CN116286791A - Method for extracting virus DNA in soil - Google Patents
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Abstract
The invention discloses a method for extracting virus DNA in soil. The extraction method comprises the following steps: s1, eluting a soil sample three times by adopting a phosphate buffer solution to obtain a suspension; the mass of the soil sample is 20g, and mitomycin C is added into the soil sample; the ratio of the soil sample to the phosphate buffer solution is 1mg:1mL; carrying out centrifugation on the suspension to obtain supernatant; filtering with vacuum filter membrane to obtain filtrate; adding DNase and RNase into the concentrated solution for enzymolysis; then EDTA is added for incubation to inactivate enzymes; concentrating and enriching the filtrate by adopting an ultrafiltration centrifuge tube to obtain virus concentrated solution; and extracting viruses from the virus concentrated solution to extract virus DNA in soil. The invention increases the leaching efficiency while reducing the sample use by increasing the elution times; adding mitomycin C to obtain intracellular virus information; the combined use of DNase and RNase ensures the effective removal of bacterial contamination in the crude virus extract.
Description
Technical Field
The invention relates to a method for extracting viral DNA in soil, belonging to the technical field of biology.
Background
Viruses are the most abundant organisms on earth, and their number is about 10 31～33 Is bacterialThe virus in the soil has important effects on the microbial community composition and the dynamics of the global ecological system, the bio-geochemical circulation, the bacterial evolution and the like by 10 times, so that the research on the virus in the soil is significant. With the continued advancement of metagenomic sequencing and bioinformatics technologies, the world of microorganisms has developed faster in "dark matter" -environmental virome research. Genome sequencing technology can extend people's knowledge of the diversity and abundance of viruses in environmental samples. The macrogenomics technology is based on direct extraction of DNA from the environment for subsequent sequencing analysis, so that it is important to obtain DNA samples with good purity and high concentration. However, compared with water media such as sewage, ocean and the like, the soil has high heterogeneity, complex structure and rich microorganisms, so that the separation of viruses and the extraction of DNA in the soil are a great challenge. Currently, there is no universal standardized extraction scheme for in-soil viral DNA.
Because of the large soil difference, the steps of soil sample size, leaching liquor type, concentration and enrichment and the like are also different in the process of extracting the soil virus DNA. (1) extract: at present, a beef extract solution, a PBS buffer solution, an SM buffer solution and the like are mainly used, wherein the beef extract solution can dissolve other organic compounds in soil while improving the soil leaching efficiency, and the follow-up experiment is interfered. And the extraction effect of SM and PBS buffer is equivalent. (2) concentrating and enriching: currently, there are mainly filtration, polyethylene glycol Precipitation (PEG) and density gradient centrifugation. The filtration method is generally combined with PEG, but has organic solvent pollution, and is suitable for large samples with high virus abundance. Cesium chloride gradient centrifugation uses expensive equipment and equipment, and is relatively complex to operate. The filtration method (ultrafiltration centrifugation) has the advantages of high speed, high efficiency, simple and convenient operation and the like, and can achieve higher concentration multiple and easily achieve the purpose of concentrating and recycling target products from diluted samples. (3) filter membrane material: currently, there are mainly mixed cellulose filters (MCE), polyethersulfone filters (PES) and Nylon filters (Nylon). The MCE has high porosity, good interception effect, good hydrophilicity and low adsorption, and is suitable for filtering operations of sterilization and particle removal; PES is also a hydrophilic membrane, has large flux and low protein adsorption force, and is suitable for terminal filtration of food industry and ultrapure water; nylon has good mechanical strength and can resist most organic solvents and alkaline solutions.
The extraction method of the soil virus DNA is mostly research referring to other environmental samples (water bodies), but the soil is highly heterogeneous, and the effect of directly applying the current method is poor. Therefore, it is necessary to optimize the method for extracting viral DNA from soil with high organic matter content and high microbial diversity.
Disclosure of Invention
The invention aims to provide a method for extracting virus DNA in soil, which has higher concentration of the extracted virus DNA and can carry out subsequent research on virus groups in soil.
The method for extracting the virus DNA in the soil provided by the invention comprises the following steps:
s1, eluting a soil sample three times by using Phosphate Buffer Solution (PBS) to obtain a suspension;
the molar concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.2-7.4;
the mass of the soil sample is 20g;
adding mitomycin C into the soil sample, wherein the mitomycin C is added in the form of a solution, and the concentration of the solution is 100 mug/mL;
the ratio of the soil sample, the phosphate buffer solution and the mitomycin C solution is as follows: 1g:1mL: 10. Mu.L;
s2, centrifuging the suspension to obtain a supernatant; filtering with 0.22 μm vacuum filter membrane to obtain filtrate; adding DNase and RNase into the filtrate for enzymolysis; then EDTA is added for incubation, and enzyme inactivation treatment is carried out;
s3, concentrating and enriching the filtrate obtained in the step S2 by adopting an ultrafiltration centrifuge tube to obtain virus concentrate;
s4, extracting viruses from the virus concentrated solution, namely extracting virus DNA in soil.
In the above extraction method, in step S1, it is found that the elution frequency affects the concentration of the extracted DNA, and when one elution is performed, the concentration of the extracted DNA is low, and when three elution is performed, the concentration of the obtained DNA is significantly increased, more than 2ng/μl, and is 2 times that of one elution; and when the elution times are continuously increased, the extraction efficiency is not obviously improved.
In the above extraction method, in step S1, it was found that the amount of the soil sample affects the concentration of the extracted DNA, and when 20g of the soil sample is used, the concentration of the DNA increases by about 50 to 90% to 3 ng/. Mu.l or more, compared to 2g of the soil sample.
In the above extraction method, in step S2, the material of the vacuum filtration membrane is Polyethersulfone (PES);
according to the research, the material of the vacuum pumping filter membrane influences the concentration of extracted DNA, the invention compares the filtering effects of the mixed cellulose filter Membrane (MCE), the polyether sulfone filter membrane (PES) and the Nylon filter membrane (Nylon), and the result shows that the polyether sulfone filter membrane has the best effect of extracting the virus DNA.
In the above extraction method, in step S2, the DNase is DNase I, and the RNase is RNase a;
adding the DNase and the RNase to a final concentration of 100U/mL, and incubating at 37 ℃ for 2 hours to degrade residual DNA and RNA derived from host bacteria;
according to the invention, by adding DNase and RNase in combination, effective removal of bacterial nucleic acid is ensured;
the EDTA was added to a final concentration of 20mmol/L and incubated at 65℃for 5min.
In the above extraction method, in step S3, the molecular weight cut-off of the ultrafiltration centrifuge tube is 100kDa.
According to research, the molecular weight cut-off of the ultrafiltration centrifuge tube influences the concentration of extracted DNA, and compared with the ultrafiltration centrifuge tube with the molecular weight of 30kDa and 50kDa, the ultrafiltration centrifuge tube with the molecular weight of 100kDa has the optimal effect on enriching virus DNA;
current methods of virus enrichment include: PEG8000 (polyethylene glycol) precipitation and cesium chloride isopycnic gradient centrifugation are adopted, wherein the PEG precipitation method is suitable for large samples with higher virus abundance, and the cesium chloride centrifugation method has higher operation technical requirements on instrument facilities and experimenters. The invention adopts the ultrafiltration centrifuge tube method to concentrate the filtrate, and has the advantages of high speed, high efficiency, simple and convenient operation and the like.
In the above extraction method, in step S4, the Kit used for virus extraction is TIANamp Virus DNA/RNA Kit purchased from the company of the biochemical technology of the root of heaven (beijing).
The extraction method has the following beneficial technical effects:
1. by increasing the elution times, the use of samples is reduced while the leaching efficiency is improved.
2. The combined use of DNase and RNase ensures the effective removal of bacterial contamination in the crude virus extract.
3. Aiming at the soil types with high organic matter content and high microorganism diversity, the experimental steps of suction filtration, ultrafiltration and the like and the screening of the kit are optimized.
4. The addition of mitomycin C induces the release of the virus encoded on the bacterial genome, thereby obtaining intracellular viral information.
Drawings
FIG. 1 is a graph showing the effect of enzyme treatment on bacterial nucleic acid removal.
FIG. 2 is a graph showing the effect of extracting 13 soil viral DNAs by the method of the present invention.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the following examples, qubit 3.0 (High Sensitivity) was used for the detection of the soil virus DNA concentration.
Example 1 extraction of viral DNA in soil
1. Soil virus elution
The pretreatment of the DNA extraction of the soil virus, namely, the elution of the soil virus to obtain a suspension is a key step, wherein the gram of the soil used and the number of times of eluting and suspending have great influence on the concentration of the finally extracted DNA.
1) Number of elution times
Firstly, trying to weigh 2g of soil sample, and preparing 20 mu L of mitomycin C (prepared into solution by sterilized normal saline with the concentration of 100 mu g/mL), and setting a control without adding mitomycin C; after further mixing with sterile PBS buffer (0.01 mol/L, pH=7.2 to 7.4) 1:1 (g: mL), the soil virus particles were eluted only 1 time to suspend them, and the extracted DNA concentration was low, 1.212 ng/. Mu.L, as shown in Table 1. The subsequent attempt to increase the elution times to 3 times resulted in a significant increase in DNA concentration of 2.423 ng/. Mu.L, which was 2-fold higher than the original, by fully contacting and mixing the virus particles with the buffer solution. While the elution times are increased to 5 times, the extraction efficiency is not improved significantly, and the workload of the subsequent ultrafiltration step is increased. Mitomycin C is an inducer that damages the DNA of bacterial cells and causes the phage to leave the dead host, and as can be seen from the data in Table 1, the addition of mitomycin C greatly increases the concentration of viral DNA.
TABLE 1 detection of viral DNA concentration at different elution times
2) Soil sample size
Different gradients (2 g, 5g, 20 g) were set and eluted 3 times according to the procedure in step 1). The concentration change was found to be insignificant when the sample was increased to 5g by measuring the DNA concentration, but when the sample amount was increased to 20g, the concentration was increased by about 50 to 90% as compared to 2g, reaching 3.526 ng/. Mu.L, and when the sample amount was increased to 30g, the concentration change was insignificant as compared to 20g, and the results are shown in Table 2. Therefore, in order to increase the virus content as much as possible without wasting samples, a final protocol of using a 20g soil sample size and performing 3 elution to soil pretreatment was selected.
TABLE 2 detection of viral DNA concentration at different soil sample amounts
2. Bacterial contamination removal
Centrifuging the suspension obtained in the step 1, retaining the supernatant, and separating microorganisms in the soil into bacteria and viruses by utilizing a vacuum filtration mode. The device is matched equipment, has strict design, can reduce loss to the greatest extent, has a good effect of removing bacterial DNA pollution, and can not reduce the diversity of soil viruses.
The invention explores the filtering effect of the filtering film with three apertures of 0.80 mu m, 0.45 mu m and 0.22 mu m, and the invention is a main consideration standard on the premise of effectively removing bacterial pollution without damaging virus diversity and ensuring higher recovery rate. The result shows that the 0.22 μm filter membrane has better effect of removing bacterial DNA pollution.
The invention then continues to explore the filtering effect of the 0.22 μm but different material filter membranes: the results of mixing the cellulose filter Membrane (MCE), the polyethersulfone filter membrane (PES) and the Nylon membrane (Nylon) show that the polyethersulfone filter membrane has the optimal filtration effect, and the results are shown in table 3.
DNase I and RNase A were added to the obtained virus concentrate to a final concentration of 100U/mL and incubated at 37℃for 2 hours to degrade the residual host-derived DNA and RNA. The enzyme was further inactivated by adding EDTA (0.5 mol/L, pH=8.0) to the concentrate and incubating at 65℃for 5min to a final concentration of 20 mmol/L. The detection result of the 16S rRNA gene is shown in FIG. 1, and compared with a Marker, the sample treated by DNase alone is positive in 16S rRNA, which indicates that the sample is polluted by residual bacterial nucleic acid. The DNA+RNase combined treatment is negative, which shows that the two enzymes can be used simultaneously to effectively remove host nucleic acid, thus obtaining the crude extract of the soil virus with higher purity.
3. Virus enrichment concentration
After the soil suspension is subjected to suction filtration, bacteria are trapped on the surface layer of the filter membrane, and viruses are filtered into a suction filtration bottle. Because of the large volume, the subsequent extraction of DNA is not facilitated, and ultrafiltration concentration and enrichment of filtrate are needed.
The ultrafiltration centrifuge tube has the advantages of high speed, high efficiency, simple and convenient operation and the like, can achieve higher concentration multiple and easily achieve the purpose of concentrating and recycling target products from diluted samples.
A comparative study was performed with ultrafiltration tubes (Millipore, USA) of 3 different molecular weight cut-offs (30, 50, 100 kDa).
The influence of the material of the filter membrane and the aperture of the ultrafiltration centrifuge tube on the concentration of the virus DNA is shown in Table 3, and as can be seen from Table 3, after the filtration of the polyether sulfone filter membrane (PES), the concentration of the virus DNA in the soil is 2.989 ng/. Mu.L, the enrichment is carried out by using the ultrafiltration centrifuge tube with 100kDa.
TABLE 3 detection of viral DNA concentration using filters of different molecular weights
4. Extraction of DNA
3 kits were selected for comparison studies, respectively: QIAprep Spin M13 Kit (Qiagen, germany), phage DNA Isolation Kit Product #46800 (Norgen Biotek, canada), TIANamp Virus DNA/RNA Kit (Tiangen, china). The same sample was used, 2 repeated experiments were performed, and three different kits were used to extract viral DNA under the same conditions as the other operations, and the results are shown in Table 4. As can be seen from Table 4, the highest extraction concentration of viral DNA in the soil was 3.126 ng/. Mu.L using TIANamp Virus DNA/RNA Kit.
TABLE 4 detection of viral DNA concentration
Wherein, the steps of extraction using TIANamp Virus DNA/RNA Kit are as follows:
(1) mu.L of Proteinase K was added to a clean 1.5mL centrifuge tube;
(2) Adding a virus solution obtained by sample pretreatment into a centrifuge tube;
(3) After incubation at 56℃for 15min, 250. Mu.L of absolute ethanol was added and vortexed for 15s to mix well with shaking. Standing at room temperature for 5min, and centrifuging briefly;
(4) Transferring all the solution in the centrifuge tube to an adsorption column CR2 (the adsorption column is placed in a collecting pipe), centrifuging for 1min at 6000 Xg, and discarding the waste liquid in the collecting pipe;
(5) Adding 500 mu L of buffer GD into an adsorption column, centrifuging at 6000 Xg for 1min, and discarding the waste liquid in a collecting pipe;
(6) Adding 600 mu L of rinsing liquid PW into an adsorption column, standing for 2min, centrifuging for 1min at 6000 Xg, and discarding the waste liquid in a collecting pipe;
(7) Repeating step (6);
(8) Adding 500 mu L absolute ethyl alcohol into the adsorption column, centrifuging for 1min at 6000 Xg, and discarding the waste liquid in the collecting pipe;
(9) Placing the adsorption column back into a collecting pipe, centrifuging for 3min at 13400 Xg, and discarding the waste liquid after the adsorption film is thoroughly dried;
(10) The column was placed in a fresh 1.5mL clean centrifuge tube, the column lid carefully opened, and left at room temperature for 3min. 50 mu L of sterile ddH is added to the center of the adsorption film in a suspending manner 2 O, left at room temperature for 5min, centrifuged at 13400 Xg for 1min, and the DNA was transferred to a centrifuge tube by centrifugation.
After the optimization of the extraction method of 4 above, 13 soil samples were used for verification, and the results are shown in fig. 2. All samples were 16S rRNA negative compared to Marker, indicating that the optimized protocol was suitable for in-soil viral DNA extraction.
Claims (5)
1. The method for extracting the virus DNA in the soil comprises the following steps:
s1, eluting a soil sample three times by adopting a phosphate buffer solution to obtain a suspension;
the molar concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.2-7.4;
the mass of the soil sample is 20g;
adding mitomycin C into the soil sample, wherein the mitomycin C is added in the form of a solution, and the concentration of the solution is 100 mug/mL;
the ratio of the soil sample, the phosphate buffer solution and the mitomycin C solution is as follows: 1g:1mL: 10. Mu.L;
s2, centrifuging the suspension to obtain a supernatant; filtering with 0.22 μm vacuum filter membrane to obtain filtrate; adding DNase and RNase into the filtrate for enzymolysis; then EDTA is added for incubation, and enzyme inactivation treatment is carried out;
s3, concentrating and enriching the filtrate obtained in the step S2 by adopting an ultrafiltration centrifuge tube to obtain virus concentrate;
s4, extracting viruses from the virus concentrated solution, namely extracting virus DNA in soil.
2. The extraction method according to claim 1, characterized in that: in step S2, the material of the vacuum filtration membrane is polyethersulfone.
3. The extraction method according to claim 1 or 2, characterized in that: in the step S2, the DNase is DNase I, and the RNase is RNase A;
the EDTA was added to a final concentration of 20mmol/L and incubated at 65℃for 5min.
4. The extraction method according to claim 3, characterized in that: in step S3, the molecular weight cut-off of the ultrafiltration centrifuge tube is 100kDa.
5. The extraction method according to claim 4, wherein: in step S4, the Kit used for virus extraction is TIANamp Virus DNA/RNA Kit purchased from Tiangen Biochemical technologies (Beijing) Inc.
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| CN117417814A (en) * | 2023-12-18 | 2024-01-19 | 中国海洋大学 | Full-automatic virus extraction system and extraction method |
| CN117417814B (en) * | 2023-12-18 | 2024-04-12 | 中国海洋大学 | Full-automatic virus extraction system and extraction method |
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