Device and method for enriching and extracting DNA of turbid water sample in filter membrane environment

Abstract

The invention relates to the technical field of aquatic organism environment DNA detection, in particular to a turbid water sample filter membrane environment DNA enrichment and extraction device and method. Through reasonable connection and arrangement of the invertible fixed bracket, the digestion vessel, the funnel, the transfer tube, the centrifugal tube, the fixed sleeve and the sucking pump, the whole structure is compact, time and labor are saved in operation, DNA extraction by multiple filter membranes of the same sample can be realized, and the efficiency of DNA extraction in a turbid water sample environment can be greatly improved; according to the invention, a plurality of filter membranes for suction filtration of the same sample are flatly laid and stacked in the digestion vessel without folding or cutting, and the digestive juice can be hermetically transferred to the centrifugal tube by inverting the fixed bracket without using a pipettor, so that the operation steps are effectively reduced, and the pollution risk is reduced; the method is convenient to operate, high in safety, low in cost, wide in application prospect and easy to popularize.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic biological environmental DNA detection, and in particular to a device and method for enriching and extracting environmental DNA from a turbid water sample filter membrane. Background Art

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

[0003] The environmental DNA test for aquatic organisms first requires the extraction of environmental DNA from water samples. One environmental DNA sample usually requires the collection of 1 to 2 liters of water samples for filtration. However, in turbid water samples, due to the presence of a large amount of suspended matter, organic matter, microorganisms and other impurities, the filtration volume of a single filter membrane will be greatly reduced, and 2 to 4 filter membranes are required to completely extract one water sample.

[0004] Currently available water sample DNA extraction kits can only extract one filter at a time. Samples with multiple filters require multiple extractions, increasing workload and costs. Furthermore, existing tissue DNA extraction methods require the filter to be shredded with scissors and the digestion solution to be transferred using a pipette, increasing contamination risks and operational complexity. Therefore, efficient and convenient combined extraction of multiple filters is a key challenge that urgently needs to be addressed in environmental DNA enrichment from turbid water samples. Summary of the Invention

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

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: 1. A turbid water sample filter membrane environmental DNA enrichment and extraction device The present invention provides a device for enriching and extracting DNA from a turbid water sample filter membrane environment, which mainly comprises: a fixed bracket 1, a suction pump 2 is fixedly installed in the middle of the fixed bracket 1, and a fixed sleeve 3 is installed at the suction port of the suction pump 2; one end of the fixed sleeve 3 is fixedly connected to a centrifuge tube 4, and the other end of the fixed sleeve 3 is sealed and inserted with a transfer tube 5; 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 cover, and after the sealing cover is opened, it can be fixedly connected to the transfer tube 5; the lower bucket cover of the funnel 6 is provided on the digestion dish 7 and is fixedly connected to the digestion dish 7. A plurality of water sample filter membranes are laid flat and stacked in the digestion dish 7, and a preset dose of lysis solution is added; The fixing bracket 1 can be handheld and turned upside down to drive the fixing sleeve 3, centrifuge tube 4, transfer tube 5, funnel 6 and digestion dish 7 to turn over together.

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

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

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

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

[0011] Preferably, the lower part of the funnel 6 is fixedly connected to the open end of the digestion vessel 7 through a threaded connection.

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

[0013] 2. A method for enriching and extracting environmental DNA from turbid water samples using filter membranes Based on the same inventive concept, the present invention also provides a method for enriching and extracting environmental DNA from a turbid water sample filter membrane, which is based on the above-mentioned environmental DNA enrichment and extraction device for a turbid water sample filter membrane, 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 sample as a sample, and filter X mL of water sample through each filter membrane, and then lay n filter membranes flat and stacked in a digestion dish; Step S3, DNA lysis and release: a preset dose of lysis solution is added to the digestion dish, and the funnel cover is placed on the open end of the digestion dish, and the sealing cover of the funnel mouth is closed. The digestion dish is then placed in a water bath at a preset temperature for a preset reaction time, with appropriate shaking during the reaction to lyse the cells and release DNA fragments; Step S4, device connection: open the sealing cover of the funnel opening, and thread and seal the funnel opening with 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, inverting the support: holding the inverted fixed support, so that the centrifuge tube, transfer tube, funnel and digestion dish are turned 180 degrees together with the fixed support; Step S6, obtaining digestion fluid: standing for a preset time of two, allowing the digestion fluid in the digestion dish to flow into the centrifuge tube through the funnel and transfer tube, then turning on the vacuum pump to fully transfer the residual digestion 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 device for centrifugation, take the supernatant, and use a DNA extraction kit to extract and purify DNA.

[0014] Preferably, in step S3, the preset dosage calculation formula of the lysate is as follows: H=X / 500+0.6×(n-1) Wherein, H is the preset dosage of the lysate, X is the amount of water sample filtered by a single filter membrane, and n is the number of filter membranes stacked flat in the digestion dish.

[0015] Preferably, in step S3, a preset ratio of proteinase K is added to the lysis solution, the digestion dish is placed in a constant temperature water bath at a preset temperature of one for DNA cleavage and release reaction, and then placed in an ice bath at a preset temperature of two for a preset time of three to terminate the cleavage 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 by rationally connecting and setting up the inverted fixed bracket, digestion dish, funnel, transfer tube, centrifuge tube, fixed sleeve and vacuum pump. It can realize the combined extraction of DNA from multiple filter membranes of the same sample, which can greatly improve the efficiency of DNA extraction in turbid water samples. 2. The present invention lays multiple filter membranes for filtering the same sample flat in a digestion dish without folding or cutting them. By inverting the fixed bracket, the digestion solution can be sealed and transferred to a centrifuge tube without the need for a pipette, effectively reducing the number of operation steps and the risk of contamination. 3. The overall operation of the present invention is convenient, safe, low-cost, has broad application prospects, and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is an overall schematic diagram of the turbid water sample filter membrane environmental DNA enrichment and extraction device in an embodiment of the present invention; Figure 2 Schematic diagram of the inverted device for enriching and extracting DNA from a turbid water sample filter membrane according to an embodiment of the present invention; Figure 3 Flow chart of the method for enriching and extracting environmental DNA from turbid water sample filter membrane in an embodiment of the present invention.

[0018] In the figure: 1-fixed bracket, 2-vacuum pump, 3-fixed sleeve, 4-centrifuge tube, 5-transfer tube, 6-funnel, 7-digestion dish, 8-one-way air outlet, 9-control valve. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is 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 for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0020] It should be pointed out that, according to the needs of implementation, the various steps / components described in this application can be split into more steps / components, or two or more steps / components or partial operations of steps / components can be combined into new steps / components to achieve the purpose of the present invention.

[0021] In the present invention, unless otherwise expressly specified or limited, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise expressly specified or limited.

[0022] Example 1. This embodiment provides a turbid water sample filter membrane environmental DNA enrichment and extraction device, which is used to extract environmental DNA from multiple filter membranes of turbid water samples. 2 to 4 filter membranes can be flatly digested without folding or breaking the filter membranes, and the digestion liquid is sealed and transferred to a centrifuge tube to solve the problem of merging multiple filter membranes of the same sample to extract DNA, improve the efficiency of environmental DNA extraction, and reduce operational pollution.

[0023] like Figure 1 As shown, it mainly includes: a fixed bracket 1, an air pump 2, a fixed sleeve 3, a centrifuge tube 4, a transfer tube 5, a funnel 6, a digestion dish 7, a one-way air outlet 8, and a control valve 9; Among them, an air pump 2 is fixedly installed in the middle of the fixed bracket 1, and a fixed sleeve 3 is installed at the air suction port of the air pump 2; one end of the fixed sleeve 3 is fixedly connected to a centrifuge tube 4, and the other end of the fixed sleeve 3 is sealed and inserted with a transfer tube 5; 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 cover, and after the sealing cover is opened, it can be fixedly connected to the transfer tube 5; the lower bucket cover of the funnel 6 is provided on the digestion dish 7 and is fixedly connected to the digestion dish 7. A plurality of water sample filter membranes are laid flat and stacked in the digestion dish 7, and a preset dose of lysis solution is added; The fixing bracket 1 can be handheld and turned upside down to drive the fixing sleeve 3, centrifuge tube 4, transfer tube 5, funnel 6 and digestion dish 7 to turn over together.

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

[0025] Furthermore, when the fixed support 1 is inverted, the fixed sleeve 3 , the centrifuge tube 4 , the transfer tube 5 , the funnel 6 and the digestion dish 7 are turned 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 through 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 pipe 5 through a threaded connection.

[0028] Furthermore, the lower part of the funnel 6 is fixedly connected to the open end of the digestion vessel 7 through 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 stacked flat in the digestion dish 7 .

[0030] Example 2, based on the same inventive concept, this embodiment 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 environmental DNA enrichment and extraction device as described above, such as Figure 3 As shown, the following steps are included: Step S1, filter membrane preparation: select n filter membranes with preset pore sizes (two filter membranes in this embodiment); Step S2, water sample collection and filtration: Collect (n × X) mL of surface water sample as a sample, and filter X mL of water sample through each filter membrane. Then, stack n filter membranes flatly in a digestion dish (in this embodiment, each filter membrane filters 500 mL of water sample, so 1 L of surface water sample is collected as a sample); Step S3, DNA lysis and release: A preset dose of lysis solution is added to the digestion dish, and the funnel cover is placed on the open end of the digestion dish. The sealing cover of the funnel mouth is closed at the same time. The digestion dish is then placed in a water bath at a preset temperature for a preset reaction time (in this embodiment, it is placed in a water bath at 58°C for 1 hour), and the digestion dish is shaken appropriately during the reaction to lyse the cells and release DNA fragments. Step S4, device connection: open the sealing cover of the funnel opening, and thread and seal the funnel opening with 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, inverting the support: holding the inverted fixed support, so that the centrifuge tube, transfer tube, funnel and digestion dish are turned 180 degrees together with the fixed support; Step S6, obtaining digestion fluid: Allow the vessel to stand for a preset time of two (30 seconds in this embodiment) to allow the digestion fluid in the digestion vessel to flow into the centrifuge tube through the funnel and transfer tube. Then, turn on the vacuum pump (repeatedly 2-3 times) to fully transfer the residual digestion fluid in the funnel and transfer tube to the centrifuge tube using vacuum pressure. In this embodiment, approximately 0.9 mL of digestion fluid can be obtained. Step S7, DNA extraction and purification: Place the centrifuge tube containing the digestion solution into a centrifuge device for centrifugation, take the supernatant, and use a DNA extraction kit to extract and purify DNA.

[0031] Furthermore, in step S3, the preset dosage calculation formula of the lysate is as follows: H=X / 500+0.6×(n-1) Wherein, H is the preset dosage of the lysate, X is the amount of water sample filtered by a single filter membrane, and n is the number of filter membranes stacked flat in the digestion dish.

[0032] Furthermore, in step S3, a preset ratio of proteinase K is added to the lysis solution, the digestion dish is placed in a constant temperature water bath at a preset temperature of one for DNA cleavage and release reaction, and then placed in an ice bath at a preset temperature of two for a preset cooling time of three (5 to 10 minutes in this embodiment) to terminate the cleavage reaction.

[0033] Example 3: This example provides a method for enriching and extracting environmental 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 amount of the lysis buffer used is 1 mL per filter membrane. For each additional filter membrane, add 0.6 mL. For two filter membranes, add 1.6 mL of lysis buffer (10 mmol / L Tris-HCl, pH 8.0; 50 mmol / L EDTA; 0.5% SDS) and add 0.06 mL of proteinase K. After the centrifuge tube is removed from the extraction device, it is centrifuged at a speed of 12,000 rpm for 5 minutes, and the supernatant is collected. 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 the existing technology or are implemented using the existing technology.

[0035] In summary: 1. The present invention has a compact overall structure and saves time and effort by rationally connecting and setting up the inverted fixed bracket, digestion dish, funnel, transfer tube, centrifuge tube, fixed sleeve and vacuum pump. It can realize the combined extraction of DNA from multiple filter membranes of the same sample, which can greatly improve the efficiency of DNA extraction in turbid water samples. 2. The present invention lays multiple filter membranes for filtering the same sample flat in a digestion dish without folding or cutting them. By inverting the fixed bracket, the digestion solution can be sealed and transferred to a centrifuge tube without the need for a pipette, effectively reducing the number of operation steps and the risk of contamination. 3. The overall operation of the present invention is convenient, safe, low-cost, has broad application prospects, and is easy to promote.

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

[0037] The present application is described with reference to the flowcharts and / or block diagrams of the methods, devices (systems), and computer program products according to the embodiments of the present application. It should be understood that each process and / or block in the flowchart and / or block diagram, as well as the combination of processes and / or blocks in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowchart and / or block diagram. Figure 1 a process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0038] It will be easily understood by those skilled in the art 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 in the scope of protection of the present invention.

Claims

1. A turbid water sample filter membrane environmental DNA enrichment and extraction device, characterized by: It comprises a fixed bracket (1), a vacuum pump (2) is fixedly installed in the middle of the fixed bracket (1), and a fixed sleeve (3) is installed at the vacuum port of the vacuum pump (2); one end of the fixed sleeve (3) is fixedly connected to a centrifuge tube (4), and the other end of the fixed sleeve (3) is sealed and inserted with a transfer tube (5); 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 cover, and after the sealing cover is opened, it can be fixedly connected to the transfer tube (5); the lower bucket cover of the funnel (6) is provided on the digestion dish (7) and is fixedly connected to the digestion dish (7), and a plurality of water sample filter membranes are laid flat and stacked in the digestion dish (7), and a preset dose of lysis solution is added; The fixed support (1) can be handheld and inverted to drive the fixed sleeve (3), centrifuge tube (4), transfer tube (5), funnel (6) and digestion dish (7) to flip together.

2. The turbid water sample filter membrane environmental DNA enrichment and extraction device 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 outlet of the air pump (2) and the fixed sleeve (3).

3. The turbid water sample filter membrane environmental DNA enrichment and extraction device according to claim 1, characterized in that: When the fixed support (1) is inverted, the fixed sleeve (3), the centrifuge tube (4), the transfer tube (5), the funnel (6) and the digestion dish (7) are turned 180 degrees together with the fixed support (1).

4. The turbid water sample filter membrane environmental DNA enrichment and extraction device according to claim 1, characterized in that: One end of the fixed sleeve (3) is fixedly connected to the open end of the centrifuge tube (4) via a threaded connection.

5. The turbid water sample filter membrane environmental DNA enrichment and extraction device according to claim 1, characterized in that: The upper funnel opening of the funnel (6) is fixedly connected to the transfer tube (5) through a threaded connection after the sealing cover is opened.

6. The turbid water sample filter membrane environmental DNA enrichment and extraction device according to claim 1, characterized in that: The lower part of the funnel (6) is fixedly connected to the open end of the digestion vessel (7) via a threaded connection.

7. The turbid water sample filter membrane environmental DNA enrichment and extraction device according to claim 1, characterized in that: The preset dosage of the lysis solution is calculated and determined based on the number of water sample filter membranes stacked flat in the digestion dish (7).

8. A method for enriching and extracting DNA from a turbid water sample filter membrane environment, based on the turbid water sample filter membrane environmental DNA enrichment and extraction device according to any one of claims 1 to 7, characterized in that: The steps include: 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 sample as a sample, and filter X mL of water sample through each filter membrane, and then lay n filter membranes flat and stacked in a digestion dish; Step S3, DNA lysis and release: adding a preset dose of lysis solution to the digestion dish, placing the funnel cover on the open end of the digestion dish, and closing the sealing cover of the funnel mouth, and then placing the digestion dish in a water bath at a preset temperature for a preset reaction time; Step S4, device connection: open the sealing cover of the funnel opening, and thread and seal the funnel opening with 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, inverting the support: holding the inverted fixed support, so that the centrifuge tube, transfer tube, funnel and digestion dish are turned over together with the fixed support; Step S6, obtaining digestion fluid: standing for a preset time of two, allowing the digestion fluid in the digestion dish to flow into the centrifuge tube through the funnel and transfer tube, then turning on the vacuum pump to fully transfer the residual digestion 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 device for centrifugation, take the supernatant, and use a DNA extraction kit to extract and purify DNA.

9. The method for enriching and extracting environmental DNA from turbid water sample filter membrane according to claim 8, characterized in that In step S3, the preset dosage calculation formula of the lysate is as follows: H=X / 500+0.6×(n-1) Wherein, H is the preset dosage of the lysate, X is the amount of water sample filtered by a single filter membrane, and n is the number of filter membranes stacked flat in the digestion dish.

10. The method for enriching and extracting environmental DNA from turbid water sample filter membrane according to claim 8, characterized in that In step S3, a preset ratio of proteinase K is added to the lysis solution, the digestion dish is placed in a constant temperature water bath at a preset temperature of one for DNA cleavage and release reaction, and then placed in an ice bath at a preset temperature of two for a preset time of three to terminate the cleavage reaction.

Images