, Tween 20 2.5% (v / v), anhydrous ethanol 40% (v / v).

[0059] (e) Nucleic acid washing solution WB2: Tris-HCl 10mM, NaCl 50mM, anhydrous ethanol 70% (v / v).

[0060] (f) Nucleic acid elution buffer EB:TE buffer.

[0061] The mass ratio of sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer PD1, nucleic acid binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB is 1:0.6:0.6:4:4:0.3.

[0062] The extraction method includes the following steps: Step 1: Sample lysis After the plant tissue sample was broken up, it was lysed with sample lysis binding solution LS, and polysaccharide and polyphenol removal solution PD1 was added. The mixture was vortexed and placed at 4°C for 1.5 min to obtain the first mixture. If liquid nitrogen is available, the specific steps for lysing the plant tissue sample by breaking it up and adding the sample lysis binding solution LS include: grinding the plant tissue into powder in liquid nitrogen, transferring it to a centrifuge tube under frozen conditions, adding the sample lysis binding solution LS, then letting it stand at 45°C for 10 minutes to complete the lysis, adding a polysaccharide and polyphenol co-precipitant, vortexing and mixing for 1 minute, and placing it in a 4°C refrigerator for 2 minutes. If liquid nitrogen is not available, the specific steps for lysing the plant tissue sample by breaking it up and adding the sample lysis binding solution LS include: cutting the plant tissue into small pieces, taking an appropriate amount and adding it to a grinding tube, adding the sample lysis binding solution LS, and heating at 92°C for 13 minutes; after heating, adding a polysaccharide and polyphenol co-precipitant in time, and vortexing for 3.2 minutes on a vortex mixer to completely homogenize the tissue.

[0063] Step 2, Nucleic acid binding: Centrifuge the first mixture, take the supernatant, add 0.6 times the volume of the supernatant of nucleic acid binding solution LB, mix well, and then use the adsorption column method or magnetic bead method to bind the nucleic acid to the solid-phase carrier. When using the adsorption column method, the first mixture is transferred to the nucleic acid adsorption column; When using the magnetic bead method, add 33 μL of nucleic acid adsorption magnetic beads to the first mixture, mix well, and let stand at room temperature for 6 minutes, mixing once every 2 minutes.

[0064] Step 3: Nucleic acid washing and purification: The solid-phase carrier bound with nucleic acid obtained in step two was washed once with protein washing buffer WB1 to remove protein impurities. The solid support was then washed twice with nucleic acid washing buffer WB2 to remove salt ions and organic reagent residues. When using the adsorption column method, the adsorption column is centrifuged at 12500 rpm for 1 min and the filtrate is discarded. Add 500 μL of protein washing buffer WB1, centrifuge at 12500 rpm for 1 min, and discard the filtrate; Add 500 μL of nucleic acid washing buffer WB2, centrifuge at 12500 rpm for 1 min, discard the filtrate; repeat once. When using the magnetic bead method, place the solid support on a magnetic rack and let it stand for 2 minutes. After the solution becomes clear, remove the supernatant. Add 500 μL of protein washing buffer WB1, resuspend the magnetic beads, place them on a magnetic rack and let them stand, then discard the supernatant. Add 500 μL of nucleic acid washing buffer WB2, resuspend the magnetic beads, place them on a magnetic rack and let stand, then discard the supernatant; repeat once.

[0065] Step 4: Nucleic acid elution: Add nucleic acid elution buffer EB to the solid support, incubate at room temperature, centrifuge or magnetically separate, and collect the elution buffer to obtain nucleic acid; When using the adsorption column method: add 70 μL of nucleic acid elution buffer EB to the adsorption column, incubate at room temperature for 3.5 min, centrifuge at 12500 rpm for 1 min, and collect the elution buffer; When using the magnetic bead method: add 70 μL of nucleic acid elution buffer EB to the magnetic beads, incubate at room temperature for 3.5 min, place on a magnetic rack and let stand, and collect the elution buffer.

[0066] Example 3: This embodiment discloses an extraction reagent capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid. The extraction reagent includes: (a) Sample lysis binding buffer LS (pH 8.5): Sodium acetate-acetic acid 80mM; Tween 20 8% (v / v); Sodium lauroyl sarcosinate 3% + SDS 1%; CTAB 2.5% (w / v), PVP 2.5% (w / v), PVPP 1.5% (w / v), PEG6000 4% (w / v); DTT 15mM, β-mercaptoethanol 1.5% (v / v); Trehalose 0.25M, Betaine 0.8M.

[0067] (b) Polysaccharide and polyphenol removal solution PD1 (pH 6.5): Tris-HCl 150mM; guanidine hydrochloride 3M; PVP 2.5% (w / v), PVPP 1.5% (w / v), ammonium aluminum sulfate 8mM.

[0068] (c) Nucleic acid binding solution LB: PEG8000 5% (w / v) + NaCl 2M.

[0069] (d) Protein washing buffer WB1: Tris-HCl 80mM, citric acid 40mM, guanidine thiocyanate 4M, NP40 4% (v / v), isopropanol 50% (v / v).

[0070] (e) Nucleic acid washing solution WB2: Tris-HCl 15mM, NaCl 80mM, anhydrous ethanol 80% (v / v).

[0071] (f) Nucleic acid elution buffer EB:TE buffer.

[0072] The mass ratio of sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer PD1, nucleic acid binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB is 1:0.5:0.5:3:3:0.2.

[0073] The extraction method includes the following steps: Step 1, Sample Lysis: After crushing the plant tissue sample, add sample lysis binding solution LS for lysis, add polysaccharide and polyphenol removal solution PD1, vortex to mix, and incubate at 5°C for 2.5 min to obtain the first mixture; wherein, if liquid nitrogen is available, the specific steps of crushing the plant tissue sample and adding sample lysis binding solution LS for lysis include: grinding the plant tissue into powder in liquid nitrogen, transferring it to a centrifuge tube under frozen conditions, adding sample lysis binding solution LS, and then incubating at 65°C for 10 min to complete lysis, adding polysaccharide and polyphenol co-precipitant, vortexing to mix for 1 min, and incubating at 4°C for 2 min; if liquid nitrogen is not available, the specific steps of crushing the plant tissue sample and adding sample lysis binding solution LS for lysis include: cutting the plant tissue into small pieces, taking an appropriate amount and adding it to a grinding tube, adding the sample lysis binding solution LS, heating at 94°C for 17 min; after heating, promptly adding polysaccharide and polyphenol co-precipitant, and vortexing for 3.8 min on a vortex mixer to completely homogenize the tissue.

[0074] Step 2, Nucleic Acid Binding: Centrifuge the first mixture, take the supernatant, add 0.8 times the volume of the supernatant of nucleic acid binding solution LB, mix well, and then bind the nucleic acid to the solid support using an adsorption column method or a magnetic bead method; wherein, when using the adsorption column method, the first mixture is transferred to the nucleic acid adsorption column; when using the magnetic bead method, 37 μL of nucleic acid adsorption magnetic beads are added to the first mixture, mixed well, and placed at room temperature for 8 min, mixing once every 3 minutes.

[0075] Step 3, Nucleic Acid Washing and Purification: Wash the solid-phase support containing nucleic acid obtained in Step 2 twice with protein washing buffer WB1 to remove protein impurities; then wash the solid-phase support twice with nucleic acid washing buffer WB2 to remove salt ions and organic reagent residues. When using the adsorption column method, centrifuge the adsorption column at 13500 rpm for 1 min and discard the filtrate; add 650 μL of protein washing buffer WB1, centrifuge at 13500 rpm for 1 min, and discard the filtrate; repeat once; add 650 μL of nucleic acid washing buffer WB2, centrifuge at 13500 rpm for 1 min, and discard the filtrate; repeat once. When using the magnetic bead method, place the solid-phase support on a magnetic rack and let it stand for 4 min until the solution is clear, then discard the supernatant; add 650 μL of protein washing buffer WB1, resuspend the magnetic beads, place it on a magnetic rack and let it stand, then discard the supernatant; repeat once; add 650 μL of protein washing buffer WB1, resuspend the magnetic beads, place it on a magnetic rack and let it stand, then discard the supernatant; repeat once. μL of nucleic acid washing buffer WB2 was used to resuspend the magnetic beads and then placed on a magnetic rack to stand. The supernatant was then discarded. This process was repeated once.

[0076] Step 4, Nucleic Acid Elution: Add nucleic acid elution buffer EB to the solid support, incubate at room temperature, centrifuge or magnetically separate, and collect the eluent to obtain nucleic acid; when using the adsorption column method: add 140 μL of nucleic acid elution buffer EB to the adsorption column, incubate at room temperature for 4.5 min, centrifuge at 13500 rpm for 1 min, and collect the eluent; when using the magnetic bead method: add 140 μL of nucleic acid elution buffer EB to the magnetic beads, incubate at room temperature for 4.5 min, place on a magnetic rack and let stand, and collect the eluent.

[0077] Example 4: This embodiment discloses an extraction reagent capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid. The extraction reagent includes: (a) Sample lysis binding buffer LS (pH 8.8): Sodium citrate-citric acid 100mM; Triton X-100 10% (v / v); Urea 5M + SDS 2%; CTAB 3% (w / v), PVP 3% (w / v), PVPP 2% (w / v), PEG6000 5% (w / v); Acetylcysteine ​​50mM, β-mercaptoethanol 2% (v / v); Trehalose 0.3M, Betaine 1M.

[0078] (b) Polysaccharide and polyphenol removal solution PD1 (pH 7.0): Sodium acetate-acetic acid 200mM; guanidine isothiocyanate 4M; PVP 3% (w / v), PVPP 2% (w / v), ammonium aluminum sulfate 10mM.

[0079] (c) Nucleic acid binding solution LB: potassium acetate 3M + isopropanol 100% (v / v) (mixed in equal volumes).

[0080] (d) Protein washing solution WB1: Tris-HCl 100mM, benzenesulfonic acid 50mM, urea 5M, Triton 100 5% (v / v), anhydrous ethanol 60% (v / v).

[0081] (e) Nucleic acid washing solution WB2: Tris-HCl 20mM, NaCl 100mM, anhydrous ethanol 85% (v / v).

[0082] (f) Nucleic acid elution buffer EB: nuclease-free water.

[0083] The mass ratio of sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer PD1, nucleic acid binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB is 0.8:0.3:0.5:3:3:0.2.

[0084] The extraction method includes the following steps: Step 1, Sample Lysis: After crushing the plant tissue sample, add sample lysis binding solution LS for lysis, add polysaccharide and polyphenol removal solution PD1, vortex to mix, and incubate at 6°C for 3 minutes to obtain the first mixture; wherein, if liquid nitrogen is available, the specific steps of crushing the plant tissue sample and adding sample lysis binding solution LS for lysis include: grinding the plant tissue into powder in liquid nitrogen, transferring it to a centrifuge tube under frozen conditions, adding sample lysis binding solution LS, and then incubating at 65°C for 10 minutes to complete lysis, adding polysaccharide and polyphenol co-precipitant, vortexing to mix for 1 minute, and incubating at 4°C for 2 minutes; if liquid nitrogen is not available, the specific steps of crushing the plant tissue sample and adding sample lysis binding solution LS for lysis include: cutting the plant tissue into small pieces, taking an appropriate amount and adding it to a grinding tube, adding the sample lysis binding solution LS, heating at 96°C for 20 minutes; after heating, promptly adding polysaccharide and polyphenol co-precipitant, vortexing on a vortex mixer for 4 minutes to completely homogenize the tissue.

[0085] Step 2, Nucleic Acid Binding: Centrifuge the first mixture, take the supernatant, add LB nucleic acid binding solution with a volume equal to the supernatant, mix well, and bind the nucleic acid to the solid support using an adsorption column method or a magnetic bead method; wherein, when using the adsorption column method, transfer the first mixture to the nucleic acid adsorption column; when using the magnetic bead method, add 40 μL of nucleic acid adsorption magnetic beads to the first mixture, mix well, and let stand at room temperature for 10 min, mixing once every 3 minutes.

[0086] Step 3, Nucleic Acid Washing and Purification: Wash the solid-phase support containing nucleic acid obtained in Step 2 twice with protein washing buffer WB1 to remove protein impurities; then wash the solid-phase support three times with nucleic acid washing buffer WB2 to remove salt ions and organic reagent residues. When using the adsorption column method, centrifuge the adsorption column at 14000 rpm for 1 min and discard the filtrate; add 750 μL of protein washing buffer WB1, centrifuge at 14000 rpm for 1 min, and discard the filtrate; repeat once; add 750 μL of nucleic acid washing buffer WB2, centrifuge at 14000 rpm for 1 min, and discard the filtrate; repeat twice. When using the magnetic bead method, place the solid-phase support on a magnetic rack and let it stand for 5 min until the solution is clear, then discard the supernatant; add 750 μL of protein washing buffer WB1, resuspend the magnetic beads, place it on a magnetic rack and let it stand, then discard the supernatant; repeat once; add 750 μL of protein washing buffer WB1, resuspend the magnetic beads, place it on a magnetic rack and let it stand, then discard the supernatant; repeat once. μL of nucleic acid washing buffer WB2 was used to resuspend the magnetic beads and then placed on a magnetic rack to stand. The supernatant was then discarded. This process was repeated twice.

[0087] Step 4, Nucleic Acid Elution: Add nucleic acid elution buffer EB to the solid support, incubate at room temperature, centrifuge or magnetically separate, and collect the eluent to obtain nucleic acid; when using the adsorption column method: add 200 μL of nucleic acid elution buffer EB to the adsorption column, incubate at room temperature for 5 min, centrifuge at 14000 rpm for 1 min, and collect the eluent; when using the magnetic bead method: add 200 μL of nucleic acid elution buffer EB to the magnetic beads, incubate at room temperature for 5 min, place on a magnetic rack and let stand, and collect the eluent.

[0088] To further illustrate the effectiveness of the extraction reagent of the present invention, the following experiments were conducted: Experimental Example 1 The nucleic acid co-extraction reaction reagents disclosed in this experimental example include lysis binding buffer LS, polysaccharide and polyphenol removal buffer PD1, nucleic acid binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB.

[0089] The reagents of this invention shall be prepared in the following manner: LS lysis binder: Tris-HCl 30mM, Triton X-100 1%, NP40 1%, Guanidine isothiocyanate 2.0M, Urea 2M, Sodium lauroyl sarcosinate 2%, SDS 0.1%, CTAB 2%, PVP 1.5%, PEG6000 4%, Acetylcysteine ​​20mM, TCEP 5mM, Trehalose 0.2M, Betaine 0.5M.

[0090] Polysaccharide and polyphenol removal solution PD1: Sodium acetate-citric acid buffer 50mM (pH=5.5), 4M guanidine hydrochloride, 2% PVP, 2% PVPP, 10mM aluminum ammonium sulfate.

[0091] Nucleic acid binding solution LB includes

[0092] 80% anhydrous ethanol, 5% PEG6000, 15% nuclease-free water.

[0093] Protein Wash Buffer WB1 includes

[0094] Tris-HCl 30mM (pH=8.0), 2.5M guanidine hydrochloride, 0.5% Triton 100, 0.5M sodium chloride, 50% anhydrous ethanol.

[0095] Nucleic acid washing solution WB2 includes

[0096] 10 mM Tris-HCl (pH=8.0), 0.05 M sodium chloride, 75% anhydrous ethanol

[0097] Nucleic acid elution buffer EB

[0098] Nuclease-free water or TE buffer.

[0099] This experimental example discloses the specific co-extraction method: Step 1 Method 1: Take an appropriate amount of plant tissue (leaves, roots, stems, seeds, etc.), grind it into a fine powder in liquid nitrogen, and then add the powder to a 1.5 mL centrifuge tube while frozen. Add 500 μL of lysis buffer (LS) and incubate at room temperature or 65 °C for 10 min.

[0100] Method 2: Take 600uL of lysis buffer LS and put it into a grinding tube. Then cut the tissue sample into small pieces (total amount not exceeding 50mg) with scissors and put them into the grinding tube. Incubate at 95℃ for 10-15min. After incubation, immediately add 300uL of polysaccharide and polyphenol removal solution PD1. Then, vortex at the maximum speed for 3-4min on a vortex mixer to completely homogenize the tissue.

[0101] Step Two Add 250 μL (50% of the lysis buffer volume) of polysaccharide and polyphenol removal solution PD1 (if step one is the same as method two for sample lysis, then PD1 is not needed). Vortex mix for 1 min and place at room temperature or 4°C for 2 min.

[0102] Step 3 If using a nucleic acid adsorption column: Centrifuge at 12000-14000 rpm for 2 minutes, then carefully transfer approximately 550 μL of the supernatant to a clean centrifuge tube. Add 250 μL of nucleic acid binding buffer (LB) and mix by inverting the tube several times. Then transfer the entire solution to the nucleic acid adsorption column for nucleic acid binding. If using magnetic beads, centrifuge at 12000rpm~14000rpm for 2 minutes on a high-speed centrifuge, or at the maximum speed on a desktop handheld centrifuge for 5-10 minutes. Transfer 550uL of supernatant to a clean centrifuge tube, add 30uL of nucleic acid adsorption magnetic beads, invert to mix, and incubate at room temperature for 5-10 minutes, inverting to mix several times every 2-3 minutes.

[0103] Step 4 If a nucleic acid adsorption column is used, then: centrifuge the adsorption column loaded with solution in step 3-A at 12000-14000 rpm for 1 min on a high-speed centrifuge. If using magnetic beads, transfer the centrifuge tube containing the solution with magnetic beads to a magnetic rack and let it stand for 1-5 minutes until the solution becomes clear. Then, remove all the liquid.

[0104] Step 5 If using a nucleic acid adsorption column: add 600 μL of protein washing buffer WB1 to the adsorption column and centrifuge at 12,000 rpm for 1 min; if using magnetic beads: add 700 μL of protein washing buffer WB1 to the magnetic bead centrifuge tube, resuspend the magnetic beads by vortexing or pipetting, then place them back on the magnetic rack and let them stand for a few minutes, then discard the solution to remove protein residue.

[0105] Furthermore, for some protein-rich samples, repeat the previous step once.

[0106] Step Six If a nucleic acid adsorption column is used, add 600 μL of nucleic acid washing buffer WB2 to the adsorption column and centrifuge at 12,000 rpm for 1 min. If using magnetic beads, add 700 μL of nucleic acid washing buffer WB2 to the magnetic bead centrifuge tube, resuspend the magnetic beads by vortexing or pipetting, then place it back on the magnetic rack and let it stand for a few minutes. Discard the solution to remove protein ions or organic reagents, etc.

[0107] Step 7: Repeat step 6.

[0108] Step 8 If using a nucleic acid adsorption column, discard the collection tube, transfer the nucleic acid adsorption column to a new 1.5 mL centrifuge tube, and leave the cap of the adsorption column open for 2-3 minutes to allow any residual ethanol to evaporate.

[0109] If using magnetic beads: Hold the centrifuge tube on the magnetic rack, use a 10µL pipette tip to remove any remaining liquid from the bottom of the centrifuge tube, then keep the cap open and let the magnetic beads air dry for 3-5 minutes until there is no reflection on the surface of the magnetic beads.

[0110] Step Nine If a nucleic acid adsorption column is used, add 50-100 μL of preheated (65°C) nuclease-free water or TE solution to the nucleic acid adsorption column, let it stand at room temperature for 2 minutes, and then centrifuge at 12000 rpm for 1 minute to collect the nucleic acid solution.

[0111] If using magnetic beads, remove the centrifuge tube from the magnetic rack, add 50-100 μL of nuclease-free water or TE solution, vortex for 1 min to resuspend the magnetic beads, incubate at room temperature for 5 min, or incubate at 60°C for 3-5 min to completely elute the nucleic acid from the magnetic beads, and finally place the centrifuge tube on the magnetic rack for a few minutes to collect the nucleic acid eluent.

[0112] Nucleic acid solutions can be stored at 4°C for a short period of time, or at -20°C for one year. For longer storage, the nucleic acid solutions can be stored at -80°C.

[0113] Experimental Example 2

[0114] Using the extraction reagent from Example 1, nucleic acid was extracted from sweet potato leaves, green rice straw, fresh corn kernels, and fir needles, respectively. Approximately 30 mg of each sample was taken, thoroughly ground with liquid nitrogen, and then nucleic acid extraction was performed following the procedures in Example 1. Finally, 100 μL of TE solution was used for elution.

[0115] The extraction results are shown in Table 1 below: Table 1

[0116] 2.5 μL was taken for agarose gel electrophoresis identification, and the results are shown above. Figure 1 .

[0117] From Table 1 and Figure 1 It can be seen that the extraction reagent in this experiment can effectively extract complete DNA from different plant varieties, with good purity, yield and integrity. The reagent of this invention can be applied to different plants and different tissues.

[0118] Experimental Example 3

[0119] To demonstrate that the extraction reagent of this invention is compatible with different operating methods and different nucleic acid adsorption and purification schemes, this experimental example was implemented.

[0120] Using the extraction reagent from Experiment Example 1, and following the operating steps of Experiment Example 1, nucleic acid extraction was performed on sweet potato leaves, green rice stalks, and fresh corn kernels using liquid nitrogen grinding, nucleic acid adsorption column and high-temperature heating, vortexing, and magnetic bead adsorption methods, respectively.

[0121] The extraction concentration and purity results are shown in Table 2 below. Figure 2

[0122] Table 2

[0123] It is evident that the reagent of this invention is compatible with different operating procedures under different experimental conditions, and the extraction effect is comparable.

[0124] Experiment Example 4

[0125] In addition to extracting the plant's own genomic DNA, this invention can simultaneously and efficiently extract epiphytic or infecting fungi, bacteria, DNA viruses, and RNA viruses. To illustrate this function, this experimental example was conducted.

[0126] Nucleic acid co-extraction was performed on leaf samples from sweet potato fields that showed signs of root rot, bacterial wilt, yellowing and curling of leaves, and spot-like chlorosis (caused by fungus Fusarium, bacteria Ralstonia solanacearum, DNA virus SPLCV, and RNA virus SPFMV, respectively).

[0127] Using the reagents from Experiment 1, nucleic acid co-extraction was performed using the high-temperature incubation and adsorption column extraction method described in Experiment 1, and finally, nucleic acid was eluted with nuclease-free water.

[0128] The eluted nucleic acids were subjected to multiplex detection using commercially available mature RT-qPCR. The primer and probe sequence results are shown in Table 3. It can be seen that this invention, in addition to extracting the plant's own genomic DNA, can simultaneously and efficiently extract epiphytic or infecting fungi, bacteria, DNA viruses, RNA viruses, etc.

[0129] Table 3

[0130] Experimental Example 5

[0131] To demonstrate that the extraction reagent of this invention is also applicable to downstream applications of rapid detection technologies such as LAMP, this experimental example was conducted.

[0132] Using the nucleic acid extracted in Experiment 4 as a sample, a commercially available pH indicator-based RT-LAMP assay was used to rapidly detect four pathogens: Fusarium oxysporum, Ralstonia solanacearum, SpLCV, and SPFMV. The results are shown in Table 4 below. Figure 3 Therefore, the extraction reagent of this invention can also be applied to downstream applications of rapid detection technologies such as LAMP.

[0133] Table 4

[0134] Experimental Example 6

[0135] To illustrate that this invention can also be applied to research on plant intervention operations, this experimental example was conducted.

[0136] In a cornfield, select a region of corn seedlings infected with gray mold and spray fungicide every 5 days. Then, take samples of the same part of the corn seedlings on days 0, 5, 10, 15 and 20 after spraying. Take 5 plants for each sample and mix them for homogenization to eliminate random factors.

[0137] Nucleic acid co-extraction was performed using the reagents from Experiment Example 1 and following the procedure outlined in Experiment Example 1.

[0138] qPCR was used to detect Botrytis cinerea, and the relative magnitude of the final Ct value was used to determine the fungicidal ability of the sprayed fungicide against Botrytis cinerea, in order to track and monitor the effect of the fungicide on Botrytis cinerea and the status of resistance. The experimental results are shown in Table 5 below.

[0139] Table 5

[0140] The results show that the extraction reagent of this invention can simultaneously and efficiently extract the target pathogens from crops. The detected Ct value is consistent with the actual pesticide spraying pattern, indicating that it can efficiently enrich the pathogen nucleic acid in epiphytic or infecting plant tissues.

[0141] Experimental Example 7

[0142] To demonstrate that the present invention can also be applied to the rapid detection of transgenic target sequences, this experimental example was conducted.

[0143] Sweet potato species were transfected using Agrobacterium-mediated transformation, integrating two palindromic sequences of the SPLCV virus into the sweet potato genome. The palindromic sequences were linked by a linker sequence to achieve conditional resistance to the SPLCV virus in sweet potatoes. Seven leaves from the F1 generation of transgenic plants were randomly selected. Nucleic acid extraction was performed using the extraction reagent from Example 1, following the steps outlined in Example 1.

[0144] LAMP primers were designed using the linker sequence, and rapid isothermal detection of transgenic target sequences was performed using a commercially available pH-indicating LAMP detection reagent.

[0145] The experimental results are shown in Figure 4 .Depend on Figure 4 It can be seen that among the seven transgenic F1 samples, the target gene was successfully detected in 5 samples, while the color remained unchanged in 2 samples, indicating that the transgene transfer in these two samples failed.

[0146] It is understood that the present invention can be applied to plant genome-related research applications such as transgenic target gene detection.

[0147] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An extraction reagent capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid, characterized in that, It consists of the following components (a) to (f), which are stored independently: sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer PD1, nucleic acid binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB; (a) Sample lysis binding buffer LS, which contains: The first pH buffer component is selected from one or more combinations of Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid, and the concentration of the first pH buffer component is 10 mM to 100 mM, and the pH is 7.5 to 8.

8. Nonionic surfactant component: selected from one or more of Triton X-100, NP40, and Tween 20, wherein the volume ratio of the nonionic surfactant component is 0.1% to 10%; Cell wall and cell lysis components: selected from at least one of guanidine isothiocyanate and guanidine hydrochloride, and a combination of one or more of urea, sodium lauroyl sarcosinate and SDS; Polysaccharide and polyphenol scavenging components: containing CTAB 0.5%~3% (w / v), PVP 0.5%~3% (w / v), PVPP 0.1%~2% (w / v), and PEG6000 1%~5% (w / v); Antioxidant components: Contains acetylcysteine ​​5 mM~50 mM, DTT 5 mM~20 mM, TCEP 5 mM~20 mM, and β-mercaptoethanol 0.1%~2% (v / v); Nucleic acid protective agent components: containing trehalose 0.1 M~0.3 M and betaine 0.1 M~1 M; (b) Polysaccharide and polyphenol removal solution PD1, which contains: The second pH buffer component is selected from one or more combinations of Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid, and the concentration of the second pH buffer component is 10 mM to 200 mM, and the pH is 5.5 to 7.

0. The liquid salt components are guanidine isothiocyanate and / or guanidine hydrochloride, with a concentration of 1 M to 4 M. Polysaccharide-polyphenol coprecipitation components: containing PVP 0.5%~3% (w / v), PVPP 0.1%~2% (w / v), and aluminum ammonium sulfate 1 mM~10 mM; (c) Nucleic acid binding solution LB, which is selected from one or more combinations of anhydrous ethanol, isopropanol, PEG6000, PEG8000, NaCl, KCl, potassium acetate, and sodium acetate; (d) Protein washing solution WB1, which contains Tris-HCl, benzenesulfonic acid and / or sodium citrate and / or citric acid, guanidine hydrochloride and / or guanidine isothiocyanate and / or guanidine thiocyanate and / or urea, Triton 100 and / or Tween 20 and / or NP40, isopropanol and / or anhydrous ethanol. (e) Nucleic acid washing solution WB2, which contains Tris-HCl, NaCl, and anhydrous ethanol; (f) Nucleic acid elution buffer EB, which is nuclease-free water or TE buffer; The mass ratio of sample lysis binding buffer LS, polysaccharide and polyphenol removal buffer PD1, nucleic acid binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB is 0.8~1:0.2~0.6:0.2~0.6:1~4:1~4:0.1~0.

3.

2. The extraction reagent according to claim 1, capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid, is characterized in that, The concentrations of guanidine isothiocyanate or guanidine hydrochloride in the cell wall and cell lysis components of the sample lysis binding solution LS are 1 M to 4 M, urea is 2 M to 5 M, sodium lauroyl sarcosinate is 1% to 4%, and SDS is 0.1% to 2%.

3. The extraction reagent according to claim 2, capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid, is characterized in that, In the nucleic acid binding solution LB, the concentrations of anhydrous ethanol are 30%–100% (v / v), isopropanol is 30%–100% (v / v), PEG6000 and PEG8000 are 1%–10%, NaCl is 0.5 M–3 M, KCl is 0.5 M–3 M, potassium acetate is 0.5 M–3 M, and sodium acetate is 0.5 M–3 M.

4. The extraction reagent according to claim 3, capable of simultaneously extracting plant pathogen DNA & RNA and host nucleic acid, is characterized in that, In the protein washing solution WB1, the concentration of Tris-HCl is 10mM~100mM; the concentrations of benzenesulfonic acid, sodium citrate, and citric acid are all 10mM~50mM; the concentrations of guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, and urea are all 1M~5M; the concentrations of Triton 100, Tween 20, and NP40 are all 0.5%~5%; and the concentrations of isopropanol and anhydrous ethanol are all 20%~60%. The nucleic acid washing solution WB2 contains Tris-HCl at a concentration of 1 mM to 20 mM, sodium chloride at a concentration of 10 mM to 100 mM, and anhydrous ethanol at a concentration of 50% to 85%.

5. A kit for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid, characterized in that, The extractant comprises the extraction reagent for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid as described in any one of claims 1 to 4; nucleic acid adsorption magnetic beads, silicon-based nucleic acid adsorption column and grinding tube.

6. A method for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid, characterized in that, The extraction reagent according to any one of claims 1 to 4 is used, comprising the following steps: Step 1: Sample lysis After the plant tissue sample was broken, it was added to the sample lysis binding buffer LS for lysis, and then the polysaccharide and polyphenol removal buffer PD1 was added. The mixture was vortexed and placed at room temperature or 3~6℃ for 1~3 min to obtain the first mixture. Step 2, Nucleic acid binding: Centrifuge the first mixture, take the supernatant, add 0.5 to 1 times the volume of the supernatant of nucleic acid binding solution LB, mix well, and then use the adsorption column method or magnetic bead method to bind the nucleic acid to the solid support. Step 3: Nucleic acid washing and purification: Wash the nucleic acid-bound solid support obtained in step two with protein washing buffer WB1 1-2 times to remove protein impurities; The solid support is then washed 1-3 times with nucleic acid washing buffer WB2 to remove salt ions and organic reagent residues. Step 4: Nucleic acid elution: Add nucleic acid elution buffer EB to the solid support, incubate at room temperature, then centrifuge or magnetically separate, collect the elution buffer to obtain nucleic acid.

7. The method for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid according to claim 6, characterized in that, In step one, if liquid nitrogen is available, the specific steps of breaking the plant tissue sample and adding the sample lysis binding solution LS for lysis include: grinding the plant tissue into powder in liquid nitrogen, transferring it to a centrifuge tube under frozen conditions, adding the sample lysis binding solution LS, and then letting it stand at room temperature or 65°C for 10 minutes to complete the lysis. After that, adding the polysaccharide and polyphenol co-precipitant, vortexing and mixing for 1 minute, and then placing it at room temperature for 5-6 minutes or in a 4°C refrigerator for 1-3 minutes. If liquid nitrogen is not available, the specific steps for lysing the plant tissue sample by breaking it up and adding the sample lysis binding solution LS include: cutting the plant tissue into small pieces, taking an appropriate amount and adding it to a grinding tube, adding the sample lysis binding solution LS, and heating at 90~96℃ for 10-20 minutes; after heating, adding a polysaccharide and polyphenol co-precipitant in time, and vortexing for 3~4 minutes on a vortex mixer to completely homogenize the tissue.

8. The method for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid according to claim 7, characterized in that, In step two, the adsorption column method or magnetic bead method for binding nucleic acids to the solid-phase support includes: When using the adsorption column method, the first mixture is transferred to the nucleic acid adsorption column; When using the magnetic bead method, add 30 μL to 40 μL of nucleic acid adsorption magnetic beads to the first mixture, mix well, and let stand at room temperature for 5-10 min, mixing once every 2-3 minutes.

9. The method for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid according to claim 7, characterized in that, In step three, washing the nucleic acid-bound solid support obtained in step two with protein washing solution WB1 1-2 times to remove protein impurities includes: When using the adsorption column method, centrifuge the adsorption column at 12000 rpm to 14000 rpm for 1 to 2 min and discard the filtrate. Add 400 μL to 750 μL of protein washing buffer WB1, centrifuge at 12000 rpm to 14000 rpm for 1 to 2 min, and discard the filtrate; optionally repeat once. Add 400 μL to 750 μL of nucleic acid washing buffer WB2, centrifuge at 12000 rpm to 14000 rpm for 1 to 2 min, discard the filtrate; repeat 1 to 2 times. When using the magnetic bead method Place the solid support on a magnetic rack and let it stand for 1-5 minutes. After the solution becomes clear, remove the supernatant. Add 400 μL to 750 μL of protein washing buffer WB1, resuspend the magnetic beads, place them on a magnetic rack and let stand, then discard the supernatant; repeat once if desired. Add 400 μL~750 μL of nucleic acid washing buffer WB2, resuspend the magnetic beads, place them on a magnetic rack and let them stand, then discard the supernatant; repeat 1-2 times.

10. The method for simultaneously extracting plant pathogen DNA & RNA and host nucleic acid according to claim 7, characterized in that, In step four, the addition of nucleic acid elution buffer EB to the solid support, followed by centrifugation or magnetic separation at room temperature, and collection of the elution buffer specifically includes: When using the adsorption column method: Add 30 μL to 200 μL of nucleic acid elution buffer EB to the adsorption column, incubate at room temperature for 3-5 min, centrifuge at 12000 rpm to 14000 rpm for 1 min, and collect the elution buffer; When using the magnetic bead method: add 30 μL to 200 μL of nucleic acid elution buffer EB to the magnetic beads, let stand at room temperature for 3-5 min, place on a magnetic rack and let stand, and collect the elution buffer.

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