A method for precise extraction and separation of intracellular and extracellular DNA in natural water bodies

Abstract

The present invention discloses a method for precisely extracting intra- and extracellular DNA in natural water bodies, belonging to the DNA extraction technology in the biological field. In this method, an extractant capable of binding to metal ions is added to the target water body containing transparent extracellular polymeric particles, so that the transparent extracellular polymeric particles are uniformly dispersed and completely broken under the action of the extractant, and the intra- and extracellular DNA on them is released into the target water body and then centrifuged to obtain a supernatant containing all intra- and extracellular DNA. The supernatant is filtered through a filter membrane, the extracellular DNA is collected in the filtrate, and the intracellular DNA is collected on the filter membrane, thus completing the precise separation of intra- and extracellular DNA in the target water body. The present invention can specifically and precisely extract intra- and extracellular DNA in natural water bodies, solve the problem of serious underestimation of extracellular DNA content by traditional extracellular DNA extraction methods in water bodies, and is of great significance for the research on microbial community structure, resistance genes, functional genes, material cycle, etc. in aquatic biological ecosystems.

Description

Technical Field

[0001] The present invention relates to a method for accurately extracting intracellular and extracellular DNA in natural water bodies, belonging to the DNA extraction technology in the biological field. Background Art

[0002] A major category of EPS - transparent exopolymer particles (TEP) is the link connecting the two major organic carbon pools of particulate organic carbon (POC) and dissolved organic carbon (DOC). TEP is an important pathway for the transformation of DOM into POM and is of great significance to the carbon cycle. TEP is composed of acidic polysaccharides, and other components also include proteins, humic acids, amino acids, metals, etc. TEP is invisible under an optical microscope but belongs to a type of EPS that can be observed after staining with Alcian blue.

[0003] Transparent exopolymer particles (TEP) are ubiquitous in the water environment. TEP has the characteristic of low density, which can prolong the residence time of solid particles in the water body, thereby affecting the carbon cycle in the water body, affecting the active elements adsorbed on the aggregates, and further affecting the nutrient element cycle in the water body. Transparent particulate matter forms large aggregates (such as "lake snow" and "marine snow") in the aquatic ecosystem using its high viscosity, promoting the sedimentation or horizontal migration of substances. At the same time, transparent exopolymer particles can provide habitats for microorganisms, thus affecting the composition and distribution of bacteria and the nutrient salt cycle in the water body.

[0004] Environmental DNA refers to DNA extracted from environmental samples such as soil, sediment, air, water bodies, etc. It is a mixed DNA from multiple different species such as microorganisms, animals, and plants, including both intracellular DNA (iDNA) of living cells shed or released into the environment from organisms and extracellular DNA (eDNA) released into the environment due to the lysis of decaying corpses and cell breakage after the death of organisms. In recent years, with the gradual maturity and development of environmental DNA technology, environmental DNA has also been used in research on aspects such as the diet analysis of aquatic organisms, population size and population dynamics, resistance genes, and functional genes.

[0005] Currently, most methods for separating intracellular and extracellular DNA in water bodies involve filtering with a 0.22 μm filter membrane. The intracellular DNA contained in cells is retained on the filter membrane, and the free extracellular DNA is filtered into the filtrate. However, studies have found that a large amount of extracellular DNA is also contained in transparent extracellular polymeric particles. At the same time, researchers have obtained through experiments that the content of extracellular DNA in transparent extracellular polymeric particles is much higher than that of intracellular DNA. The particle size of transparent extracellular polymeric particles is larger than 0.22 μm. Using a 0.22 μm filter membrane filtration method will retain the transparent extracellular polymeric particles on the filter membrane, thus classifying the extracellular DNA therein as intracellular DNA. Therefore, the existing method of separating intracellular and extracellular DNA by passing through a 0.22 μm filter membrane will underestimate the content of extracellular DNA and overestimate the content of intracellular DNA.

[0006] Therefore, there is an urgent need for an extractant that can destroy transparent extracellular polymeric particles but not biological membranes and DNA, so as to release the intracellular and extracellular DNA contained in the transparent extracellular polymeric particles, and provide a method for accurately extracting intracellular and extracellular DNA in natural water bodies. Summary of the Invention

[0007] The purpose of the present invention is to solve the problem that traditional DNA extraction methods underestimate the content of extracellular DNA, and provide a method for accurately extracting intracellular and extracellular DNA in natural water bodies. This method can uniformly disperse the transparent extracellular polymeric particles in seawater and fresh water, thereby releasing the intracellular and extracellular DNA therein. Then, through filtration with a 0.22 μm filter membrane, the extracellular DNA is collected in the filtrate, and the intracellular DNA remains on the filter membrane. The extracellular DNA is extracted from the filtrate, and the intracellular DNA is extracted from the filter membrane, so as to specifically and accurately extract the intracellular and extracellular DNA in the water body.

[0008] The specific technical solution adopted by the present invention is as follows:

[0009] The present invention provides a method for accurately extracting intracellular and extracellular DNA in natural water bodies, specifically as follows:

[0010] An extractant capable of binding to metal ions is added to the target water body containing transparent extracellular polymeric particles to obtain a mixed solution. The mixed solution is shaken to allow the extractant to fully react with the transparent extracellular polymeric particles in the target water body. After the transparent extracellular polymeric particles in the target water body are completely ruptured and the intracellular and extracellular DNA thereon is completely released into the target water body, centrifugation is performed to obtain a supernatant containing all intracellular and extracellular DNA. The above supernatant is filtered through a 0.22 μm filter membrane, the extracellular DNA is collected in the filtrate, and the intracellular DNA is collected on the filter membrane, completing the accurate separation of intracellular and extracellular DNA in the target water body containing transparent extracellular polymeric particles.

[0011] The above extractant is one of ethylenediaminetetraacetic acid, sodium pyrophosphate or sodium hexametaphosphate.

[0012] Preferably, the above extractant is mixed with the target water body in a volume ratio of 1:(1-10).

[0013] Preferably, the concentration of the above ethylenediaminetetraacetic acid is 0.001 mM - 10 mM;

[0014] Preferably, the concentration of the above sodium pyrophosphate is 1 mM - 20 mM.

[0015] Preferably, the concentration of the above sodium hexametaphosphate is 0.01 mM - 15 mM.

[0016] Preferably, the conditions for the above shaking treatment are: the shaking speed is 150 - 250 rpm, the shaking time is 1 - 3 h, and the shaking temperature is 4 - 10 °C.

[0017] Preferably, the conditions for the above centrifugation treatment are: the centrifugation speed is 6000 - 12000 rpm, the centrifugation time is 10 - 30 min, and the centrifugation temperature is 4 - 25 °C.

[0018] Preferably, the above filtration treatment uses the vacuum filtration method.

[0019] Preferably, the extracellular DNA in the above filtrate is extracted using the extractant of the free circulating nucleic acid extraction kit for blood.

[0020] Preferably, the intracellular DNA on the above filter membrane is extracted using the soil genomic DNA extraction kit.

[0021] The present invention has the following beneficial effects compared with the prior art:

[0022] (1) The extractant provided by the present invention has high cost performance, is easy to obtain, has less pollution, and has no destructive effect on biological membranes and DNA. It will not cause the intracellular DNA in cells to be released as extracellular DNA due to the rupture of cell membranes, nor will it damage DNA molecular fragments and affect the detection results;

[0023] (2) The extraction method provided by the present invention is applicable to the extraction of intracellular and extracellular DNA in various water bodies such as fresh water and sea water, and has good extraction effects;

[0024] (3) The extraction method provided by the present invention releases all the extracellular DNA in the transparent extracellular polymeric particles in the water body, can accurately quantify the content of extracellular DNA in the water body, solves the problem that the content of extracellular DNA in the water body was underestimated in the past, and is applicable to the research on the estimation of aquatic biomass, the feeding habits of animals, population size and population dynamics, resistance genes, functional genes, etc. Description of the Drawings

[0025] Figure 1Intracellular and extracellular DNA contents measured after treating the target water body with different extractants. Detailed implementation manners

[0026] The present invention will be further described and explained below in conjunction with the detailed implementation manners. The technical features of each implementation manner in the present invention can be combined correspondingly on the premise that there is no conflict with each other.

[0027] Example 1

[0028] This example provides an extraction method using ethylenediaminetetraacetic acid (EDTA) with a concentration of 0.1 mM as an extractant. The specific steps are as follows:

[0029] (1) Add ethylenediaminetetraacetic acid (EDTA) with a concentration of 0.1 mM to 500 mL of river water containing transparent extracellular polymeric particles to obtain a mixed solution. The volume ratio of EDTA to river water is 1:2.

[0030] (2) Shake the mixed solution in step (1) to make EDTA react fully with the transparent extracellular polymeric particles in the river water. The shaking treatment is carried out at 25 °C and 200 rpm for 3 h.

[0031] (3) Wait until the transparent extracellular polymeric particles in the river water are completely broken and the intracellular and extracellular DNA on them are completely released into the river water, and then carry out centrifugation treatment. The above centrifugation treatment is carried out at 25 °C and 8000 rpm for 20 min to obtain a supernatant containing all intracellular and extracellular DNA.

[0032] (4) Filter the supernatant in step (3) using a 0.22 μm filter membrane and a vacuum filtration device to collect extracellular DNA in the filtrate and intracellular DNA on the filter membrane, and complete the precise separation of intracellular and extracellular DNA in the river water.

[0033] (5) Use a free DNA extraction kit (Blood Free Circulating Nucleic Acid Extraction Kit (Magnetic Bead Method), purchased from Wuxi Baimaige Biotechnology Co., Ltd.) to extract extracellular DNA in the filtrate of step (4).

[0034] (6) Use a soil genomic DNA extraction kit (Fast Spin Kit for Soil, MP Biomedicals, USA) to extract intracellular DNA on the filter membrane of step (4).

[0035] Example 2

[0036] This example provides an extraction method using ethylenediaminetetraacetic acid (EDTA) with a concentration of 1 mM as an extractant. The specific steps are as follows:

[0037] (1) Add ethylenediaminetetraacetic acid (EDTA) with a concentration of 1 mM to 500 mL of river water containing transparent extracellular polymeric particles to obtain a mixed solution. The volume ratio of EDTA to river water is 1:2.

[0038] (2) Shake the mixed solution in step (1) so that EDTA fully reacts with the transparent extracellular polymeric particles in the river water. The shaking treatment is carried out at 25 °C and 200 rpm for 3 h.

[0039] (3) Wait until the transparent extracellular polymeric particles in the river water are completely ruptured and all the intracellular and extracellular DNA on them is completely released into the river water, and then perform centrifugation. The above centrifugation is carried out at 25 °C and 8000 rpm for 20 min to obtain a supernatant containing all intracellular and extracellular DNA.

[0040] (4) Filter the supernatant in step (3) using a 0.22 μm filter membrane and a vacuum filtration device to collect extracellular DNA in the filtrate and intracellular DNA on the filter membrane, thus completing the precise separation of intracellular and extracellular DNA in the river water.

[0041] (5) Use a free DNA extraction kit (Blood Free Circulating Nucleic Acid Extraction Kit (Magnetic Bead Method), purchased from Wuxi Biomarker Biotechnology Co., Ltd.) to extract extracellular DNA from the filtrate in step (4).

[0042] (6) Use a soil genomic DNA extraction kit (Fast Spin Kit for Soil, MP Biomedicals, USA) to extract intracellular DNA from the filter membrane in step (4).

[0043] Example 3

[0044] This example provides an extraction method using sodium pyrophosphate (Na4P2O7) with a concentration of 1 mM as an extractant. The specific steps are as follows:

[0045] (1) Add sodium pyrophosphate (Na4P2O7) with a concentration of 1 mM to 500 mL of river water containing transparent extracellular polymeric particles to obtain a mixed solution. The volume ratio of sodium pyrophosphate to river water is 1:5.

[0046] (2) Shake the mixed solution in step (1) so that sodium pyrophosphate fully reacts with the transparent extracellular polymeric particles in the river water. The shaking treatment is carried out at 25 °C and 200 rpm for 3 h.

[0047] (3) After the transparent extracellular polymeric particle in the river water is completely ruptured and all the intracellular and extracellular DNA on it is completely released into the river water, centrifugation is carried out. The above centrifugation is carried out at 25 °C and 8000 rpm for 20 min to obtain a supernatant containing all the intracellular and extracellular DNA.

[0048] (4) The supernatant in step (3) is filtered using a 0.22 μm filter membrane and a vacuum filtration device. The extracellular DNA is collected in the filtrate, and the intracellular DNA is collected on the filter membrane, thus completing the precise separation of the intracellular and extracellular DNA in the river water.

[0049] (5) An extracellular DNA extraction kit (Blood Cell-Free Circulating Nucleic Acid Extraction Kit (Magnetic Bead Method), purchased from Wuxi Biomarker Biotechnology Co., Ltd.) is used to extract the extracellular DNA in the filtrate of step (4).

[0050] (6) A soil genomic DNA extraction kit (Fast Spin Kit for Soil, MP Biomedicals, USA) is used to extract the intracellular DNA on the filter membrane in step (4).

[0051] Example 4

[0052] This example provides an extraction method using sodium pyrophosphate (Na4P2O7) with a concentration of 10 mM as an extractant. The specific steps are as follows:

[0053] (1) Sodium pyrophosphate (Na4P2O7) with a concentration of 10 mM is added to 500 mL of river water containing transparent extracellular polymeric particles to obtain a mixed solution. The volume ratio of sodium pyrophosphate to river water is 1:5.

[0054] (2) The mixed solution in step (1) is shaken to allow sodium pyrophosphate to react fully with the transparent extracellular polymeric particles in the river water. The shaking is carried out at 25 °C and 200 rpm for 3 h.

[0055] (3) After the transparent extracellular polymeric particle in the river water is completely ruptured and all the intracellular and extracellular DNA on it is completely released into the river water, centrifugation is carried out. The above centrifugation is carried out at 25 °C and 8000 rpm for 20 min to obtain a supernatant containing all the intracellular and extracellular DNA.

[0056] (4) The supernatant in step (3) is filtered using a 0.22 μm filter membrane and a vacuum filtration device. The extracellular DNA is collected in the filtrate, and the intracellular DNA is collected on the filter membrane, thus completing the precise separation of the intracellular and extracellular DNA in the river water.

[0057] (5) Use a free DNA extraction kit (Blood Free Circulating Nucleic Acid Extraction Kit (Magnetic Bead Method), purchased from Wuxi Biomarker Biotechnology Co., Ltd.) to extract the extracellular DNA in the filtrate of step (4).

[0058] (6) Use a soil genomic DNA extraction kit (Fast Spin Kit for Soil, from MP Biomedicals, USA) to extract the intracellular DNA on the filter membrane of step (4).

[0059] Example 5

[0060] This example provides an extraction method using sodium hexametaphosphate ((NaPO3)6) with a concentration of 0.5 mM as the extractant. The specific steps are as follows:

[0061] (1) Add sodium hexametaphosphate ((NaPO3)6) with a concentration of 0.5 mM to 500 mL of river water containing transparent extracellular polymeric particles to obtain a mixed solution. The volume ratio of sodium pyrophosphate to river water is 1:1.

[0062] (2) Shake the mixed solution in step (1) so that sodium hexametaphosphate reacts fully with the transparent extracellular polymeric particles in the river water. The shaking treatment is carried out at 25 °C and 200 rpm for 3 h.

[0063] (3) Wait until the transparent extracellular polymeric particles in the river water are completely broken, and after all the intracellular and extracellular DNA on them is completely released into the river water, perform centrifugation. The above centrifugation is carried out at 25 °C and 8000 rpm for 20 min to obtain a supernatant containing all intracellular and extracellular DNA.

[0064] (4) Filter the supernatant in step (3) using a 0.22 μm filter membrane and a vacuum filtration device to collect the extracellular DNA in the filtrate and the intracellular DNA on the filter membrane, thus completing the precise separation of intracellular and extracellular DNA in the river water.

[0065] (5) Use a free DNA extraction kit (Blood Free Circulating Nucleic Acid Extraction Kit (Magnetic Bead Method), purchased from Wuxi Biomarker Biotechnology Co., Ltd.) to extract the extracellular DNA in the filtrate of step (4).

[0066] (6) Use a soil genomic DNA extraction kit (Fast Spin Kit for Soil, from MP Biomedicals, USA) to extract the intracellular DNA on the filter membrane of step (4).

[0067] Example 6

[0068] This embodiment provides an extraction method using sodium hexametaphosphate ((NaPO3)6) with a concentration of 5 mM as an extractant. The specific steps are as follows:

[0069] (1) Add sodium hexametaphosphate ((NaPO3)6) with a concentration of 5 mM to 500 mL of river water containing transparent extracellular polymeric particles to obtain a mixed solution. The volume ratio of sodium pyrophosphate to river water is 1:1.

[0070] (2) Subject the mixed solution in step (1) to a shaking treatment to allow sodium hexametaphosphate to fully react with the transparent extracellular polymeric particles in the river water. The shaking treatment is carried out at 25 °C and 200 rpm for 3 h.

[0071] (3) Wait until the transparent extracellular polymeric particles in the river water are completely ruptured and the intracellular and extracellular DNA on them is completely released into the river water, and then carry out a centrifugation treatment. The above centrifugation treatment is carried out at 25 °C and 8000 rpm for 20 min to obtain a supernatant containing all intracellular and extracellular DNA.

[0072] (4) Filter the supernatant in step (3) using a 0.22 μm filter membrane and a vacuum filtration device to collect extracellular DNA in the filtrate and intracellular DNA on the filter membrane, thus completing the precise separation of intracellular and extracellular DNA in the river water.

[0073] (5) Use a free DNA extraction kit (Blood Free Circulating Nucleic Acid Extraction Kit (Magnetic Bead Method), purchased from Wuxi Biomarker Biotechnology Co., Ltd.) to extract the extracellular DNA in the filtrate of step (4).

[0074] (6) Use a soil genomic DNA extraction kit (Fast Spin Kit for Soil, MP Biomedicals, USA) to extract the intracellular DNA on the filter membrane of step (4).

[0075] Comparative Example

[0076] This comparative example provides an extraction method without using an extractant. The specific steps are as follows:

[0077] (1) Subject 500 mL of river water to a shaking treatment. The shaking treatment is carried out at 25 °C and 200 rpm for 3 h.

[0078] (2) Subject the shaken river water to a centrifugation treatment. The centrifugation treatment is carried out at 25 °C and 8000 rpm for 20 min to obtain a supernatant containing intracellular and extracellular DNA.

[0079] (3) Filter the supernatant in step (2) using a 0.22 μm filter membrane and a vacuum filtration device to collect extracellular DNA in the filtrate and intracellular DNA on the filter membrane, thus completing the separation of intracellular and extracellular DNA in river water.

[0080] (4) Use a free DNA extraction kit (Blood Free Circulating Nucleic Acid Extraction Kit (Magnetic Bead Method), purchased from Wuxi Baimaige Biotechnology Co., Ltd.) to extract the extracellular DNA in the filtrate of step (3).

[0081] (5) Use a soil genomic DNA extraction kit (Fast Spin Kit for Soil, MP Biomedicals, USA) to extract the intracellular DNA on the filter membrane of step (3).

[0082] Intracellular DNA (stained with Hoechst 33342 and showing blue fluorescence under fluorescence) and extracellular DNA (stained with DDAO and showing red fluorescence under fluorescence) in the transparent extracellular polymeric particles photographed by a fluorescence microscope. The results show that the red fluorescence is significantly higher than the blue fluorescence, indicating that the extracellular DNA in the transparent extracellular polymeric particles is significantly more than the intracellular DNA.

[0083] To verify the concentrations of intracellular and extracellular DNA extracted in each example and comparative example, the intracellular and extracellular DNA extracted in Examples 1 - 6 and the comparative example were respectively detected for concentration using a NanoDrop ND - 1000 ultra - micro spectrophotometer, and the results are as Figure 1 shown.

[0084] The content of intracellular DNA (iDNA) and extracellular DNA (eDNA) obtained in the comparative example were 18.03 μg / L and 10.13 μg / L respectively. The content of intracellular DNA (iDNA) and extracellular DNA (eDNA) obtained in Example 1 were 5.53 μg / L and 31.04 μg / L respectively; the content of intracellular DNA (iDNA) and extracellular DNA (eDNA) obtained in Example 2 were 6.59 μg / L and 34.35 μg / L respectively; the content of intracellular DNA (iDNA) and extracellular DNA (eDNA) obtained in Example 3 were 2.98 μg / L and 39.68 μg / L respectively; the content of intracellular DNA (iDNA) and extracellular DNA (eDNA) obtained in Example 4 were 7.09 μg / L and 31.60 μg / L respectively; the content of intracellular DNA (iDNA) and extracellular DNA (eDNA) obtained in Example 5 were 6.43 μg / L and 33.22 μg / L respectively; the content of intracellular DNA (iDNA) and extracellular DNA (eDNA) obtained in Example 6 were 4.00 μg / L and 36.81 μg / L respectively.

[0085] Compared with the comparative example, in Example 1, the dispersed extracellular polymeric substance (EPS) particles in water can reduce the intracellular DNA content by 69% and increase the extracellular DNA content by 63%; in Example 2, the intracellular DNA content can be reduced by 76% and the extracellular DNA content can be increased by 73%; in Example 3, the intracellular DNA content can be reduced by 83% and the extracellular DNA content can be increased by 89%; in Example 4, the intracellular DNA content can be reduced by 61% and the extracellular DNA content can be increased by 65%; in Example 5, the intracellular DNA content can be reduced by 64% and the extracellular DNA content can be increased by 69%; in Example 6, the intracellular DNA content can be reduced by 78% and the extracellular DNA content can be increased by 80%.

[0086] In summary, the extraction method provided by the present invention can release all the extracellular DNA in the extracellular polymeric substance (EPS) particles in water, accurately quantify the extracellular DNA content in water, solve the problem that the extracellular DNA content in water has been underestimated in the past, and is applicable to the research on the estimation of aquatic biomass, the feeding habits analysis of animals, population size and population dynamics, resistance genes, functional genes, etc.

[0087] The above-described embodiments are only a preferred solution of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical fields can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by adopting equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for accurately extracting intra- and extracellular DNA in natural water bodies, characterized in that, The specific steps are as follows: An extractant capable of binding metal ions is added to the target water body containing transparent extracellular polymeric particles to obtain a mixed solution; the mixed solution is shaken to allow the extractant to fully react with the transparent extracellular polymeric particles in the target water body; After the transparent extracellular polymeric particles in the target water body are completely ruptured and the intracellular and extracellular DNA on them is completely released into the target water body, centrifugation is carried out to obtain a supernatant containing all intracellular and extracellular DNA; the supernatant is filtered through a 0.22 μm filter membrane, the extracellular DNA is collected in the filtrate, and the intracellular DNA is collected on the filter membrane, completing the precise separation of intracellular and extracellular DNA in the target water body containing transparent extracellular polymeric particles; The extractant is one of ethylenediaminetetraacetic acid, sodium pyrophosphate or sodium hexametaphosphate that has no destructive effect on biological membranes and DNA; the extractant and the target water body are mixed in a volume ratio of 1: (1-10); The concentration of the ethylenediaminetetraacetic acid is 0.001 mM - 10 mM; the concentration of the sodium pyrophosphate is 1 mM - 20 mM; the concentration of the sodium hexametaphosphate is 0.01 mM - 15 mM.

2. The method for accurately extracting intracellular and extracellular DNA in natural water bodies according to claim 1, wherein The conditions for the shaking treatment are: the shaking speed is 150 - 250 rpm, the shaking time is 1 - 3 h, and the shaking temperature is 4 - 10 °C.

3. The method for precise extraction of intracellular and extracellular DNA in natural water bodies according to claim 1, characterized in that, The conditions for the centrifugation treatment are: the centrifugation speed is 6000 - 12000 rpm, the centrifugation time is 10 - 30 min, and the centrifugation temperature is 4 - 25 °C.

4. The method for accurately extracting intracellular and extracellular DNA in natural water bodies according to claim 1, wherein The filtration treatment uses a vacuum filtration method.

5. The method for accurately extracting intra- and extracellular DNA in natural water bodies according to claim 1, wherein, The extracellular DNA in the filtrate is extracted using a blood free circulating nucleic acid extraction kit.

6. The method for accurately extracting intracellular and extracellular DNA in natural water bodies according to claim 1, characterized in that The intracellular DNA on the filter membrane is extracted using a soil genomic DNA extraction kit.

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