A method for removing extracellular DNA / extracellular antibiotic resistance genes in red soil

Abstract

The application discloses a method for removing extracellular DNA / antibiotic resistance genes in red soil, and belongs to the field of environmental pollution treatment. The application mainly solves the problem of extracellular DNA / antibiotic resistance gene pollution in red soil. The application is based on an extracellular DNA (eDNA) remover, a chemical reagent, which is used for degrading the extracellular DNA in the red soil, and then removing antibiotic resistance genes (eARGs) contained in the extracellular DNA, so as to help people correctly evaluate the risk of resistance gene transmission.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of environmental pollution control, and particularly relates to a method for removing extracellular DNA / extracellular antibiotic resistance genes in red soil. BACKGROUND

[0002] Since the 20th century, antibiotics have been widely used in livestock farming, agriculture, medical and health fields, playing an important role in the treatment of human and livestock diseases and the promotion of plant and animal growth. In the livestock breeding industry, antibiotics enter the animal body, part of which is absorbed and utilized, but most of which is excreted outside the body in the form of metabolites. Long-term use and abuse of antibiotics in animal breeding can make animal manure an important source of antibiotics and resistance genes.

[0003] Red soil is developed through desilication and aluminization and biological enrichment, and has great production potential. However, factors such as high temperature and rainfall can cause rapid decomposition of soil organic matter in red soil, greatly reducing the contents of N, P, K and other nutrients in red soil, and thus reducing the productivity of red soil. Therefore, in agricultural production, organic fertilizer is often applied to red soil to improve soil fertility, improve soil quality and increase crop yield. The application of livestock manure as organic fertilizer to soil can greatly increase the abundance of antibiotics and resistance genes in red soil, accelerate the spread and diffusion of resistance genes among bacteria in the environment, and cause environmental resistance spread risk.

[0004] Most of the microorganisms in livestock manure come from animal intestines. The pH values of animal intestines and soil are different, so the microorganisms derived from animal intestines cannot survive in soil, resulting in a large number of dead intestinal microorganisms in soil. Microorganisms usually lyse after death, causing the release of cytoplasmic contents, including extracellular antibiotic resistance genes (eARGs). We usually evaluate resistance risk as a whole by taking intracellular antibiotic resistance genes (iARGs) and extracellular antibiotic resistance genes (eARGs) together, but extracellular antibiotic resistance genes (eARGs) are a potential resistance spread risk. It will only cause real resistance spread risk when it is transformed into cells to become intracellular antibiotic resistance genes (iARGs). eDNA remover is a chemical reagent that can remove extracellular DNA (eDNA) and extracellular antibiotic resistance genes (eARGs) in red soil. The chemical reagent can degrade extracellular DNA (eDNA) in red soil, thereby preventing the release of cytoplasmic contents, reducing the abundance of extracellular antibiotic resistance genes in red soil, and helping us correctly understand the resistance spread risk caused by antibiotic resistance genes in red soil. SUMMARY

[0005] In response to the problem of extracellular DNA / extracellular antibiotic resistance gene contamination in red soil, the present invention provides a method for removing extracellular DNA / extracellular antibiotic resistance genes in red soil.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] The present invention provides a method for removing extracellular DNA / extracellular antibiotic resistance genes in red soil. The method uses an eDNA remover to degrade extracellular DNA (eDNA) in red soil and antibiotic resistance genes (eARGs) present in the extracellular DNA, thereby achieving the purpose of eliminating extracellular DNA (eDNA) and extracellular antibiotic resistance genes (eARGs) in red soil.

[0008] In a specific embodiment, the method is achieved by the following specific steps:

[0009] (1) Prepare eDNA removal agent and set aside;

[0010] (2) Weighing red soil, adding the eDNA remover prepared in step (1), mixing well, and letting it stand at room temperature;

[0011] Among them, the mass-to-volume ratio of red soil and eDNA is 1:0.5~1.5.

[0012] In a specific embodiment, the mass-to-volume ratio of the red soil to the eDNA is 1:1. In a specific embodiment, the eDNA remover primarily comprises the following substances: 10-30 mM citric acid-sodium citrate buffer, 5-15 mM L-ascorbic acid, 1-3 mM copper sulfate pentahydrate, 0.2-2 mM ethylenediaminetetraacetic acid, and 0.01-0.035 mM hydrogen peroxide.

[0013] Preferably, the eDNA remover mainly comprises the following substances: 20 mM citric acid-sodium citrate buffer, 10 mM L-ascorbic acid, 2 mM copper sulfate pentahydrate, 1 mM ethylenediaminetetraacetic acid, and 0.025 mM hydrogen peroxide.

[0014] In a specific embodiment, in step (ii), the red soil can be sieved according to actual conditions and then treated with an eDNA remover.

[0015] Beneficial effects of the present invention:

[0016] (1) Under the action of eDNA remover, the removal rate of extracellular DNA in red soil exceeded 95%.

[0017] (2) eDNA removers do not affect the intracellular DNA of living bacteria.

[0018] (3) The eDNA removal agent can degrade the extracellular DNA (eDNA) in the red soil, thereby removing the extracellular antibiotic resistance genes (eARGs) carried in the eDNA and reducing the abundance of the extracellular antibiotic resistance genes in the red soil. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The gfp gene copy number change in the soil precipitate and supernatant based on the PVPP method;

[0020] Figure 2 The gfp gene copy number change in the eDNA in the red soil system;

[0021] Figure 3 The gfp gene copy number change in the eDNA in the red soil system;

[0022] Figure 4 The gfp gene copy number change in the eDNA in different soil systems;

[0023] Figure 5 The gfp gene copy number change in the iDNA in the red soil system;

[0024] Figure 6 The antibiotic resistance gene abundance change in the red soil applied with organic fertilizer. DETAILED DESCRIPTION

[0025] The application will be further described in detail below with reference to the examples. The reagents or instrument devices not specified by the manufacturer are all regarded as conventional products that can be purchased in the market.

[0026] The application claims a method for removing the eDNA / eARGs in the red soil, which is realized by the following steps:

[0027] (I) Prepare the eDNA removal agent for use;

[0028] (II) Weigh 0.3 g of the red soil into a 10 mL centrifuge tube, add 3 mL of the eDNA removal agent, vortex, and prepare a soil suspension; stand at room temperature for 10 min.

[0029] The following examples illustrate the preferred specific embodiments of the application, but the application is not limited thereto.

[0030] Example 1: The eDNA removal agent removes the eDNA in the red soil

[0031] The red soil samples without any fertilizer (SCon), red soil with organic fertilizer (SM) and red soil with inorganic fertilizer (SNPK) were collected, passed through a 2 mm sieve and homogenized. 0.3 g of the homogenized soil sample was weighed and placed in a 10 mL transparent centrifuge tube. 10 μl of the sample was added, carrying 4.3 × 10 7 The pET29a plasmid DNA containing the gfp gene was resuspended in 3 mL of sterile water / eDNA remover and allowed to stand for 10 minutes. The mixture was centrifuged at 10,000 g for 2 minutes, and the supernatant was discarded. An equal volume of 0.1 M sodium phosphate buffer (pH 7.4) was added and the mixture was centrifuged at 10,000 g for 2 minutes, and the supernatant was discarded. This was repeated three times. Soil DNA was extracted using the DNeasy PowerSoil Pro Kit (Qiagen). The gfp gene fragment in the plasmid DNA was amplified by fluorescent quantitative PCR (Table 1), and its gene copy number was calculated. The changes in gfp gene copy number in the plasmid DNA before and after treatment with the eDNA remover were compared ( Figure 3 ).

[0032] The results are as follows Figure 3 As shown, after the soil was treated with the eDNA remover, the gfp gene copy number was significantly reduced compared with the control group, and the removal rate was above 95%, indicating that the eDNA remover can effectively remove extracellular DNA in red soil.

[0033] Comparative Example 1: Elution of extracellular DNA from red soil by PVPP method

[0034] Red soil samples were collected, passed through a 2 mm sieve, and homogenized. 0.2 g of soil was weighed into a 2 mL transparent centrifuge tube and 1 mL of 0.1 M PBS buffer was added. 10 μl of 4.3 × 10 7 The pET29a plasmid DNA containing the gfp gene was vortexed for 5 minutes and centrifuged at 14000g for 3 minutes. The supernatant was purified by PCR product recovery kit (purchased from Axygen) to recover DNA, and the precipitate was purified by DNeasy PowerSoil Pro Kit (purchased from Qiagen). The gfp gene fragment in the plasmid DNA was amplified by fluorescence quantitative PCR (Table 1), and the gfp gene copy number was calculated. The changes in gfp gene copy number in the supernatant and soil precipitate were compared ( Figure 1 ).

[0035] The results are as follows Figure 1 As shown in Figure 2, the gfp gene copy number in the soil sediment was significantly higher than that in the supernatant, indicating that more than 95% of the exogenous plasmid DNA added to the red soil was adsorbed by the soil. Figure 3 In contrast, PVPP could not effectively elute eDNA from red soil.

[0036] Table 1 gfp gene primers

[0037]

[0038] Comparative Example 2 PMA method combined with extracellular DNA in red soil

[0039] Weigh 0.3 g of the homogenized red soil sample into a 10 mL transparent centrifuge tube, add 3 mL of 0.01 M PBS buffer; then add 10 ul of 4.3 x 10 7 The gfp gene pET29a plasmid DNA with a gene copy was added with different concentrations (0, 30, 40 and 50 uM) of PMA reagent under room temperature and light-avoiding conditions, and mixed well; the sample was placed in dark conditions for 4 min, and then placed 20 cm directly below a 650 W halogen lamp for 30 s of light and 30 s of darkness for a total of 4 cycles; centrifuged at 10000 g for 5 min, and the supernatant was discarded; soil DNA was extracted using a DNeasy PowerSoil Pro Kit (purchased from Qiagen company); the gfp gene fragment was amplified by fluorescent quantitative PCR method, and the gene copy number was calculated to compare the change of gene copy number before and after PMA treatment Figure 2 ), to verify whether PMA can bind the extracellular DNA in red soil.

[0040] The results are shown in Figure 2 There was no significant difference in the gfp gene copy number in the soil treated with PMA compared with CK. Compared with Figure 3 PMA cannot effectively bind the eDNA in the red soil sample.

[0041] Comparative Example 3 Removal effect of eDNA removal agent on extracellular DNA in different soils

[0042] Soil samples from Anhui (AH), Henan (HN) and Shandong (SD) were collected, sieved through a 2 mm sieve, and homogenized; 0.3 g of the homogenized soil sample was placed in a 10 mL transparent centrifuge tube, 10 ul of 4.3 x 10 7 The gfp gene pET29a plasmid DNA with a gene copy was resuspended in 3 mL of sterile water / eDNA removal agent, and stood for 10 min; centrifuged at 10000 g for 2 min, and the supernatant was discarded; an equal volume of 0.1 M sodium phosphate buffer (pH = 7.4) was added, centrifuged at 10000 g for 2 min, and the supernatant was discarded, repeated three times; soil DNA was extracted using a DNeasy PowerSoil Pro Kit (purchased from Qiagen). The gfp gene fragment in the plasmid DNA was amplified by fluorescent quantitative PCR method (Table 1), and the gene copy number was calculated to compare the change of gfp gene copy number in the plasmid DNA before and after eDNA removal agent treatmentFigure 4 ).

[0043] Results are shown in Table 2. The gfp gene copy number in the soil samples treated with the eDNA removal agent did not decrease significantly compared with CK. Compared with the eDNA removal agent which can effectively remove more than 95% of the eDNA in red soil, the experiment showed that the eDNA removal agent cannot effectively remove the eDNA in the soil samples from Anhui, Shandong and Henan. Figure 4

[0044] Example 2 Effect of eDNA removal agent on intracellular DNA in red soil

[0045] Red soil samples (SCon) without any fertilizer, red soil samples (SM) with organic fertilizer and red soil samples (SNPK) with inorganic fertilizer were collected, sieved through a 2 mm sieve and homogenized. Eight types of bacteria carrying gfp gene fragments were cultured according to the culture conditions (Table 2). Equal amounts of bacterial liquid were placed in 2 mL centrifuge tubes and washed with sterile water three times to prepare a mixed bacterial population. After homogenization, 0.3 g of soil sample was placed in a 10 mL centrifuge tube, 10 ul of mixed bacterial liquid carrying 3x10 7 gene copies of gfp gene was added, and the mixture was resuspended in 3 mL of sterile water / eDNA removal agent and allowed to stand for 10 min. Centrifugation was performed at 10000 g for 2 min, and the supernatant was discarded. An equal volume of 0.1 M sodium phosphate buffer (pH = 7.4) was added, and centrifugation was performed at 10000 g for 2 min. The supernatant was discarded, and the process was repeated three times. Soil DNA was extracted using a DNeasy PowerSoil Pro Kit (purchased from Qiagen). The gfp gene fragment in the intracellular DNA was amplified by fluorescence quantitative PCR method (Table 1), and the gene copy number was calculated to compare the change in gfp gene copy number in the intracellular DNA before and after treatment with the eDNA removal agent. Figure 5

[0046] Results are shown in Table 2. The gfp gene copy number in the soil samples treated with the eDNA removal agent did not decrease significantly compared with CK. Compared with the eDNA removal agent which can effectively remove more than 95% of the eDNA in red soil, the experiment showed that the eDNA removal agent cannot effectively remove the eDNA in the soil samples from Anhui, Shandong and Henan. Figure 5

[0047] Table 2 Bacterial population information

[0048]

[0049] Example 3

[0050] ​​​A method for removing extracellular DNA / antibiotic resistance genes in red soil is carried out according to the following steps: sampling the red soil sample to which organic fertilizer is applied for 30 days, passing through a 2mm sieve, and uniformizing treatment; weighing 0.3g of the soil sample after homogenization and placing in a 10mL centrifuge tube, adding 3mL of sterile water / eDNA removing agent, mixing, and standing for 10min; centrifuging at 10000g for 2min, and discarding the supernatant; adding 3mL of 0.1M sodium phosphate buffer (pH=7.4), mixing thoroughly, centrifuging at 10000g for 2min, discarding the supernatant, and repeating three times; extracting soil DNA by using a DNeasy PowerSoil Pro Kit (purchased from Qiagen); and comparing the abundance of antibiotic resistance genes (Table 3) and the change of antibiotic species in the red soil before and after eDNA removing agent treatment by using a metagenomic sequencing method. Figure 6 ) and antibiotic species (Table 3) in the red soil before and after eDNA removing agent treatment.

[0051] The results are shown in Figure 6 and Table 3, and the abundance and species of antibiotic resistance genes in the red soil sample after eDNA removing agent treatment have obvious differences compared with CK, indicating that the eDNA removing agent can effectively remove extracellular DNA and extracellular antibiotic resistance genes in the red soil.

[0052] Table 3 Change of antibiotic species in red soil to which organic fertilizer is applied

[0053]

[0054] The protection scope of the present application is not limited to the above embodiments. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and the appended claims are used as the protection scope.

Claims

1. A method for removing extracellular DNA and antibiotic resistance genes in extracellular DNA in red soil, characterized in that: The extracellular DNA in the red soil and the antibiotic resistance genes in the extracellular DNA are degraded by the extracellular DNA remover, thereby achieving the purpose of removing the extracellular DNA in the red soil and the antibiotic resistance genes in the extracellular DNA; The method is achieved through the following specific steps: (1) Prepare the extracellular DNA removal agent and set aside; (2) Weigh red soil, add the extracellular DNA remover prepared in step (1), mix well, and let stand at room temperature; Among them, the mass volume ratio of red soil and extracellular DNA remover is 1:0.5~1.5; The extracellular DNA removal agent mainly includes the following substances: 10-30 mM citric acid-sodium citrate buffer, 5-15 mM L-ascorbic acid, 1-3 mM copper sulfate pentahydrate, 0.2-2 mM ethylenediaminetetraacetic acid, and 0.01-0.035 mM hydrogen peroxide.

2. The method for removing extracellular DNA and antibiotic resistance genes in extracellular DNA in red soil according to claim 1, characterized in that: The mass-to-volume ratio of the red soil and the extracellular DNA remover is 1:

1.

3. The method for removing extracellular DNA and antibiotic resistance genes in extracellular DNA in red soil according to claim 1, wherein: The extracellular DNA removal agent mainly includes the following substances: 20 mM citric acid-sodium citrate buffer, 10 mM L-ascorbic acid, 2 mM copper sulfate pentahydrate, 1 mM ethylenediaminetetraacetic acid, and 0.025 mM hydrogen peroxide.

4. The method for removing extracellular DNA and antibiotic resistance genes in extracellular DNA in red soil according to claim 1, characterized in that: In the step (ii), the red soil is sieved and then treated with an extracellular DNA remover.

5. The method for removing extracellular DNA and antibiotic resistance genes in extracellular DNA in red soil according to claim 4, characterized in that: The sieving was performed using a 2 mm sieve.

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