A reagent combination, a reagent kit and a method for extracting microbial genomic DNA in soil

Abstract

This invention relates to a reagent combination, extraction kit, and extraction method for extracting microbial genomic DNA from soil, belonging to the field of biotechnology. The reagent combination for extracting microbial genomic DNA from soil of this invention includes individually packaged grinding beads I, grinding beads II, lysis buffer, inhibitor removal, binding buffer, magnetic beads, rinsing solution I, rinsing solution II, and elution buffer. In this invention, soil samples are ground and heated after adding grinding beads of two particle sizes and lysis buffer, which effectively lyses and releases nucleic acids from soil microorganisms. The inhibitor removal agent in this invention reduces the interaction between impurities in the sample and nucleic acids, and the flocculant in the inhibitor removal agent effectively settles impurities such as humic acid in the soil. The binding buffer in this invention promotes the binding of nucleic acid molecules to magnetic beads. The two rinsing solutions in this invention effectively remove impurities such as proteins and some small molecule compounds during the extraction process.

Description

Technical Field

[0001] This invention relates to a reagent combination, extraction kit, and extraction method for extracting microbial genomic DNA from soil, belonging to the field of biotechnology. Background Technology

[0002] Soil ecosystems are the most important components of the Earth's biosphere, and soil microorganisms are an important part of soil ecosystems. The richness and diversity of microorganisms are of great significance to the protection of the soil environment. For example, some microorganisms can improve the liquefaction resistance of sandy soil and help to stabilize sandy soil. In addition, the presence of certain beneficial microorganisms in the soil can help improve soil fertility and promote plant growth.

[0003] Therefore, identifying soil microbial species and exploring the interactions between microorganisms and soil is of great significance for sustainable ecological development and environmental protection. Identifying soil microbial species, especially bacteria, requires sequencing the soil microorganisms. A common method is sequencing the 16S amplicon of bacteria. Before performing 16S amplicon sequencing, nucleic acid extraction from the soil microorganisms is necessary. The ability to obtain high-quality genomic DNA has a significant impact on identifying soil microbial species and determining whether ecological restoration is required.

[0004] Currently, the main method for extracting genomic DNA from soil microorganisms involves mechanically disrupting cells to lyse the microorganisms in the soil before extracting genomic DNA. However, this method cannot completely lyse Gram-positive bacteria, and the extracted nucleic acids contain inhibitors such as humic acid, resulting in low nucleic acid purity and easily inhibiting downstream PCR detection, library construction, and sequencing.

[0005] Chinese patent CN200510120584.7 discloses a method for rapid extraction of small amounts of total DNA from soil. Using soil as the material, it employs a combination of quartz sand, silica powder, agitation, and cetyltrimethylammonium bromide (CTAB) to lyse cells and release DNA. This patent uses grinding of quartz sand and silica, which has low grinding efficiency, and both quartz sand and silica require sterilization and drying before grinding, which is cumbersome. Furthermore, CTAB cannot fully lyse cells in the soil.

[0006] Chinese patent CN201010536640.6 discloses a method for rapidly extracting total DNA from soil. The method provided by this invention comprises the following steps: (1) lysing a soil sample using the SDS-lysozyme method to obtain a lysate; (2) precipitating the DNA in the lysate and collecting the precipitate; (3) dissolving the precipitate and performing agarose gel electrophoresis to recover and purify the DNA bands on the agarose gel, thereby obtaining total soil DNA. In this patent, the phrase "lysing a soil sample using the SDS-lysozyme method" implies that lysozyme may break down the gDNA of some Gram-negative bacteria in the soil into small DNA fragments. Furthermore, the pretreatment of the soil sample is minimal, lacking operations such as grinding and heating, and therefore cannot fully lyse Gram-negative and Gram-positive bacteria, fungi, etc., in the soil.

[0007] Chinese patent CN 201010548527.X provides a method for effectively extracting genomic DNA and total RNA from crop rhizosphere soil microorganisms. The method includes first removing impurities such as humic substances that severely interfere with nucleic acid extraction from soil samples using aluminum sulfate; then breaking down soil microbial cells with glass beads; followed by adding SDS and LiCl solutions to lyse cells and release free nucleic acids; extraction with a mixture of phenol, chloroform, and isoamyl alcohol; and finally, precipitation of the extract with isopropanol and sodium acetate to obtain soil microbial metagenomic DNA and total RNA. However, this patent uses aluminum sulfate to remove inhibitors such as humic acid from the soil. Experiments have shown that aluminum ions are unstable in solution and easily precipitate. Furthermore, the sample pretreatment in this patent is insufficient.

[0008] Chinese patent CN201811285548.X discloses a highly efficient and economical method for extracting soil microbial DNA. The method includes the following steps: Step 1: Obtaining a microbial suspension; weighing 1 gram of soil, adding 2 ml of suspension buffer C1, adding 1 gram of glass beads with a particle size of 0.09-0.12 mm, adding 500 μL of humic acid adsorbent buffer C2, adding 10 μL of proteinase K (10 mg / mL), mixing and shaking; Step 2: Obtaining microbial DNA; Step 3: Obtaining crude DNA; placing the substance obtained in the previous step in a 60°C water bath while stirring, then centrifuging at high speed at 4°C, collecting the first supernatant; Step 4: Obtaining purified DNA. This patent uses only one particle size of grinding beads for grinding microorganisms in the soil, resulting in insufficient grinding. Furthermore, it uses a column-based method for nucleic acid extraction and employs toxic substances such as chloroform.

[0009] In summary, existing technologies have the following problems: incomplete lysis, with Gram-positive bacteria not being completely lysed; the addition of lysozyme to the lysis buffer in some experiments can easily break down microbial gDNA, preventing the acquisition of complete microbial genomic DNA; and the removal of inhibitors such as humic acid in soil using aluminum ions for precipitation in other patents, but the unstable nature of aluminum ions in liquids can lead to the precipitation of aluminum hydroxide or other aluminum salts during the placement of the inhibitor removal agent, affecting the removal of humic acid and ultimately impacting the concentration and purity of the extracted nucleic acid.

[0010] Based on the above-mentioned existing technologies, there is a need to provide a new combination of reagents, an extraction kit, and an extraction method for extracting microbial genomic DNA from soil. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a combination of reagents, an extraction kit, and an extraction method for extracting microbial genomic DNA from soil. The extraction method of this invention yields nucleic acid with high concentration and good purity. Furthermore, this method can effectively reduce the content of inhibitors such as humic acid in nucleic acids, which is beneficial for subsequent 16S amplicon sequencing of soil microorganisms.

[0012] This invention incorporates the following improvements: In sample pretreatment, two types of grinding beads with different particle sizes, an optimized lysis buffer formulation, and heating are used together to lyse the sample, which can fully lyse microorganisms in the soil, including Gram bacteria and fungi, without interrupting gDNA (no lysozyme is added). Fe and Ca ions are used to remove inhibitors, which are more stable than Al ions and improve nucleic acid yield.

[0013] The technical solution to the technical problem in this invention is as follows:

[0014] In a first aspect of the invention, a reagent combination for extracting microbial genomic DNA from soil is provided.

[0015] The reagent combination includes individually packaged grinding beads one, grinding beads two, lysis buffer, inhibitor removal buffer, binding buffer, magnetic beads, rinsing buffer one, rinsing buffer two, and elution buffer:

[0016] in,

[0017] The grinding beads are zirconia beads with a diameter of 0.1 mm. Preferably, the grinding beads are 0.1 mm zirconia beads from BioSpec.

[0018] The second grinding bead is a glass bead with a diameter of 0.5 mm. Preferably, the grinding bead is a 0.5 mm glass bead from BioSpec.

[0019] The lysis buffer comprises 0.01-0.2M Tris-HCl, 0.01-0.2M EDTA, 0.05-0.5M NaCl, 0.05-0.5M KCl, and 1-10M NaSCN, with a pH of 7.5-9.5. Preferably, the lysis buffer comprises 0.1M Tris-HCl, 0.15M EDTA, 0.25M NaCl, 0.1M KCl, and 4M NaSCN, with a pH of 8.5.

[0020] The deinhibitor component comprises: 0.05-0.5M CaCl2, 0.05-0.5M FeCl3, 10-30% glacial acetic acid, 1-5% PVP, 0.01-0.5M Tris-HCl, and 0.05%-2% sodium polypropylene sulfonate, with a pH value of 5.0-7.0. Preferably, the deinhibitor component comprises: 0.05M CaCl2, 0.25M FeCl3, 12% glacial acetic acid, 0.2M Tris-HCl, 2% PVP, and 2% sodium polypropylene sulfonate, with a pH value of 6.0.

[0021] The binding solution comprises: 0.05-0.2M Tris-HCl, 0.1-10M guanidine isothiocyanate, 2-20mM EDTA, and 20-40% isopropanol. Preferably, the binding solution comprises: 0.1M Tris-HCl, 6M guanidine isothiocyanate, 0.01M EDTA, and 30% isopropanol, and the pH of the binding solution is 7.5.

[0022] The magnetic beads are: 70-80% by volume superparamagnetic microspheres, with a particle size between 15-45 nm, and the pH of the magnetic bead solution is 5.0-6.0. Preferably, the magnetic bead composition is a magnetic bead solution containing 70% by volume superparamagnetic microspheres and 30% by volume nuclease-free water, with a pH of 5.4; the magnetic bead solution is Promega magnetic beads.

[0023] The first rinsing solution comprises: 0.1-0.5M Tris-HCl, 0.1-2M sodium chloride, 0.5-10M guanidine hydrochloride, 0.01-0.1M EDTA, and 30-70% anhydrous ethanol, with a pH of 6.0-8. Preferably, the first rinsing solution comprises: 0.15M Tris-HCl, 0.8M NaCl, 4M guanidine hydrochloride, 0.01M EDTA, and 60% anhydrous ethanol, with a pH of 8.0.

[0024] The second rinsing solution comprises 0.1-0.5M Tris-HCl and 75%-90% anhydrous ethanol, and has a pH value of 6.0-8.0. Preferably, the second rinsing solution comprises 0.16M Tris-HCl and 80% anhydrous ethanol, and has a pH value of 8.0.

[0025] The eluent comprises enzyme-free water, and the pH value of the eluent is 6.0-8.0. Preferably, the eluent is enzyme-free water, and the pH value of the eluent is 7.0.

[0026] In this invention, all percentages (%) refer to weight / volume percentages (w / v%), expressed in g / mL.

[0027] In this invention, the functions of grinding bead one, grinding bead two, lysis buffer, inhibitor removal buffer, binding buffer, magnetic beads, rinsing solution one, rinsing solution two, and elution solution are as follows:

[0028] The function of grinding beads one and grinding beads two: The two types of grinding beads work together to mechanically break down (physically pyrolyze) the materials in the soil. The two sizes of grinding beads (grinding beads one and grinding beads two) can grind more thoroughly and pyrolyze more thoroughly.

[0029] The function of lysis buffer: Lysis buffer is used to (chemically) lyse microbial cells, releasing nucleic acids.

[0030] The role of deinhibitors: After cell lysis in microorganisms, not only nucleic acids are released, but also some metabolites (which can inhibit PCR amplification). Deinhibitors bind with these metabolites (such as humic acid) to form flocculent substances, which can then be removed by centrifugation.

[0031] The function of the binding solution is to promote the binding of nucleic acids released by microbial cells to magnetic beads.

[0032] The function of magnetic beads: to bind with nucleic acids and then settle and separate them.

[0033] The purpose of rinsing solution one and rinsing solution two is to remove some salt ions (from lysis buffer, binding buffer, etc.) from the surface of nucleic acids bound to the magnetic beads.

[0034] The function of the elution buffer is to separate magnetic beads and nucleic acids, with the nucleic acids ultimately dissolving in the elution buffer.

[0035] In a second aspect of the invention, a kit comprising a reagent combination as described in the first aspect is provided.

[0036] In a third aspect of the invention, a method for extracting soil microbial genomic DNA is provided using a reagent combination as described in the first aspect or a kit as described in the second aspect.

[0037] The extraction method includes the following steps: a certain mass of soil is weighed, and grinding beads of different particle sizes (bead 1 and bead 2) and lysis buffer are added. After mechanical grinding, heat treatment, and lysis buffer treatment, the microorganisms in the soil are completely lysed, releasing gDNA. Then, the supernatant 1 is collected by centrifugation, an inhibitor is added, and after vortexing, the supernatant 2 is collected by centrifugation. Binding buffer and magnetic beads are added to the supernatant 2 for incubation, so that the nucleic acid binds to the magnetic beads, and the magnetic beads are precipitated. The magnetic bead precipitate is washed twice by rinsing buffer 1 and rinsing buffer 2, leaving the magnetic bead precipitate. The magnetic bead precipitate is eluted with elution buffer to obtain the genomic DNA of microorganisms in the soil.

[0038] The soil can be any type of soil, such as red soil, loess, black soil, sandy soil, etc.

[0039] Preferably, the method is as follows:

[0040] 1) Weigh approximately 0.2g of soil sample into a clean 2mL screw-capped grinding tube, and add approximately 0.1g of grinding bead 1, approximately 0.1g of grinding bead 2, and 700uL of lysis buffer to the tube. Place the grinding tube in a grinder and grind for 5 minutes at 1600rpm. After grinding, place the grinding tube in a constant temperature mixer at 1000rpm and 75℃ for 5 minutes. After processing, centrifuge the grinding tube under the following conditions: 12000rpm for 2 minutes.

[0041] 2) Transfer the supernatant 1 after centrifugation, and add the inhibitor removal agent at a volume ratio of 3:1 (supernatant 1 to inhibitor removal agent); the steps include: taking about 600 uL of supernatant 1, adding 200 uL of inhibitor removal agent to a clean 2 mL EP tube, vortexing briefly, centrifuging at 12000 rpm for 2 min, and retaining supernatant 2, about 450 uL;

[0042] 3) Take supernatant 2 and add it to a clean 2 mL centrifuge tube containing an equal volume of binding solution, and add about 400 μg of magnetic beads; in this invention, the concentration of the magnetic bead suspension is 20 mg / mL, that is, add 20 μL of magnetic bead suspension; incubate the binding solution, magnetic beads and supernatant 2 at room temperature for 10 min to allow the nucleic acid to bind to the magnetic beads;

[0043] 4) The volume ratio of the first rinsing solution and the second rinsing solution is 1:1. The number of rinsing cycles for both the first rinsing solution and the second rinsing solution is 2. The first rinsing is performed twice with the first rinsing solution, and the second rinsing is performed twice with the second rinsing solution. The volume ratio of the rinsing solution for the four rinsing cycles is 1:1:1:1. The volume of the rinsing solution is 700uL.

[0044] 5) The eluent is preheated before use at 56°C for 5 min; the elution operation includes resuspending the magnetic beads in the eluent, then mixing them at 56°C, 1200 rpm for 5 min on a constant temperature mixer to remove the magnetic beads and obtain a soil microbial genomic DNA solution.

[0045] In a fourth aspect, a method for detecting soil microorganisms is also provided.

[0046] The detection method first involves extracting soil microbial genomic DNA using the reagent combination described in the first aspect, the kit described in the second aspect, or the extraction method described in the third aspect, and then performing PCR detection using specific primers and / or probes for the target gene.

[0047] Preferably, the PCR detection method is real-time quantitative PCR detection.

[0048] Preferably, the target gene is the 16S gene.

[0049] Preferably, the specific primers for the 16S gene include:

[0050] F: CCTACGGGAGGCAGCAG

[0051] R: ATTACCGCGGCTGCTGG.

[0052] The present invention has the following technical effects:

[0053] This invention utilizes a grinding and heating process involving the addition of two different particle sizes of grinding beads and a lysis buffer to soil samples. This process effectively lyses and releases nucleic acids from soil microorganisms. The inhibitor in this invention contains a blocking agent that reduces the interaction between impurities and nucleic acids in the sample, while the flocculant effectively settles impurities such as humic acid in the soil. The binding solution in this invention promotes the binding of nucleic acid molecules to magnetic beads. The two rinsing solutions in this invention effectively remove impurities such as proteins and some small molecule compounds from the extraction process. Experiments show that the soil microbial genomic DNA extracted by this method has high concentration, purity, and integrity. Attached Figure Description

[0054] Figure 1 The images shown are gel images of soil microbial genomic DNA from Example 2 and Comparative Example 5.

[0055] Figure 2 A diagram for quantitative real-time amplification of the bacterial 16S gene. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0057] This invention provides a soil microbial genomic DNA extraction reagent and its application. Those skilled in the art can refer to the content of this article and appropriately modify the process parameters to achieve the desired results.

[0058] The reagents and materials used in this invention are all commercially available products. The invention is further illustrated below with reference to specific examples:

[0059] Example 1

[0060] In this embodiment, the reagent combination for extracting microbial genomic DNA from soil includes the following individually packaged reagent components:

[0061] 1) Grinding bead one, which is a 0.1mm diameter zirconia bead from BioSpec.

[0062] 2) Grinding bead two, which is a BioSpec glass bead with a diameter of 0.5mm.

[0063] 3) The lysis buffer consists of 0.1M Tris-HCl, 0.15M EDTA, 0.25M NaCl, 0.1M KCl and 4M NaSCN, with a pH of 8.5.

[0064] 4) Deinhibitor removal, with the following components: 0.05M CaCl2, 0.25M FeCl3, 12% glacial acetic acid, 0.2M Tris-HCl, 2% PVP and 2% sodium polypropylene sulfonate. The pH value for deinhibitor removal is 6.0.

[0065] 5) The binding solution consists of 0.1M Tris-HCl, 6M guanidine isothiocyanate, 0.01M EDTA and 30% isopropanol, with a pH of 7.5.

[0066] 6) Magnetic beads: Promega's magnetic bead solution. The magnetic bead suspension consists of 70% (v / v) superparamagnetic microspheres, 30% (v / v) nuclease-free water, and a pH of 5.4. (The magnetic beads used in this invention are the same commercially available magnetic beads used in patent CN114107289A.)

[0067] 7) Rinse solution one, with the following components: 0.15M Tris-HCl, 0.8M NaCl, 4M guanidine hydrochloride, 0.01M EDTA and 60% anhydrous ethanol, pH 8.0.

[0068] 8) Rinse solution two, consisting of 0.16M Tris-HCl and 80% anhydrous ethanol. pH value is 8.0.

[0069] 9) The eluent is composed of enzyme-free water and has a pH of 7.0.

[0070] Example 2

[0071] Soil microbial genomic DNA was extracted using the reagents and consumables described in Example 1. The extraction method included the following steps:

[0072] 1) Sample preparation: Five different types of surface soil were randomly selected from the area of ​​119°54'35"E and 32°23'59"N. 0.2g of each sample was transferred to a clean 2mL screw-capped centrifuge tube.

[0073] 2) Add 0.1g of grinding bead 1, 0.1g of grinding bead 2 and 700uL of lysis solution to the grinding tube in step 1 in sequence.

[0074] 3) Place the grinding tube from step 2 into a grinder for grinding. Set the grinding conditions to 1600 rpm for 5 minutes. After grinding, place the grinding tube into a constant temperature mixer and process it at 1000 rpm, 75°C for 5 minutes.

[0075] 4) After the treatment, the above grinding tubes are centrifuged at 12,000 rpm for 2 minutes, and the supernatant is taken as approximately 600 μL.

[0076] 5) Take 1600uL of supernatant, add 200uL of deinhibitor to a clean 2mL EP tube according to the ratio of supernatant 1 to deinhibitor volume of 3:1, vortex, centrifuge at 12000rpm for 2min, and take 2450uL of supernatant.

[0077] 6) Transfer the supernatant 2 to a clean 2mL EP tube, add an equal volume of binding solution and 20uL of magnetic bead suspension (the concentration of the magnetic bead suspension is 20mg / mL, which is about 400ug of magnetic beads), and incubate at room temperature for 10min.

[0078] 7) Place the EP tube from step 6 on a magnetic rack until the solution becomes clear, remove the supernatant with a pipette, and remove the EP tube.

[0079] 8) Add 700uL of rinsing solution 1 to the EP tube in step 7, mix thoroughly, vortex for 5s, centrifuge briefly, place the EP tube on a magnetic rack until the solution is clear, and discard the supernatant.

[0080] 9) Repeat step 8 once.

[0081] 10) Add 700uL of rinsing solution II to the EP tube in step 9, mix thoroughly, vortex for 5s, centrifuge briefly, place the EP tube on a magnetic rack until the solution is clear, and discard the supernatant.

[0082] 11) Repeat step 10 once.

[0083] 12) Place the centrifuge tubes from step 11 on the magnetic rack, and place the entire magnetic rack in a clean bench to air dry until the surface of the magnetic beads is smooth and translucent.

[0084] 13) Add 100 μL of preheated elution buffer (65°C) to the centrifuge tube from step 12, and gently pipette the magnetic beads 50 times or vortex for 2 minutes to fully resuspend the magnetic beads. Heat at 65°C for 5 minutes, then place the centrifuge tube on a magnetic rack until the solution is clear. Transfer the supernatant to a new 1.5 mL EP tube. This is the purified soil microbial genome, which can be stored at -20°C.

[0085] The DNA content and purity determination in the elution buffer and the results of agarose gel electrophoresis are shown in Table 1:

[0086] Table 1. Determination of DNA content and purity in the eluent.

[0087] Concentration (ng / uL) A260 / 280 A260 / 230 Sample 1 25.5 1.84 0.67 Sample 2 33.1 1.82 0.66 Sample 3 69 1.75 0.72 Sample 4 71.9 1.8 0.79

[0088] Figure 1 The gel electrophoresis results of the four samples extracted from Table 1 show that the gDNA bands are highly specific, have high nucleic acid integrity, and are of good quality.

[0089] Experimental results: The kit of the present invention can extract DNA with high purity, high content (>25ng / uL), good integrity and clear electrophoretic bands.

[0090] The DNA in the elution buffer was used for quantitative real-time PCR detection.

[0091] The primer and probe sequences used for detection are as follows:

[0092] Specific primers for the 16S gene include:

[0093] F: CCTACGGGAGGCAGCAG (SEQ ID NO: 1)

[0094] R: ATTACCGCGGCTGCTGG (SEQ ID NO: 2)

[0095] The fluorescent dye used for amplifying 16S was Ultra SYBR Mixture (Catalog No.: CW0957) from Kangwei Century.

[0096] The reaction system for real-time PCR detection is prepared as follows:

[0097] 1. Quantitative fluorescence amplification of 16S

[0098]

[0099]

[0100] The quantitative PCR amplification reaction program is configured as follows:

[0101] 1. Quantitative fluorescence amplification of 16S (using ABI 7500)

[0102]

[0103] The PCR detection results of the extracted products are as follows:

[0104] Amplification of 16S:

[0105] Gene name / Ct value 16S Sample 1 22.44 Sample 2 23.65 Sample 3 22.66 Sample 4 23.52 water 30.12

[0106] Figure 2 This is a quantitative fluorescence image of the amplified bacterial 16S gene.

[0107] The 16S gene is a widely distributed gene in bacterial genomes. The ability to amplify the 16S gene using quantitative fluorescence indicates that the bacterial genome has been extracted. Water serves as the control group. Figure 2 Corresponding to the table for amplified 16S.

[0108] Experimental results: Soil microbial genomes purified using the reagents of this invention can be used for the detection of bacterial 16S genes.

[0109] Example 3

[0110] The extent of soil microbial lysis during pretreatment was examined: the heating lysis process was removed during pretreatment; in the comparative example, only one type of grinding bead (0.1 mm) was used during pretreatment. The remaining reagents and steps were the same as in Examples 1 and 2. Following the preferred scheme of the above examples, four soil samples were randomly selected, divided into three equal parts, and extracted. The concentration and purity of the extracted products were then measured. The results are as follows:

[0111]

[0112] Experimental results: The grinding bead combination in this invention can fully lyse microorganisms in the soil and increase the concentration of nucleic acid extraction.

[0113] Example 4

[0114] Deinhibitors contain components such as inhibitors (sodium polypropylene sulfonate) and flocculants (calcium chloride and ferric chloride), and these two components play important roles in deinhibitors.

[0115] In this embodiment, the effects of the inhibitor and flocculant in the de-inhibitor formulation on soil genome extraction were investigated. In the comparative example, no inhibitor or flocculant was added. The remaining reagents and steps were the same as in Examples 1 and 2. Following the preferred scheme of the above embodiments, four soil samples were randomly selected, divided into three equal parts, and extracted. The concentration and purity of the extracted products were then measured. The results are as follows:

[0116]

[0117]

[0118] Experimental results: The combination of blocking agent and flocculant in this invention can significantly improve the concentration and purity of nucleic acids extracted from soil microorganisms.

[0119] Comparative Example 5

[0120] Soil microorganisms were extracted using a commercially available soil extraction kit and compared with the extraction method of the present invention.

[0121] The four soil samples from Example 2 were simultaneously extracted using the Qiager kit (#51804, which is a fecal and intestinal microbial extraction kit) and the extraction reagents and steps described in this invention.

[0122] The concentration and purity of the extracts obtained using the universal microbial extraction kit and Comparative Example 2 were tested according to the above method, and the results are as follows:

[0123]

[0124] The results are as follows Figure 1 As shown, the soil microbial genomic nucleic acids extracted using the extraction method of this invention have higher concentrations, better purity, and better integrity than those extracted using commercially available extraction kits.

[0125] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A reagent combination for extracting microbial genomic DNA from soil, characterized in that, The reagent components are included in the following individually packaged containers: 1) Grinding bead one, which is a zirconia bead with a diameter of 0.1 mm; 2) Grinding bead two, which is a glass bead with a diameter of 0.5mm; 3) The lysis buffer consists of 0.1M Tris-HCl, 0.15M EDTA, 0.25M NaCl, 0.1M KCl and 4M NaSCN, with a pH of 8.

5. 4) Deinhibitor removal, the ingredients are: 0.05M CaCl2, 0.25M FeCl3, 12% glacial acetic acid, 0.2M Tris-HCl, 2% PVP and 2% sodium polypropylene sulfonate, the pH value of deinhibitor removal is 6.0; 5) The binding solution consists of 0.1M Tris-HCl, 6M guanidine isothiocyanate, 0.01M EDTA and 30% isopropanol, with a pH of 7.

5. 6) Magnetic beads: The composition is a magnetic bead solution containing 70% by volume superparamagnetic microspheres and 30% by volume nuclease-free water, pH 5.4; 7) Rinse solution one, with the following components: 0.15M Tris-HCl, 0.8M NaCl, 4M guanidine hydrochloride, 0.01M EDTA and 60% anhydrous ethanol, pH 8.0; 8) Rinse solution two, composed of 0.16 M Tris-HCl and 80% anhydrous ethanol; pH value is 8.0; 9) The eluent is composed of enzyme-free water and has a pH of 7.

0.

2. A kit for extracting microbial genomic DNA from soil, characterized in that, It contains the reagent combination as described in claim 1.

3. A method for extracting genomic DNA from soil microorganisms, characterized in that, Extraction was performed using the reagent combination as described in claim 1.

4. The method according to claim 3, characterized in that, The extraction method includes the following steps: a certain mass of soil is weighed, and grinding beads of different particle sizes (bead 1 and bead 2) and lysis buffer are added. After mechanical grinding, heat treatment, and lysis buffer treatment, the microorganisms in the soil are completely lysed, releasing gDNA. Then, the supernatant 1 is collected by centrifugation, an inhibitor is added, and after vortexing, the supernatant 2 is collected by centrifugation. Binding buffer and magnetic beads are added to the supernatant 2 for incubation, so that the nucleic acid binds to the magnetic beads, and the magnetic beads are precipitated. The magnetic bead precipitate is washed twice by rinsing buffer 1 and rinsing buffer 2, leaving the magnetic bead precipitate. The magnetic bead precipitate is eluted with elution buffer to obtain the genomic DNA of microorganisms in the soil.

5. A method for detecting soil microorganisms, characterized in that, First, soil microbial genomic DNA is extracted using the reagent combination described in claim 1, and then PCR detection is performed using specific primers and / or probes for the target gene.

6. The detection method according to claim 5, characterized in that, The PCR detection method is real-time quantitative PCR.

7. The detection method according to claim 5, characterized in that, The target gene is the 16S gene.

8. The detection method according to claim 7, characterized in that, The specific primers for the 16S gene include: F: CCTACGGGAGGCAGCAG; R: ATTACCGCGGCTGCTGG.

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