Environmental DNA extraction kit based on magnetic nanoparticles as well as extraction method and application of environmental DNA extraction kit

Abstract

The invention discloses an environmental DNA (deoxyribonucleic acid) extraction kit based on magnetic nanoparticles as well as an extraction method and application of the environmental DNA extraction kit. The environment DNA extraction kit comprises a nanoparticle solution, pH test paper, a Tris solution, nuclease-free water and diluted hydrochloric acid. By using the extraction kit, enrichment and extraction are carried out according to the designed magnetic nanoparticle dosage and eluent volume, the environmental DNA in the water body can be enriched and extracted, the process steps are simple and convenient, the required field sampling equipment is less, the use of toxic organic reagents is avoided, meanwhile, the method also has the advantage of easy realization of automation, eDNA can be obtained from the water body on a large scale, and the method is suitable for industrial production. And the extraction effect is good. The quality and purity of the eDNA extracted by the method are superior to those of the eDNA extracted by the traditional method, the extraction effect is good, and reliable theoretical and technical bases are provided for optimization of an environmental DNA technology and establishment of a unified standard.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and in particular relates to an environmental DNA extraction kit based on magnetic nanoparticles, an extraction method and application thereof. Background Art

[0002] Biological monitoring is an important prerequisite for ecological environment management and protection. At present, with the rapid development of molecular biology technology, species detection methods have begun to transition from traditional morphological classification to molecular biology, especially environmental DNA technology. Environmental DNA (eDNA) technology refers to the enrichment and extraction of DNA left by different species in water environment samples, and species detection through molecular labeling, sequencing and other technologies. As an emerging biological monitoring technology in water environment, environmental DNA technology has been widely used in biodiversity surveys, invasive species monitoring and other aspects, with advantages such as accurate and sensitive detection and little interference with the ecosystem. However, there are many methods for enriching and extracting environmental DNA in water bodies, but there is no unified standard. Traditional environmental DNA technology requires eDNA enrichment through large volumes of water samples during sampling, which is very cumbersome and time-consuming; the sampling process requires carrying a large amount of equipment and tools; the extraction of environmental DNA generally requires the addition of toxic organic reagents; the environmental DNA extraction process is small-scale and difficult to achieve efficient automation. Therefore, it is necessary to establish a fast and simple environmental DNA extraction method to optimize the process of water environmental DNA technology and promote the application of this technology in ecology and other fields.

[0003] Nanoparticles are artificially made microparticles with a size between 1 and 100 nanometers. They are in the form of metal particles, carbon particles, silica particles, etc. They are widely used in medicine, environment and other fields. At present, nanoparticles can be transformed in various ways to obtain a structure that simulates the virus spike to achieve binding with other molecules. Therefore, how to use the characteristics of this nanoparticle to capture eDNA in the water environment is of great significance. In addition, it is necessary to study two key issues: what kind of transformation should be carried out based on this nanoparticle and how to design the enrichment and extraction process design. Summary of the invention

[0004] To solve the above technical problems, the present invention provides an environmental DNA extraction kit based on magnetic nanoparticles, and its extraction method and application. The present invention designs a magnetic silica nanoparticle with a simulated virus spike structure, and then designs a rapid extraction kit, which can efficiently and conveniently enrich and extract environmental DNA in water, so as to be used for subsequent library construction, sequencing analysis, etc.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The invention provides an environmental DNA extraction kit based on magnetic nanoparticles, which comprises a nanoparticle solution, a pH test paper, an eluent and a regulator.

[0006] Furthermore, the concentration of the nanoparticle solution is 10 mg / ml to 20 mg / ml.

[0007] Furthermore, the eluent is a Tris solution and nuclease-free water; the pH of the Tris solution is 12-14, and the concentration is 1 mM-1.5 mM.

[0008] Furthermore, the regulator is dilute hydrochloric acid.

[0009] The present invention also provides a method for extracting water environmental DNA using the environmental DNA extraction kit, which comprises the following steps: (1) Collect water samples, add nanoparticle solution and mix well to adsorb environmental DNA; (2) recovering the nanoparticles obtained in step (1); (3) Using an eluent to elute the nanoparticles of step (2), and using a regulator to adjust the pH value of the solution to neutral to obtain environmental DNA.

[0010] Furthermore, the recovery method in step (2) is a centrifugal method or a magnetic guidance method.

[0011] Furthermore, the recovery step of the centrifugation method is: centrifuge a 50 ml centrifuge tube at 4200 rpm / min for 30 minutes, aspirate the supernatant, wash the centrifuged material precipitate with 500 μL Nuclease-free Water, and transfer the liquid to a new 1.5 ml centrifuge tube.

[0012] Furthermore, the recovery steps of the magnetic guidance method are as follows: placing the centrifuge tube on the magnetic rack for 10 minutes, the nanoparticles will gather on the side wall of the centrifuge tube, aspirating the supernatant in the center of the centrifuge tube and transferring it to a new 50ml centrifuge tube, retaining about 0.5ml of seawater at the bottom of the old centrifuge tube, oscillating the material adhering to the tube wall, and transferring the liquid to a new 1.5ml centrifuge tube. Placing the new centrifuge tube on the magnetic rack for 10 minutes, aspirating the supernatant in the same way, retaining about 0.5ml of seawater at the bottom of the centrifuge tube, oscillating the material adhering to the tube wall, and transferring the liquid to the original 1.5ml centrifuge tube.

[0013] Furthermore, when the centrifugal method is used for recovery, the amount of the nanoparticle solution added is 200 μL~250 μL; or when the magnetic guidance method is used, the amount of the nanoparticle solution added is 400 μL~500 μL.

[0014] Furthermore, the volume ratio of the eluent to the nanoparticle solution is 1:1-2.

[0015] The invention also provides application of the environmental DNA extraction kit in biodiversity survey or invasive species monitoring.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention uses magnetic nanoparticles to prepare a water body environmental DNA extraction kit, and uses the kit and its technical process to extract water body environmental DNA, so that high-quality environmental DNA can be extracted from the water body. The method is simple and fast, avoids the cumbersome steps of collecting water bodies and filtering in traditional environmental DNA technology, requires less field sampling equipment, avoids the use of toxic organic reagents, and also has the advantage of easy automation, can obtain eDNA from water bodies on a large scale, and has a good extraction effect. The quality and purity of the eDNA extracted by the present invention are better than those of traditional methods, and the extraction effect is good, which provides a reliable theoretical and technical basis for the optimization of environmental DNA technology and the establishment of unified standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The relationship between the amount of different nanoparticles used and the quality of extracted eDNA in the centrifugation method; Figure 2 The relationship between different elution volumes and the quality of extracted eDNA; Figure 3 This is a comparison of the effects of extracting eDNA by centrifugation and magnetic guidance. Figure 4 This is a comparison of the effects of eDNA extraction using liquid phase separation and magnetic nanoparticles. Figure 5 This is a comparison of the purity of eDNA extracted by magnetic nanoparticle method and liquid phase separation method. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to. Example 1

[0024] The environmental DNA extraction process and method based on magnetic nanoparticles are as follows: 1. Magnetic nanoparticles adsorb eDNA 1. Collect water samples Use 50ml centrifuge tubes to collect 50ml of water sample from the sampling location.

[0025] 2. Adding Nanoparticles Gently shake the 10 mg / ml nanoparticle solution, add the appropriate amount of nanoparticles to a 50 ml centrifuge tube (add 200 μL of nanoparticles for centrifugation method and 400 μL of nanoparticles for magnetic guidance method), and mix gently.

[0026] 3. Binding of nanoparticles to eDNA Run the centrifuge tube on a shaker or suspension instrument at low speed (10 rpm / min) for 10 to 30 minutes at room temperature (the running time can be appropriately extended as needed).

[0027] 2. Recycling of Magnetic Nanoparticles 1. Choose a method There are two ways to recover the eDNA-adsorbed nanoparticles in the centrifuge tube: centrifugation and magnetic guidance.

[0028] 2. Centrifugation Centrifuge the 50ml centrifuge tube at 4200rpm / min for 30 minutes, aspirate the supernatant, wash the precipitated material with 500μL Nuclease-free Water, and transfer the liquid to a new 1.5ml centrifuge tube. (This step is not required if the magnetic guidance method is selected).

[0029] 3. Magnetic guidance method Place the centrifuge tube on the magnetic rack for 10 minutes. The nanoparticles will gather on the side wall of the centrifuge tube. Pipette the supernatant in the center of the centrifuge tube and transfer it to a new 50ml centrifuge tube. Keep about 0.5ml of seawater at the bottom of the old centrifuge tube, shake the material adhering to the tube wall, and transfer the liquid to a new 1.5ml centrifuge tube. Place the new centrifuge tube on the magnetic rack for 10 minutes. Pipette the supernatant in the same way, keep about 0.5ml of seawater at the bottom of the centrifuge tube, shake the material adhering to the tube wall, and transfer the liquid to the original 1.5ml centrifuge tube. (This step is not performed if the centrifugation method is selected).

[0030] 3. Washing, Preservation and Elution of Magnetic Nanoparticles 1. Washing Nanoparticles Place the 1.5 ml centrifuge tube on the magnetic stand for 1 min, remove the supernatant, wash with 500 μL Nuclease-free Water, and finally add 500 μL Nuclease-free Water again.

[0031] 2. Preservation of Nanoparticles Store the 1.5 ml centrifuge tube in an ice box and proceed to the next step in the laboratory.

[0032] 3. Elute eDNA from magnetic nanoparticles Place the 1.5ml centrifuge tube on the magnetic rack for 1 minute, remove the supernatant, add 200μL of 1mM Tris solution (pH=about 13), and run at low speed (about 10 rpm / min) on a shaker or suspension instrument at room temperature for 30 minutes. Place the 1.5ml centrifuge tube on the magnetic rack for 1 minute, take the supernatant and add it to a new 1.5ml centrifuge tube.

[0033] 4. Remove impurities from the supernatant If the solution is turbid, you can choose to centrifuge the solution at 6000 rpm / min for 3 minutes. Impurities will precipitate at the bottom of the centrifuge tube. Take the supernatant and add it to a new 1.5ml centrifuge tube.

[0034] 5. Obtaining eDNA Solution Use dilute hydrochloric acid (HCl) to adjust the pH of the solution to neutral (7-8), which is the extracted eDNA solution. The eDNA sample is stored at -80°C. Example 2

[0035] Environmental DNA in water was extracted by magnetic nanoparticles, the appropriate amount of nanoparticles and the appropriate volume of elution solution were tested, and the effect of this method on extracting eDNA was compared with that of liquid phase separation.

[0036] 1. Explore the appropriate amount of magnetic nanoparticles for extracting eDNA by centrifugation Experimental content: Multiple groups of 50ml seawater were collected from the experimental animal breeding room, and the seawater eDNA was extracted by centrifugation using magnetic nanoparticles. The control group included positive control, negative control 1, and negative control 2; the experimental group included five groups, and the dosage of 10mg / ml nanoparticles was 50, 100, 150, 200, and 250μL respectively. The quality of eDNA extracted between multiple groups was compared to determine the appropriate dosage of nanoparticles. The experimental design is shown in Table 1.

[0037] Table 1 Experimental design and changes in concentration and quality of eDNA extracted in each group

[0038] The experimental results are as follows Figure 1 As shown in the figure: In the control group, eDNA was extracted from the positive control group, and almost no eDNA was extracted from the negative control group. In the experimental group, the quality of eDNA extracted increased continuously from 50μL to 250μL nanoparticle dosage, but reached a plateau when the nanoparticle dosage was 200μL. There was no significant difference in the quality of eDNA extracted by nanoparticles with dosages of 200μL and 250μL (P > 0.05). Taking all factors into consideration, the appropriate dosage of nanoparticles is 200μL.

[0039] 2. Explore the appropriate eluent ratio for extracting eDNA using magnetic nanoparticles by centrifugation Experimental content: 50ml of seawater was collected from the experimental animal breeding room, and eDNA was extracted from seawater by centrifugation using magnetic nanoparticles. The control group used enzyme-free water to elute the nanoparticles, and the experimental group used 1mM Tris solution (pH=13) to elute. The three groups were divided into three groups, and the volume ratio of eluent to nanoparticles was 1:1, 2:3, and 2:1, respectively. The quality of eDNA extracted between multiple groups was compared to determine the appropriate eluent ratio. The experimental design is shown in Table 2.

[0040] Table 2 Design of Experiment 2 and changes in concentration and quality of eDNA extracted in each group

[0041] The experimental results are as follows Figure 2 As shown in the figure, almost no eDNA was extracted from the control group; in the experimental group, the volume ratio of nanoparticles to eluent increased slightly from 1:1 to 1:2, but there was no significant difference in the quality of eDNA extracted between the experimental groups (P > 0.05). Taking all factors into consideration, the appropriate eluent ratio is 1:1.

[0042] 3. Comparison of the effects of magnetic guidance and centrifugation in extracting eDNA to determine the appropriate amount of nanoparticles Experimental content: Multiple groups of 50ml seawater were collected from the experimental animal breeding room, and magnetic nanoparticles were used to extract seawater eDNA by magnetic guidance and centrifugation. The control group was to extract seawater eDNA by centrifugation to recover nanoparticles (the amount of nanoparticles was 200μL), and the experimental group was to extract seawater eDNA by magnetic guidance to recover nanoparticles. According to the amount of nanoparticles, they were divided into four groups, and the amount of nanoparticles was 200, 300, 400, and 500μL respectively. The quality of eDNA extracted between multiple groups was compared to compare the eDNA extraction effect of centrifugation and magnetic guidance, and to determine the appropriate amount of nanoparticles for magnetic guidance. The experimental design is shown in Table 3.

[0043] Table 3 Design of Experiment 3 and changes in concentration and quality of eDNA extracted in each group

[0044] The experimental results are as follows Figure 3 As shown in the figure, in the magnetic guidance method, the amount of nanoparticles increased to 400 μL, and the quality of extracted eDNA reached a plateau. In this experiment, the quality of eDNA extracted by centrifugation was greater than that of each group of magnetic guidance method, and when the amount of nanoparticles in the magnetic guidance method was 400 μL, the difference in the quality of eDNA extracted by centrifugation and magnetic guidance was very significant (P < 0.01). Taking all factors into consideration, the eDNA extraction effect of nanoparticles recovered by centrifugation is better than that of magnetic guidance method, and the appropriate amount of nanoparticles for magnetic guidance method is 400 μL.

[0045] 4. Comparison of eDNA extraction effects between magnetic nanoparticles and liquid phase separation Experimental content: 50 ml of seawater was collected from the experimental animal breeding room, and the quality and purity of the extracted eDNA were compared to compare the effects of using magnetic nanoparticles and liquid phase separation to extract eDNA from seawater. Liquid phase separation method: 50 ml of water sample was filtered through a filter membrane, and eDNA was extracted mainly by phenol chloroform extraction method, ethanol sodium acetate precipitation method, etc. The experimental design is shown in Table 4.

[0046] Table 4 Experimental 4 design and changes in concentration, quality and purity of eDNA extracted in each group

[0047] The experimental results are as follows Figure 4 and Figure 5 As shown in the figure: The difference in the quality of eDNA extracted by magnetic nanoparticles and liquid phase separation is very significant (P < 0.01), and the average eDNA quality extracted by magnetic nanoparticles is greater than that by liquid phase separation. The average values ​​of A260 / 280 of eDNA extracted by magnetic nanoparticles and liquid phase separation are both lower than 1.8, but the average value of A260 / 280 of magnetic nanoparticles is higher than that of liquid phase separation, and the purity of eDNA extracted by magnetic nanoparticles is better than that of liquid phase separation. Taking all factors into consideration, the eDNA extraction effect of magnetic nanoparticles is better than that of liquid phase separation in terms of quality and purity.

[0048] Example 3 A kit for extracting environmental DNA from water bodies: The kit (stored at 4°C) includes: 1. Nanoparticle solution for extracting environmental DNA from water bodies, with a concentration of 10 mg / ml; 2. pH test paper, used to detect the pH value of the solution; 3. 1mM Tris solution (pH=13 or so), used to elute the eDNA from the magnetic nanoparticles; 4. Dilute hydrochloric acid (HCl), used to adjust the pH of the eDNA solution to neutral (7-8); 5. Nuclease-free Water.

[0049] The method of using the kit is as follows: Use a 50ml centrifuge tube to collect 50ml of water sample from the sampling site. Gently shake the 10mg / ml nanoparticle solution, add the appropriate amount of nanoparticles to the 50ml centrifuge tube (add 200μL nanoparticles for centrifugal method and 400μL nanoparticles for magnetic guidance method), and mix gently. Run the centrifuge tube on a shaker or suspension instrument at 10 rpm / min for 10~30min at room temperature.

[0050] The magnetic nanoparticles were recovered by centrifugation or magnetic guidance. The recovery steps of the centrifugation method are as follows: centrifuge the 50ml centrifuge tube at 4200rpm / min for 30 minutes, aspirate the supernatant, wash the precipitated material with 500μL Nuclease-free Water, and transfer the liquid to a new 1.5ml centrifuge tube. The recovery steps of the magnetic guidance method are as follows: place the centrifuge tube on the magnetic rack for 10 minutes, the nanoparticles will gather on the side wall of the centrifuge tube, aspirate the supernatant in the center of the centrifuge tube and transfer it to a new 50ml centrifuge tube, retain about 0.5ml of seawater at the bottom of the old centrifuge tube, oscillate the material adhering to the tube wall, and transfer the liquid to a new 1.5ml centrifuge tube. Place the new centrifuge tube on the magnetic rack for 10 minutes, aspirate the supernatant in the same way, retain about 0.5ml of seawater at the bottom of the centrifuge tube, oscillate the material adhering to the tube wall, and transfer the liquid to the original 1.5ml centrifuge tube.

[0051] Place the 1.5ml centrifuge tube on the magnetic rack for 1min, remove the supernatant, wash with 500μL Nuclease-free Water, and finally add 500μL Nuclease-free Water again. Store the 1.5ml centrifuge tube in an ice box. Place the 1.5ml centrifuge tube on the magnetic rack for 1min, remove the supernatant, add 200μL 1mM Tris solution (pH=about 13), and run it at 10 rpm / min for 30min at room temperature on a shaker or suspension instrument. Place the 1.5ml centrifuge tube on the magnetic rack for 1min, take the supernatant and add it to a new 1.5ml centrifuge tube. If the solution is turbid, choose to centrifuge the solution at 6000rpm / min for 3min in a centrifuge. Impurities will precipitate at the bottom of the centrifuge tube. Take the supernatant and add it to a new 1.5ml centrifuge tube. Use dilute hydrochloric acid (HCl) to adjust the pH of the solution to neutral (7-8), which is the extracted eDNA solution. The eDNA sample is stored at -80℃.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An environmental DNA extraction kit based on magnetic nanoparticles, characterized in that: The environmental DNA extraction kit comprises a nanoparticle solution, a pH test paper, an eluent and a regulator.

2. The environmental DNA extraction kit according to claim 1, characterized in that The concentration of the nanoparticle solution is 10 mg / ml to 20 mg / ml.

3. The environmental DNA extraction kit according to claim 1, characterized in that The eluent is a Tris solution and nuclease-free water; the pH of the Tris solution is 12-14, and the concentration is 1 mM-1.5 mM.

4. The environmental DNA extraction kit according to claim 1, characterized in that The regulator is dilute hydrochloric acid.

5. The method for extracting water environmental DNA using the environmental DNA extraction kit according to claim 1, characterized in that: The extraction method comprises the following steps: (1) Collect water samples, add nanoparticle solution and mix well to adsorb environmental DNA; (2) recovering the nanoparticles obtained in step (1); (3) Using an eluent to elute the nanoparticles of step (2), and using a regulator to adjust the pH value of the solution to neutral to obtain environmental DNA.

6. The method according to claim 5, characterized in that The recovery method in step (2) is centrifugation or magnetic guidance.

7. The method according to claim 6, characterized in that When the centrifugal method is used for recovery, the amount of the nanoparticle solution added is 200 μL~250 μL; or when the magnetic guidance method is used, the amount of the nanoparticle solution added is 400 μL~500 μL.

8. The method according to claim 5, characterized in that The volume ratio of the eluent to the nanoparticle solution is 1:1-2.

9. Use of the environmental DNA extraction kit according to claim 1 in biodiversity surveys or invasive species monitoring.

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