A turbid water-like filter membrane environment DNA enrichment extraction device and method

Abstract

The present application relates to the technical field of DNA detection of aquatic environment, and particularly relates to a turbid water sample filter membrane environment DNA enrichment and extraction device and method. The present application is characterized by reasonable connection and setting of an invertible fixing support, a digestion dish, a funnel, a transfer tube, a centrifugal tube, a fixing sleeve and a suction pump, compact overall structure, time and labor saving operation, combined extraction of DNA from multiple filter membranes of the same sample, and greatly improved efficiency of turbid water sample environment DNA extraction. The present application lays multiple filter membranes of the same sample extracted by suction filtration in the digestion dish in a flat and stacked manner, without folding or cutting, and through the invertible fixing support, the digestion liquid can be sealed and transferred to the centrifugal tube, without using a pipette, effectively reducing the operation steps and reducing the pollution risk. The present application is convenient to operate as a whole, high in safety, low in cost, wide in application prospect, and easy to popularize.

Description

Technical Field

[0001] This invention relates to the field of aquatic organism environmental DNA detection technology, specifically to a device and method for enriching and extracting environmental DNA from turbid water samples using a filter membrane. Background Technology

[0002] Environmental DNA (eDNA) refers to DNA extracted from environmental samples and released by organisms into the environment. Organisms release their DNA into their environment through various pathways, including skin, mucus, urine, feces, blood, sperm and eggs, and decaying corpses. Soil, water, and air samples all contain large amounts of eDNA. Analyzing eDNA can provide information on the species, abundance, and distribution of various biological groups in aquatic ecosystems, such as microorganisms, algae, fish, and mammals, and has broad application prospects in aquatic ecological monitoring, biodiversity assessment, and water quality monitoring.

[0003] The first step in aquatic organism environmental DNA testing is to extract environmental DNA from the water sample. An environmental DNA sample usually requires 1 to 2 liters of water to be collected and filtered. However, in turbid water samples, due to the presence of a large number of suspended solids, organic matter and microorganisms, the filtration volume of a single filter membrane is greatly reduced, requiring 2 to 4 filter membranes to complete the filtration of a single water sample.

[0004] Current water sample DNA extraction kits can only extract from one filter membrane at a time. Samples with multiple filter membranes require multiple extractions, increasing workload and cost. Furthermore, existing tissue DNA extraction methods require cutting the filter membrane into small pieces with scissors and transferring the digestion solution using a pipette, increasing the risk of contamination and operational difficulty. Therefore, how to efficiently and conveniently perform combined extraction from multiple filter membranes is a crucial issue that urgently needs to be addressed for the enrichment of environmental DNA in turbid water samples. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an environmental DNA enrichment and extraction device and method for turbid water sample filter membranes, which can realize the extraction of DNA from multiple filter membranes of the same sample, save time and effort, greatly improve the efficiency of environmental DNA extraction, and reduce operational pollution.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: I. A device for enriching and extracting DNA from turbid water samples using a filter membrane. This invention provides a DNA enrichment and extraction device for turbid water samples using a filter membrane, mainly comprising: a fixed support 1, a vacuum pump 2 fixedly installed in the middle of the fixed support 1, and a fixed sleeve 3 installed at the vacuum port of the vacuum pump 2; a centrifuge tube 4 fixedly connected to one end of the fixed sleeve 3, and a transfer tube 5 sealed and inserted into the other end of the fixed sleeve 3; one end of the transfer tube 5 passes through the fixed sleeve 3 and extends into the centrifuge tube 4, and the other end of the transfer tube 5 is used to connect to a funnel 6. The upper funnel opening of the funnel 6 is provided with a sealing cap, and after the sealing cap is opened, it can be fixedly connected to the transfer tube 5; the lower funnel body of the funnel 6 is placed on the digestion dish 7 and fixedly connected to the digestion dish 7. Multiple water sample filter membranes are laid flat and stacked inside the digestion dish 7, and a preset dose of lysis solution is added. The fixed support 1 can be held upside down by hand to rotate the fixed sleeve 3, centrifuge tube 4, transfer tube 5, funnel 6 and digestion dish 7 together.

[0007] Preferably, the air pump 2 is provided with a one-way air outlet 8, and a control valve 9 is provided at the connection between the air pump 2 and the fixed sleeve 3.

[0008] Preferably, when the fixed support 1 is inverted, the fixed sleeve 3, centrifuge tube 4, transfer tube 5, funnel 6 and digestion dish 7 are rotated 180° together with the fixed support 1.

[0009] Preferably, one end of the fixing sleeve 3 is fixedly connected to the open end of the centrifuge tube 4 via a threaded connection.

[0010] Preferably, after the sealing cover is opened, the upper funnel opening of the funnel 6 is fixedly connected to the transfer tube 5 via a threaded connection.

[0011] Preferably, the lower part of the funnel 6 is fixedly connected to the opening end of the digestive dish 7 via a threaded connection.

[0012] Preferably, the preset dose of the lysis buffer is calculated and determined based on the number of water sample filter membranes laid flat and stacked in the digestion dish 7.

[0013] II. A method for DNA enrichment and extraction from turbid water samples using a filter membrane environment Based on the same inventive concept, the present invention also provides a method for enriching and extracting DNA from a turbid water sample filter membrane environment, based on the turbid water sample filter membrane environment DNA enrichment and extraction device described above, comprising the following steps: Step S1, Filter membrane preparation: Select n filter membranes with preset pore sizes; Step S2, water sample collection and filtration: Collect (n×X) mL of surface water as a sample, and filter X mL of water sample from each filter membrane. Then, stack the n filter membranes flat in a digestion dish. Step S3, DNA lysis and release: Add a preset dose of lysis buffer to the digestion dish, place the funnel body over the open end of the digestion dish, and close the sealing cap of the funnel mouth. Then place the digestion dish in a water bath at a preset temperature for a preset time, shaking and agitating appropriately during the reaction to lyse the cells and release DNA fragments. Step S4, Device Connection: Open the sealing cap of the funnel opening and thread-seal the funnel opening to the transfer tube port of the extraction device, so that the funnel opening is connected to the centrifuge tube through the transfer tube; Step S5, invert the support: Hold the inverted support and rotate the centrifuge tube, transfer tube, funnel and digestion dish 180° together with the support. Step S6, Obtaining digestive fluid: Let stand for a preset time of two to allow the digestive fluid in the digestion dish to flow into the centrifuge tube through the funnel and transfer tube. Then turn on the vacuum pump to transfer the remaining digestive fluid in the funnel and transfer tube to the centrifuge tube through vacuum pressure. Step S7, DNA extraction and purification: Centrifuge the centrifuge tube containing the digestion solution into a centrifuge apparatus, collect the supernatant, and extract and purify the DNA using a DNA extraction kit.

[0014] Preferably, in step S3, the preset dosage of the lysis solution is calculated using the following formula: H = X / 500 + 0.6 × (n-1) In the formula, H is the preset dose of the lysis solution, X is the amount of water sample filtered by a single filter membrane, and n is the number of filter membranes laid flat in the digestion dish.

[0015] Preferably, in step S3, a preset proportion of proteinase K is added to the lysis buffer. After the digestion dish is placed in a constant temperature water bath at a preset temperature one for DNA lysis and release reaction, it is then placed in an ice bath at a preset temperature two for a preset time three to cool and terminate the lysis reaction.

[0016] Compared with the prior art, the present invention has the following main advantages: 1. The present invention has a compact overall structure and saves time and effort in operation by reasonably connecting and setting up an invertable fixing support, digestion dish, funnel, transfer tube, centrifuge tube, fixing sleeve and air pump. It can realize the extraction of DNA from multiple filter membranes of the same sample, which can greatly improve the efficiency of DNA extraction in turbid water environment. 2. This invention lays multiple filter membranes of the same sample flat and stacks them in a digestion dish without folding or cutting them. Furthermore, by using an inverted fixing bracket, the digestion liquid can be sealed and transferred to centrifuge tubes without the need for a pipette, effectively reducing the number of operation steps and lowering the risk of contamination. 3. The invention is easy to operate, highly safe, and low in cost. It has broad application prospects and is easy to promote. Attached Figure Description

[0017] Figure 1 This is an overall schematic diagram of the DNA enrichment and extraction device for turbid water sample filtration membrane environment in an embodiment of the present invention; Figure 2 This is a schematic diagram of the DNA enrichment and extraction device for turbid water sample filter membrane environment in an embodiment of the present invention when it is inverted; Figure 3 This is a flowchart of the method for enriching and extracting DNA from turbid water samples using a filter membrane, as described in this embodiment of the invention.

[0018] In the diagram: 1-fixed bracket, 2-vacuum pump, 3-fixed sleeve, 4-centrifuge tube, 5-transfer tube, 6-funnel, 7-digestion dish, 8-one-way vent, 9-control valve. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0020] It should be noted that, depending on the implementation needs, the various steps / components described in this application can be broken down into more steps / components, or two or more steps / components or parts of the operation of steps / components can be combined into new steps / components to achieve the purpose of this invention.

[0021] In this invention, unless otherwise expressly specified and limited, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.

[0022] Example 1: This example provides a device for enriching and extracting environmental DNA from multiple filter membranes of turbid water samples. It can flatten and digest 2-4 filter membranes without folding or breaking them. The digestion solution is then sealed and transferred to a centrifuge tube. This solves the problem of combining multiple filter membranes for DNA extraction from the same sample, improves the efficiency of environmental DNA extraction, and reduces operational contamination.

[0023] like Figure 1 As shown, it mainly includes: a fixed bracket 1, a vacuum pump 2, a fixed sleeve 3, a centrifuge tube 4, a transfer tube 5, a funnel 6, a digestion dish 7, a one-way vent 8, and a control valve 9. The fixed bracket 1 is fixedly installed with an air pump 2 in the middle, and a fixed sleeve 3 is installed at the air pump port of the air pump 2; a centrifuge tube 4 is fixedly connected to one end of the fixed sleeve 3, and a transfer tube 5 is sealed and inserted into the other end of the fixed sleeve 3; one end of the transfer tube 5 passes through the fixed sleeve 3 and extends into the centrifuge tube 4, and the other end of the transfer tube 5 is used to connect to a funnel 6. The upper funnel opening of the funnel 6 is provided with a sealing cap, and after the sealing cap is opened, it can be fixedly connected to the transfer tube 5; the lower funnel body of the funnel 6 is placed on the digestion dish 7 and fixedly connected to the digestion dish 7. Multiple water sample filter membranes are laid flat and stacked inside the digestion dish 7, and a preset dose of lysis solution is added. The fixed support 1 can be held upside down by hand to rotate the fixed sleeve 3, centrifuge tube 4, transfer tube 5, funnel 6 and digestion dish 7 together.

[0024] Furthermore, the air pump 2 is provided with a one-way air outlet 8, and a control valve 9 is provided at the connection between the air pump 2's air inlet and the fixed sleeve 3.

[0025] Furthermore, when the fixed support 1 is inverted, the fixed sleeve 3, centrifuge tube 4, transfer tube 5, funnel 6 and digestion dish 7 are rotated 180° together with the fixed support 1.

[0026] Furthermore, one end of the fixed sleeve 3 is fixedly connected to the open end of the centrifuge tube 4 via a threaded connection.

[0027] Furthermore, after the sealing cover is opened, the upper funnel opening of the funnel 6 is fixedly connected to the transfer tube 5 via a threaded connection.

[0028] Furthermore, the lower part of the funnel 6 is fixedly connected to the opening end of the digestive dish 7 via a threaded connection.

[0029] Furthermore, the preset dosage of the lysis solution is calculated and determined based on the number of water sample filter membranes laid flat and stacked in the digestion dish 7.

[0030] Example 2, based on the same inventive concept, also provides a method for enriching and extracting DNA from a turbid water sample filter membrane environment, based on the turbid water sample filter membrane environment DNA enrichment and extraction device described above, such as... Figure 3 As shown, it includes the following steps: Step S1, Filter membrane preparation: Select n filter membranes with a preset pore size (2 filter membranes in this embodiment). Step S2, water sample collection and filtration: Collect (n×X) mL of surface water as a sample, and filter X mL of water from each filter membrane. Then, lay the n filter membranes flat in a digestion dish (in this embodiment, each filter membrane filters 500 mL of water, so 1 L of surface water needs to be collected as a sample). Step S3, DNA lysis and release: Add a preset dose of lysis buffer to the digestion dish, place the funnel body over the open end of the digestion dish, and close the sealing cap of the funnel mouth. Then place the digestion dish in a water bath at a preset temperature for a preset time (in this embodiment, it is placed in a 58°C water bath for 1 hour for digestion). During this time, shake and mix appropriately to lyse the cells and release DNA fragments. Step S4, Device Connection: Open the sealing cap of the funnel opening and thread-seal the funnel opening to the transfer tube port of the extraction device, so that the funnel opening is connected to the centrifuge tube through the transfer tube; Step S5, invert the support: Hold the inverted support and rotate the centrifuge tube, transfer tube, funnel and digestion dish 180° together with the support. Step S6, obtaining digestive fluid: let stand for a preset time of two (30 seconds in this embodiment) to allow the digestive fluid in the digestion dish to flow into the centrifuge tube through the funnel and transfer tube. Then turn on the vacuum pump (repeatedly turn it on 2-3 times) to fully transfer the remaining digestive fluid in the funnel and transfer tube to the centrifuge tube through vacuum pressure. In this embodiment, about 0.9 mL of digestive fluid can be obtained. Step S7, DNA extraction and purification: Centrifuge the centrifuge tube containing the digestion solution into a centrifuge apparatus, collect the supernatant, and extract and purify the DNA using a DNA extraction kit.

[0031] Furthermore, in step S3, the preset dosage of the lysis solution is calculated using the following formula: H = X / 500 + 0.6 × (n-1) In the formula, H is the preset dose of the lysis solution, X is the amount of water sample filtered by a single filter membrane, and n is the number of filter membranes laid flat in the digestion dish.

[0032] Furthermore, in step S3, a preset proportion of proteinase K is added to the lysis buffer. After the digestion dish is placed in a constant temperature water bath at a preset temperature one to carry out the DNA lysis and release reaction, it is then placed in an ice bath at a preset temperature two to cool for a preset time three (5-10 minutes in this embodiment) to terminate the lysis reaction.

[0033] Example 3: This example provides a method for enriching and extracting DNA from turbid water samples using a filter membrane, specifically using a mixed cellulose ester filter membrane with a pore size of 0.45 μm. The lysis buffer was used at a rate of 1 mL per filter membrane, with an additional 0.6 mL added for each additional filter membrane. For two filter membranes, 1.6 mL of lysis buffer was added. The lysis buffer consisted of 10 mmol / L Tris-HCl, pH 8.0; 50 mmol / L EDTA; 0.5% SDS, and contained 0.06 mL of proteinase K. After the centrifuge tubes are removed from the extraction device, they are centrifuged at 12,000 rpm for 5 minutes. The supernatant is then collected and DNA is extracted and purified using an existing DNA extraction kit.

[0034] Furthermore, all parts of this application that are not described in detail are the same as or implemented using existing technology.

[0035] In summary: 1. The present invention has a compact overall structure and saves time and effort in operation by reasonably connecting and setting up an invertable fixing support, digestion dish, funnel, transfer tube, centrifuge tube, fixing sleeve and air pump. It can realize the extraction of DNA from multiple filter membranes of the same sample, which can greatly improve the efficiency of DNA extraction in turbid water environment. 2. This invention lays multiple filter membranes of the same sample flat and stacks them in a digestion dish without folding or cutting them. Furthermore, by using an inverted fixing bracket, the digestion liquid can be sealed and transferred to centrifuge tubes without the need for a pipette, effectively reducing the number of operation steps and lowering the risk of contamination. 3. The present invention is easy to operate, highly safe, and low in cost. It has broad application prospects and is easy to promote.

[0036] Those skilled in the art will understand that embodiments of this application can be provided as methods, systems, or computer program products. Therefore, this application can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, this application can take the form of a computer program product embodied on one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0037] This application is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this application. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart... Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0038] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for enriching and extracting DNA from a turbid water sample filter membrane, based on a turbid water sample filter membrane environmental DNA enrichment and extraction device, characterized in that: The extraction device includes a fixed support (1), a vacuum pump (2) is fixedly installed in the middle of the fixed support (1), and a fixed sleeve (3) is installed at the suction port of the vacuum pump (2); a centrifuge tube (4) is fixedly connected to one end of the fixed sleeve (3), and a transfer tube (5) is sealed and inserted at the other end of the fixed sleeve (3); one end of the transfer tube (5) passes through the fixed sleeve (3) and extends into the centrifuge tube (4), and the other end of the transfer tube (5) is used to connect to a funnel (6); The upper funnel opening of the funnel (6) is provided with a sealing cap, and after the sealing cap is opened, it can be fixedly connected to the transfer tube (5); the lower funnel body of the funnel (6) is placed on the digestion dish (7) and fixedly connected to the digestion dish (7); multiple water sample filter membranes are laid flat and stacked inside the digestion dish (7), and a preset dose of lysis solution is added. The fixed support (1) can be held upside down by hand so that the fixed sleeve (3), centrifuge tube (4), transfer tube (5), funnel (6) and digestion dish (7) can be rotated together; The extraction method includes the following steps: Step S1, Filter membrane preparation: Select n filter membranes with preset pore sizes; Step S2, water sample collection and filtration: Collect (n×X) mL of surface water as a sample, and filter X mL of water sample from each filter membrane. Then, stack the n filter membranes flat in a digestion dish. Step S3, DNA lysis and release: Add a preset dose of lysis buffer to the digestion dish, place the funnel body over the open end of the digestion dish, close the sealing cap of the funnel opening, and then place the digestion dish in a water bath at a preset temperature for a preset time. The formula for calculating the preset dosage of the lysis buffer is as follows: H = X / 500 + 0.6 × (n-1) In the formula, H is the preset dose of the lysis solution, X is the amount of water sample filtered by a single filter membrane, and n is the number of filter membranes laid flat and stacked in the digestion dish. Step S4, Device Connection: Open the sealing cap of the funnel opening and thread-seal the funnel opening to the transfer tube port of the extraction device, so that the funnel opening is connected to the centrifuge tube through the transfer tube; Step S5, invert the support: Hold the inverted support and rotate the centrifuge tube, transfer tube, funnel and digestion dish together with the support. Step S6, Obtaining digestive fluid: Let stand for a preset time of two to allow the digestive fluid in the digestion dish to flow into the centrifuge tube through the funnel and transfer tube. Then turn on the vacuum pump to transfer the remaining digestive fluid in the funnel and transfer tube to the centrifuge tube through vacuum pressure. Step S7, DNA extraction and purification: Place the centrifuge tube containing the digestion solution into a centrifuge apparatus for centrifugation, collect the supernatant, and use a DNA extraction kit to extract and purify the DNA.

2. The method for enriching and extracting DNA from turbid water samples using a filter membrane according to claim 1, characterized in that, The air pump (2) is provided with a one-way air outlet (8), and a control valve (9) is provided at the connection between the air pump (2) and the fixed sleeve (3).

3. The method for enriching and extracting DNA from turbid water samples using a filter membrane according to claim 1, characterized in that, One end of the fixed sleeve (3) is threaded to the open end of the centrifuge tube (4).

4. The method for enriching and extracting DNA from turbid water samples using a filter membrane according to claim 1, characterized in that, After the upper funnel opening of the funnel (6) is opened, it is threadedly connected to the transfer tube (5).

5. The method for enriching and extracting DNA from turbid water samples using a filter membrane according to claim 1, characterized in that, The lower part of the funnel (6) is threadedly connected to the opening end of the digestive dish (7).

6. The method for enriching and extracting DNA from turbid water samples using a filter membrane according to claim 1, characterized in that... In step S3, a preset proportion of proteinase K is added to the lysis buffer. After the digestion dish is placed in a constant temperature water bath at a preset temperature one for DNA lysis and release reaction, it is then placed in an ice bath at a preset temperature two for a preset time three to terminate the lysis reaction.

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