Method for extracting macro virus genome DNA (Deoxyribose Nucleic Acid) in surface sediment

Abstract

The invention relates to a method for extracting macro virus genome DNA (deoxyribonucleic acid) in surface sediment, which comprises the following steps: (1) mixing a sample with an extraction buffer solution, oscillating by a low-temperature shaking table for more than 2.5 hours, and centrifuging to take supernate; (2) enabling the supernate to pass through a filter membrane with the pore diameter of 0.22 mu m, mixing the obtained filtrate with a ferric chloride solution, and incubating for 1 hour; (3) centrifuging the mixed solution to obtain a flocculated precipitate; (4) adding an ascorbic acid mixed solution, uniformly mixing, and oscillating and incubating in a low-temperature shaking table for more than 8 hours; (5) adding a DNase I endonuclease solution, and removing extracellular DNA in the environment; and (6) extracting by using a kit according to a soil DNA extraction method, wherein the obtained DNA is the virome DNA. According to the method, virus DNA with relatively high purity can be obtained, virus nucleic acid fragment data with relatively high quality can be obtained after sequencing, and research on virus diversity and ecological functions in the environment is met.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology technology, and specifically relates to a method for extracting macrovirus genomic DNA from environmental surface sediments. Background Technology

[0003] Existing virus research mostly uses metagenomics technology, which directly extracts all microbial genomic DNA from environmental samples, namely metagenomic DNA, which contains DNA of viruses, bacteria, fungi, protists, etc. After metagenomic sequencing, virus data is screened out from the large amount of sequencing data obtained for targeted analysis and research. The proportion of available virus group data is small, and the sequencing efficiency is low.

[0004] If targeted environmental virome research is conducted and viral DNA is extracted from environmental samples separately, due to the high complexity of surface sediments such as soil, sediment, and dust, the current methods for extracting viral DNA are mostly concentrated in water environments, such as oceans, rivers, and lakes. There is no systematic method for extracting and isolating viral group DNA from surface sediments. Therefore, how to effectively extract macroviral genomic DNA from environmental surface sediments has become a technical problem that technicians in this field need to solve urgently. SUMMARY OF THE INVENTION

[0005] Aiming at the deficiency of existing technologies and the need to study the distribution patterns and ecological functions of viruses in multiple environments, the present invention provides a method for extracting macrovirome DNA from surface sediments. This method can enrich and retain virus particles from complex solid components and remove DNA from other microbial sources, ultimately obtaining high-quality and high-purity viral DNA, which meets the requirements of direct library construction and sequencing on the third-generation sequencing platform or sequencing after whole genome amplification.

[0006] The specific plan is as follows:

[0007] A method for extracting macrovirus genomic DNA from surface sediments, comprising the following steps:

[0008] (1) Mix the sample with the extraction buffer, shake it on a low-temperature shaker for more than 2.5 hours, and centrifuge to obtain the supernatant;

[0009] (2) Passing the supernatant obtained in step (1) through a filter membrane with a pore size of 0.22 μm, the obtained filtrate is mixed with a ferric chloride solution and incubated for 1 hour;

[0010] (3) centrifuging the mixed solution obtained in step (2) to obtain a flocculent precipitate;

[0011] (4) adding the ascorbic acid mixture to the precipitate obtained in step (3), mixing well, and incubating on a low-temperature shaker for more than 8 hours;

[0012] (5) Add DNase I endonuclease solution to remove extracellular DNA in the environment;

[0013] (6) Extract using a kit according to the soil DNA extraction method, and the obtained DNA is the virome DNA.

[0014] Furthermore, in step (5), add DNase I endonuclease solution and 10× reaction buffer, incubate in a water bath at 37 °C for 30 minutes; then add the reaction termination solution and incubate in a water bath at 65 °C for 10 minutes.

[0015] Furthermore, the concentration of the DNase I endonuclease solution is 3 units / μL, and it is stored in a solution with a volume ratio of glycerol to water of 1:1 containing 50 mM Tris acetate (pH 7.5), 10 mM potassium chloride;

[0016] The 10× reaction buffer is 100 mM Tris hydrochloride (pH 7.5 at 25 °C), 100 mM magnesium chloride, 1 mM calcium chloride;

[0017] The reaction termination solution is 200 mM ethylenediaminetetraacetic acid.

[0018] Furthermore, the DNase I endonuclease solution, 10× reaction buffer, and reaction termination solution are all reagents in the Beijing TransGen Biotech DNase I kit.

[0019] Furthermore, the sample in step (1) is a surface sediment sample, which can be various types of soil (including but not limited to urban green belt soil, forest soil, farmland soil), sediment, dust, or dustfall surface sediment samples;

[0020] The sample is a fresh sample within 24 hours of sampling, or a sample stored at 4 °C for no more than 30 days; the mass of the sample is 50 - 300 g, and specific point values can be selected as 50, 100, 150, 200, 300, etc.;

[0021] If the biomass contained in the sample (such as a dust sample) is too small, the sediment sample obtained after centrifugation in step (1) can be added to a new extraction buffer again, and the steps of shaking in a shaker and centrifugation can be repeated to obtain more supernatant.

[0022] Furthermore, the extraction buffer in step (1) contains Solution 1 and Solution 2:

[0023] Solution 1 is 0.01 M phosphate buffered saline, and the preparation method of the 0.01 M phosphate buffered saline is: take 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, dissolve in 1 L of ultrapure water, sterilize by autoclaving, and store at room temperature;

[0024] Solution II is mitomycin solution, and the preparation method of the mitomycin solution is as follows: Dissolve mitomycin in sterilized ultrapure water to obtain a 1 μg / mL mitomycin solution, and store it at -20 °C.

[0025] The preparation method of the extraction buffer is as follows: Add 800 μL of Solution II to 1 L of Solution I, prepare it immediately before use, and it can be temporarily stored at 4 °C and used up within 48 hours.

[0026] Furthermore, the preparation method of the ferric chloride solution in step (2) is as follows: Take 4.83 g of ferric chloride hexahydrate, dissolve it in 100 mL of ultrapure water, filter it through a 0.22 μm water-based filter membrane, and store it at 4 °C.

[0027] Furthermore, the volume of the ferric chloride solution added in step (2) is equal to the volume of the ascorbic acid mixture in step (4), and the added solution volume is 0.7 - 3 mL; specific values can be selected as 0.7, 1, 1.5, 2, 2.5, 3, etc., and the added volume is adjusted according to the amount of the added sample and the biomass contained in the sample.

[0028] Furthermore, the preparation method of the ascorbic acid mixture in step (4) is as follows: Take 1.51 g of tris(hydroxymethyl)aminomethane, 3.72 g of ethylenediaminetetraacetic acid, 4.07 g of magnesium chloride hexahydrate, 3.52 g of ascorbic acid, and 3 mL of 5 mol / L sodium chloride solution, dissolve them in 80 mL of ultrapure water, and it can be heated to 60 °C on a heating pad to promote dissolution; then adjust the mixture to pH = 6 with 5 mol / L sodium hydroxide or 5 mol / L hydrochloric acid solution, filter it through a 0.22 μm water-based filter membrane, and store it at 4 °C.

[0029] Furthermore, the kit in step (6) is the DNeasy PowerSoil Pro Kit from Qiagen.

[0030] Beneficial effects:

[0031] The DNA extracted by the present invention meets the requirements of the third-generation sequencing platform for DNA quality, or meets the sequencing requirements after whole-genome amplification, does not contain microbial DNA such as bacteria, fungi or protozoa, can obtain virus DNA with higher purity, and can obtain higher-quality virus nucleic acid fragment data after sequencing, meeting the research on virus diversity and ecological functions in the environment.

[0032] The extraction method of the present invention is safe, efficient, highly operable, requires simple equipment, and the reagents are economical and environmentally friendly, suitable for large-scale promotion and application. Description of the drawings

[0033] Figure 1 It is the electrophoresis detection diagram of the viral metagenomic DNA of the soil sample in Example 1;

[0034] Figure 2 is the electrophoresis detection diagram of the viral group DNA of the sediment sample in Example 2;

[0035] Figure 3 This is the electrophoresis detection diagram of the virus group DNA in the dust sample of Example 3. Specific implementation method

[0036] The following is a detailed description of the embodiments of the present invention, which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary, and the examples of the embodiments are intended to be used to explain the present invention and are not to be construed as limitations of the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in the field or in the product manual shall be followed. The reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased commercially.

[0037] The present invention will now be further described in conjunction with the accompanying drawings and specific implementation methods.

[0038] Example 1:

[0039] In this embodiment 1, soil is used as a sample to extract macrovirus group DNA. A total of 18 sample groups are set up: green belt soil 1-6, forest soil 1-6, vegetable field soil 1-6. The extraction steps of each sample group are as follows:

[0040] (1) Weigh 100 g of soil sample into a 500 mL plastic centrifuge bottle, add 300 mL of extraction buffer at a volume ratio of 1:3, mix well, shake at 200 rpm in a 4°C shaker for more than 2.5 hours, and then centrifuge at 4°C 4000g for 15 minutes;

[0041] Use the peristaltic pump and the plate-type one-way filter together to directly extract the supernatant from the centrifuge bottle after the previous centrifugation;

[0042] The sample of the present invention is preferably a fresh sample within 24 hours of sampling, or a sample stored at 4°C for no more than 30 days. The sample mass can be 50-300g, and this embodiment 1 samples 100g.

[0043] (2) Filter the supernatant obtained in step (1) through a mixed cellulose lipid membrane with a pore size of 0.22 μm and a diameter of 50 mm. Collect the filtrate in a 500 mL conical flask, add 1.5 mL of ferric chloride solution, and shake and mix thoroughly for 1 minute to obtain a mixed solution 1, which is then left to stand at room temperature for more than 1 hour.

[0044] (3) Transfer one portion of the mixture into a 50 mL centrifuge tube, centrifuge at 6000 g for 10 minutes at 4°C, and discard the supernatant; ​​

[0045] (4) Integrate the sediments obtained from the same sample in step (3) into the same centrifuge tube, add 1.5 mL of ascorbic acid mixture, and shake in a shaker at 4 °C at 200 rpm for more than 8 hours to obtain mixture 2;

[0046] The volume of the ascorbic acid mixture used in the present invention should be equal to the volume of the ferric chloride solution added in step (2). During the specific extraction process, adjust the added volume according to the sample amount and the biomass contained in the sample. The solution volume ranges from 0.7 - 3 mL, and specific point values can be selected as 0.7, 1, 1.5, 2, 2.5, 3, etc. The volume of the ascorbic acid mixture added in Example 1 of the present invention is 1.5 mL.

[0047] (5) Add 10 μL of DNase I endonuclease solution and 10 μL of 10× reaction buffer to mixture 2, and incubate in a water bath at 37 °C for 30 minutes; then add 5 μL of reaction termination solution and incubate in a water bath at 65 °C for 10 minutes to obtain mixture 3;

[0048] (6) Extract DNA from mixture 3 using the DNeasy PowerSoil Pro Kit soil kit according to the instructions. The kit used in Example 1 of the present invention is the DNeasy PowerSoil Pro Kit from Qiagen.

[0049] Of course, the DNeasy PowerSoil Pro Kit from Qiagen is only the kit used in the experimental process of the present invention. During the actual operation process, other kits with similar functions are also applicable to the present invention.

[0050] The samples of the present invention can be various types of soil (including but not limited to urban green belt soil, forest soil, farmland soil), sediments, dust, or surface sediment samples such as dustfall. In Example 1 of the present invention, soil is used as the sample.

[0051] As a preferred embodiment of the present invention, if the biomass contained in the sample is too small, the sediment sample obtained after centrifugation in step (1) can be added to a new extraction buffer again, and the steps of shaking and centrifugation in the shaker can be repeated to obtain more supernatant.

[0052] As a preferred embodiment of the present invention, the extraction buffer in step (1) comprises solution one and solution two:

[0053] The solution one is 0.01 M phosphate buffered saline solution. The preparation method of the 0.01 M phosphate buffered saline solution is: take 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, make up the volume to 1 L with ultrapure water, perform high-temperature and high-pressure sterilization treatment, and store at room temperature;

[0054] The second solution is mitomycin solution, and the preparation method of the mitomycin solution is as follows: Dissolve mitomycin in sterilized ultrapure water to obtain a 1 μg / mL mitomycin solution, and store it at -20°C;

[0055] The preparation method of the extraction buffer is as follows: Add 800 μL of the second solution to 1 L of the first solution, prepare it immediately before use, and it can be temporarily stored at 4°C and used up within 48 hours.

[0056] As a preferred embodiment of the present invention, the preparation method of the ferric chloride solution in step (2) is as follows: Take 4.83 g of ferric chloride hexahydrate, make up the volume to 100 mL with ultrapure water, and store it at 4°C after passing through a 0.22 μm water-based filter membrane.

[0057] As a preferred embodiment of the present invention, the preparation method of the ascorbic acid mixture in step (4) is as follows: Take 1.51 g of tris(hydroxymethyl)aminomethane, 3.72 g of ethylenediaminetetraacetic acid, 4.07 g of magnesium chloride hexahydrate, 3.52 g of ascorbic acid, and 3 mL of 5 mol / L sodium chloride solution, and dissolve them in 80 mL of ultrapure water (heating to 60°C on a heating pad can promote dissolution); then adjust the mixture to pH = 6 with 5 mol / L sodium hydroxide or 5 mol / L hydrochloric acid solution, and store it at 4°C after passing through a 0.22 μm water-based filter membrane.

[0058] As a preferred embodiment of the present invention, the concentration of the DNase I endonuclease solution in step (5) is 3 units / μL, and it is stored in a solution with a volume ratio of glycerol to water of 1:1 of 50 mM tris(hydroxymethyl)aminomethane acetate with a pH of 7.5 and 10 mM potassium chloride;

[0059] To make the DNase I endonuclease start to act, a reaction buffer needs to be added. The 10× reaction buffer is 100 mM tris(hydroxymethyl)aminomethane hydrochloride (pH = 7.5 at 25°C), 100 mM magnesium chloride, and 1 mM calcium chloride; the reaction termination solution is 200 mM ethylenediaminetetraacetic acid.

[0060] As a preferred embodiment of the present invention, the DNase I endonuclease solution, the reaction buffer, and the reaction termination solution are all reagents in the Beijing TransGen Biotech DNase I Kit.

[0061] Of course, the Beijing TransGen Biotech DNase I Kit is only the kit used in the experimental process of the present invention. In the actual operation process, other kits with similar functions are also applicable to the present invention.

[0062] As a preferred embodiment of the present invention, if the final amount of DNA obtained is insufficient, the quality of the extraction sample can be increased, and the amounts of the ferric chloride solution and the ascorbic acid mixture can be increased accordingly.

[0063] Of course, the specific dosages of the reagents in Example 1 are just examples. During actual operation, the amounts of the reagents can be increased or decreased as needed without changing the final concentrations of each component.

[0064] Test results:

[0065] To prove the effectiveness of Example 1, the inventor had the experimental results of Example 1 quality-checked.

[0066] Quality inspection unit: Guangdong Megagenomics Technology Co., Ltd.

[0067] The quality inspection conclusion was that it could be used for sequencing. The specific results are shown in Table 1 and Figure 1 :

[0068] Table 1 is a summary table of the viral metagenomic DNA concentrations, qualities, and sequencing suggestions for 18 soil samples in Example 1. The detection used two methods for detecting DNA concentration, namely nanodrop and qubit. A260 / 280 reflects the nucleic acid content, and A260 / 230 reflects the protein content.

[0069] Those skilled in the art know that it is extremely difficult to extract viral DNA. In Example 1, A260 / 280 was as high as 1.59 and A260 / 230 was as high as 0.65. It can be seen that the purity of the viral DNA extracted by the present invention is relatively high, meeting the requirements of the third-generation sequencing platform for DNA quality.

[0070] Figure 1 is the electrophoretic detection diagram of the viral metagenomic DNA of 18 soil samples in Example 1. Among them, Marker is DL15000, and the loading amounts of DNA and Marker are both 2 μL; the electrophoresis conditions are: 1% agarose gel, 120 V, 30 min.

[0071] Table 1 Viral Metagenomic DNA Concentrations, Qualities, and Sequencing Suggestions for Soil Samples

[0072]

[0073] Example 2

[0074] In Example 2, sediment was used as the sample to extract metaviral DNA. A total of 16 sample groups were set up: surface sediments 1-8 and deep sediments 1-8. The extraction steps for each sample group are as follows:

[0075] (1) Weigh 50 g of sediment sample into a 500 mL plastic centrifuge bottle, add 150 mL of extraction buffer according to a volume ratio of 1:3, mix well, and shake in a 4°C shaker at a rotation speed of 200 revolutions for more than 2.5 hours. Then centrifuge at 4°C and 4000 g for 15 minutes;

[0076] Connect a peristaltic pump with a plate-type one-way filter, and directly suck out the supernatant from the centrifuge bottle after the previous centrifugation step.

[0077] (2) Filter the supernatant obtained in step (1) with a mixed cellulose ester membrane with a pore size of 0.22 μm and a diameter of 50 mm. The obtained filtrate is collected in a 500 mL conical flask, and 1 mL of ferric chloride solution is added. After shaking well for 1 minute, a mixed solution 1 is obtained. Let it stand at room temperature for more than 1 hour.

[0078] (3) Aliquot the mixed solution 1 into 50 mL centrifuge tubes, centrifuge at 6000 g for 10 minutes at 4 °C, and discard the supernatant.

[0079] (4) Integrate the sediments obtained from the same sample in step (3) into the same centrifuge tube, add 1 mL of ascorbic acid mixed solution, and shake at 200 rpm in a shaker at 4 °C for more than 8 hours to obtain a mixed solution 2.

[0080] (5) Add 10 μL of DNase I endonuclease solution and 10 μL of 10× reaction buffer to the mixed solution 2, and incubate in a water bath at 37 °C for 30 minutes; then add 5 μL of reaction termination solution and incubate in a water bath at 65 °C for 10 minutes to obtain a mixed solution 3.

[0081] (6) Extract DNA from the mixed solution 3 using the DNeasy PowerSoil Pro Kit soil kit according to the instructions.

[0082] The specific reagents and extraction steps not mentioned in this Example 2 are the same as those in Example 1.

[0083] Detection results:

[0084] To prove the effect of this Example 2, the inventor performed quality inspection on the experimental results of this Example 2.

[0085] Quality inspection unit: Guangdong Megagenomics Technology Co., Ltd.

[0086] The quality inspection conclusion is that it can be used for sequencing. The specific results are shown in Table 2 and Figure 2 :

[0087] Table 2 is a summary table of the viral metagenomic DNA concentration, quality, and sequencing suggestions for 16 sediment samples in Example 2. Among them, nanodrop and qubit are two methods for detecting DNA concentration, A260 / 280 reflects the nucleic acid content, and A260 / 230 reflects the protein content.

[0088] Those skilled in the art know that it is extremely difficult to extract viral DNA. In Example 2, the A260 / 280 is as high as 1.53 and the A260 / 230 is as high as 0.22. It can be seen that the purity of the viral DNA extracted by the present invention is relatively high, meeting the requirements of the third-generation sequencing platform for DNA quality.

[0089] Figure 2 It is the electrophoretic detection map of the virome DNA of 16 sediment samples in Example 2. Among them, the Marker is DL15000, and the loading amounts of DNA and Marker are both 2 μL; the electrophoresis conditions are: 1% agarose gel, 120 V, 30 min.

[0090] Table 2 Concentrations, qualities of the virome DNA of sediment samples and sequencing suggestions

[0091]

[0092] Example 3

[0093] In this Example 3, dustfall is used as the sample to extract the metavirome DNA, and a total of 6 sample groups are set: surface sediments 1-6. The extraction steps for each sample group are as follows:

[0094] (1) Mix the sample with the extraction buffer, shake it on a low-temperature shaker for more than 2.5 hours, and centrifuge to obtain the supernatant:

[0095] ① Weigh 50 g of the dustfall sample into a 500 mL plastic centrifuge bottle, add 150 mL of the extraction buffer according to the volume ratio of 1:3, mix well, and shake it on a shaker at 4°C with a rotation speed of 200 rpm for more than 2.5 hours to obtain mixture 1;

[0096] ② Place 4-5 layers of sterilized gauze on the mouth of a 500 mL glass conical flask, filter mixture 1 into the conical flask, and temporarily store the obtained filtrate at 4°C;

[0097] ③ Slowly rinse the precipitate filtered by the gauze in step ② back into the plastic centrifuge bottle in step ① with 150 mL of the extraction buffer, mix well, and shake it on a shaker at 4°C with a rotation speed of 200 rpm for 2 hours to obtain mixture 2;

[0098] ④ Mix mixture 1 and mixture 2 in the centrifuge bottle, and centrifuge at 4000 g for 15 minutes at 4°C;

[0099] ⑤ Combine a peristaltic pump with a plate-type one-way filter, and directly suck out the supernatant from the centrifuge bottle after the previous centrifugation;

[0100] (2) Filter the supernatant obtained in step (1) with a mixed cellulose ester membrane with a pore size of 0.22 μm and a diameter of 50 mm. Collect the obtained filtrate in a clean 500 mL conical flask, add 1 mL of ferric chloride solution, and shake well for 1 minute to obtain mixture 3, and let it stand at room temperature for more than 1 hour;

[0101] (3) Aliquot mixture 3 into 50 mL centrifuge tubes, centrifuge at 6000 g at 4 °C for 10 minutes, and discard the supernatant;

[0102] (4) Integrate the sediments obtained from the same sample in step (3) into the same centrifuge tube, add 1 mL of ascorbic acid mixture, and shake at 200 rpm in a shaker at 4 °C for more than 8 hours to obtain mixture 4;

[0103] (5) Add 10 μL of DNase I endonuclease solution and 10 μL of 10× reaction buffer to mixture 4, and incubate in a water bath at 37 °C for 30 minutes; then add 5 μL of reaction termination solution and incubate in a water bath at 65 °C for 10 minutes to obtain mixture 5;

[0104] (6) Extract DNA from mixture 5 using the DNeasy PowerSoil Pro Kit soil kit according to the method described in the instruction manual.

[0105] The specific reagents and extraction steps not mentioned in this Example 3 are the same as those in Example 1.

[0106] Detection results:

[0107] To prove the effect of this Example 3, the inventor conducted quality inspection on the experimental results of this Example 3.

[0108] Quality inspection unit: Guangdong Megagenomics Technology Co., Ltd.

[0109] The quality inspection conclusion is that it can be used for sequencing. The specific results are shown in Table 3 and Figure 3 :

[0110] Table 3 is a summary table of the viral metagenome DNA concentration, quality, and sequencing suggestions for 6 dustfall samples in Example 3. Among them, nanodrop and qubit are two methods for detecting DNA concentration, A260 / 280 reflects the nucleic acid content, and A260 / 230 reflects the protein content.

[0111] Those skilled in the art know that it is extremely difficult to extract viral DNA. The A260 / 280 of this Example 2 is as high as 1.69, and the A260 / 230 is as high as 0.38. It can be seen that the purity of the viral DNA extracted by the present invention is relatively high and meets the requirements of the third-generation sequencing platform for DNA quality.

[0112] Figure 3It is the electrophoretic detection diagram of the virome DNA of 6 dust fall samples in Example 3. Among them, Marker is DL15000, the DNA loading amount is 3 μL, and the Marker loading amount is 2 μL; the electrophoresis conditions are: 1% agarose gel, 120 V, 30 min.

[0113] Table 3 Concentration, Quality and Sequencing Suggestions of Virome DNA in Dust Fall Samples

[0114]

[0115]

[0116] Although the present invention has been specifically shown and described in conjunction with the preferred embodiments, those skilled in the art should understand that various changes can be made to the present invention in terms of form and details without departing from the spirit and scope of the present invention defined by the appended claims, and all of them fall within the protection scope of the present invention.

Claims

1. A method for extracting macroviral genomic DNA from surface sediments, characterized in that: The following steps are involved: (1) Mix the sample with the extraction buffer, shake on a low-temperature shaker for more than 2.5 hours, and centrifuge to obtain the supernatant; (2) passing the supernatant obtained in step (1) through a filter membrane with a pore size of 0.22 μm, and mixing the obtained filtrate with a ferric chloride solution and incubating for 1 hour; (3) centrifuging the mixed solution obtained in step (2) to obtain a flocculent precipitate; (4) adding the ascorbic acid mixture to the precipitate obtained in step (3), mixing well, and incubating on a low temperature shaker for more than 8 hours; (5) adding DNase I endonuclease solution to remove extracellular DNA in the environment; (6) Extract the DNA using a kit according to the soil DNA extraction method, and the resulting DNA is the viral group DNA.

2. The extraction method according to claim 1, characterized in that Step (5) adding DNase I endonuclease solution and 10× reaction buffer, and incubating in a water bath at 37° C. for 30 minutes; then adding reaction stop solution, and incubating in a water bath at 65° C. for 10 minutes.

3. The extraction method according to claim 2, characterized in that The concentration of DNase I endonuclease solution was 3 units / μL and was stored in a solution of 50 mM Tris acetate, 10 mM potassium chloride, and a 1:1 volume ratio of glycerol to water at pH 7.5; 10× reaction buffer was 100 mM Tris-HCl, 100 mM MgCl2, 1 mM CaCl2 at pH 7.5 at 25°C; The reaction stop solution was 200 mM EDTA.

4. The extraction method according to claim 3, characterized in that The DNase I endonuclease solution, 10× reaction buffer and reaction stop solution are all reagents in the Beijing Quanshijin Biological DNase I kit.

5. The extraction method according to claim 1, characterized in that The sample in step (1) is a surface sediment sample; the sample is a fresh sample within 24 hours of sampling, or a sample stored at 4° C. for no more than 30 days; the sample mass is 50-300 g; If the biomass contained in the sample is too little, the sediment sample obtained after centrifugation in step (1) can be added with new extraction buffer again, and the shaking and centrifugation steps in the shaker can be repeated to obtain more supernatant.

6. The extraction method according to claim 1, characterized in that The extraction buffer of step (1) comprises solution 1 and solution 2: The first solution is a 0.01M phosphate buffered saline solution, and the preparation method of the 0.01M phosphate buffered saline solution is as follows: 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate, and 0.24g of potassium dihydrogen phosphate are added to 1L of ultrapure water, sterilized at high temperature and high pressure, and stored at room temperature; The second solution is a mitomycin solution, and the preparation method of the mitomycin solution is as follows: dissolving mitomycin in sterilized ultrapure water to obtain a 1 μg / mL mitomycin solution, and storing it at -20°C; The preparation method of the extraction buffer is as follows: 800 μL of solution 2 is added to 1L of solution 1, and the solution is prepared immediately before use, and can be temporarily stored at 4° C. and used up within 48 hours.

7. The extraction method according to claim 1, characterized in that The preparation method of the ferric chloride solution in step (2) is as follows: 4.83 g of ferric chloride hexahydrate is taken, fixed to volume in 100 mL of ultrapure water, filtered through a 0.22 μm water filter membrane, and stored at 4°C.

8. The extraction method according to claim 1, characterized in that The volume of the ferric chloride solution added in step (2) is equal to the volume of the ascorbic acid mixed solution in step (4), and the volume of the added solution is 0.7-3 mL.

9. The extraction method according to claim 1, characterized in that The preparation method of the ascorbic acid mixed solution in step (4) is as follows: 1.51 g of tris(hydroxymethyl)aminomethane, 3.72 g of ethylenediaminetetraacetic acid, 4.07 g of magnesium chloride hexahydrate, 3.52 g of ascorbic acid, and 3 mL of 5 mol / L sodium chloride solution are dissolved in 80 mL of ultrapure water, and the mixture can be heated to 60° C. on a heating pad to promote dissolution; then, the mixed solution is adjusted to a pH of 6 with 5 mol / L sodium hydroxide or 5 mol / L hydrochloric acid solution, and stored at 4° C. after passing through a 0.22 μm water filter membrane.

10. The extraction method according to claim 1, characterized in that The kit for step (6) is DNeasy Power Soil Pro Kit from Qiagen.

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