An environmental DNA filtering filter element, enricher, enrichment device and sampling method

Abstract

The application belongs to the technical field of environmental monitoring, and relates to an environmental DNA filtering filter core, an enriching device, an enriching device and a sampling method. In view of the technical problem that the filtering device in the prior art has a small volume of environmental DNA enrichment, low enrichment efficiency and easy sample pollution, and thus a small number of detected species, the application provides an environmental DNA filtering filter core, which comprises filter layers and two head end covers, the filter layers are located between the two head end covers and connected with the two head end covers, the filter layers are folded for multiple times, so that the cross section of the filter layers is arranged in a wave shape, and the number of folding layers of the filter layers is 40-65. The enrichment rate of the sample is increased, a high elution rate can be maintained, and the number of detected species is increased. The application further provides an environmental DNA enriching device and an enriching device, which realize multi-channel, large-capacity and rapid simultaneous enrichment of samples, and reduce sample pollution. The application further provides an environmental DNA sampling method, which adopts continuous underway sampling and is suitable for large-basin water environment.

Description

Technical Field

[0001] This invention relates to the field of environmental monitoring technology, and more specifically, to an environmental DNA sample filter cartridge, enrichment device, and sampling method. Background Technology

[0002] Environmental DNA (eDNA) refers to the sum of DNA fragments that can be directly extracted from environmental samples, including mixtures of DNA from different species such as microorganisms, animals, and plants. This DNA can originate from intracellular DNA released into the environment through skin, urine, feces, mucus, etc., or from extracellular DNA released into the environment after cell death and lysis. Environmental DNA macrobarcoding technology refers to the high-throughput identification of multiple species using DNA isolated from environmental samples (such as soil, sediment, and water). Traditional sampling methods for ecological research often present challenges in collecting samples from some species, being time-consuming and labor-intensive. Environmental DNA allows for non-invasive sampling from soil, water, or air, including animal hair, shed cells, feces, urine, blood, sperm, eggs, roots, leaves, and fruits, enabling successful genetic testing. Compared to traditional direct sampling, this method is less costly, less affected by weather conditions, and allows for the collection of larger quantities of samples.

[0003] Compared to traditional morphological monitoring methods, environmental DNA technology offers a new technical means to rapidly and accurately grasp changes in aquatic communities and the effectiveness of ecological restoration measures, potentially supplementing or even replacing traditional morphological biomonitoring methods. However, current environmental DNA technology faces challenges in large-scale river studies due to the large water body size and low DNA concentrations of protected species. Adopting a reasonable environmental DNA sampling strategy is a prerequisite for rapid and accurate monitoring. Membrane filtration has been successfully applied to environmental DNA sampling in the Yangtze River, using 0.5L–1.5L of water filtered through a 0.45-micron polyethersulfone (PES) membrane for environmental DNA enrichment. Conventional environmental DNA filtration methods using small water samples (<6L) have proven ineffective for monitoring low-density target species such as rare fish species. Larger water sample volumes can increase the concentration of target DNA in the sample and improve the detection results of environmental DNA.

[0004] The application of environmental DNA technology in aquatic biodiversity monitoring is becoming increasingly mature. However, the total environmental DNA concentration of the samples used for assessing species is significantly affected by species distribution, abundance, and environmental factors in practice. Currently, the industry mostly uses negative pressure pumps combined with flat sheet membranes (0.45μm~5μm) for sample enrichment. This method suffers from slow enrichment speed, small enrichment volume, a shortage of professional portable sampling equipment, and is particularly problematic for large water bodies such as oceans, frequent sample enrichment, and monitoring of special species (such as rare and endangered species, invasive species), which is time-consuming, labor-intensive, and has low enrichment efficiency. The accuracy of the survey and assessment is also easily questioned. Furthermore, the availability of mature sampling equipment is limited due to industry development; the available equipment also generally carries the risk of cross-contamination between samples and suffers from high costs for sampling consumables. Summary of the Invention

[0005] 1. The technical problem that the invention aims to solve

[0006] Existing filtration devices suffer from limitations such as small enrichment volume, low enrichment efficiency, and susceptibility to contamination, resulting in a limited number of detected species. This application addresses these issues by providing an environmental DNA filter cartridge that increases both enrichment and elution rates. Furthermore, this application provides an enricher and enrichment device that enables rapid, simultaneous enrichment of samples across multiple channels and with large capacity, reducing contamination and improving sample representativeness. Additionally, this application provides an environmental DNA sampling method that detects more species through continuous mobile sampling.

[0007] 2. Technical Solution

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0009] An environmental DNA filter cartridge includes a filter layer and two end caps. The filter layer is located between and connected to the two end caps. The filter layer is folded multiple times to make the cross-section of the filter layer wavy. The number of folded layers of the filter layer is 40 to 65.

[0010] Furthermore, the filter layer has 50 to 55 folded layers.

[0011] Preferably, the filter membrane in the filter layer has a filter resolution of 0.45 μm to 5 μm.

[0012] Furthermore, the filter element includes a support plate structure; the enrichment rate of the filter element is ≥80%, and the elution rate of the filter element is ≥50%.

[0013] Preferably, the enrichment rate of the filter element is ≥90%, and the elution rate of the filter element is ≥85%.

[0014] Furthermore, the filter element includes a supportless structure; the enrichment rate of the filter element is ≥80%, and the elution rate of the filter element is ≥60%.

[0015] Preferably, the enrichment rate of the filter element is ≥90%, and the elution rate of the filter element is ≥95%.

[0016] An environmental DNA enrichment device includes an outer casing, a filter element, a first port, and a second port. The filter element is disposed inside the outer casing, and the first port and the second port are respectively disposed on the water inlet side and the water outlet side of the outer casing. The filter element is the aforementioned environmental DNA filter element.

[0017] An environmental DNA enrichment device includes a housing, a first pump, a second pump, a main switch, a first switch, a second switch, and an enricher. The main switch, the first switch, and the second switch are disposed on the surface of the housing. The first pump and the second pump are disposed on the housing. Both the first pump and the second pump are peristaltic pumps. The enricher is connected to the first pump and / or the second pump. The enricher is the aforementioned environmental DNA enricher.

[0018] Furthermore, the pipeline of the first pump includes a first inlet pipe and a first outlet pipe, the first inlet pipe being connected to the inlet end of the first pump, the first outlet pipe being connected to the outlet end of the first pump, and the enrichment device being connected in the middle of the first inlet pipe or the first outlet pipe; the pipeline of the second pump includes a second inlet pipe and a second outlet pipe, the second inlet pipe being connected to the inlet end of the second pump, the second outlet pipe being connected to the outlet end of the second pump, and the enrichment device being connected in the middle of the second inlet pipe or the second outlet pipe.

[0019] Furthermore, it also includes a battery, a power display screen, and a charging port; the battery is disposed inside the housing, and the power display screen and the charging port are disposed on the surface of the housing; the main switch is electrically connected to the battery, the power display screen, the first switch, and the second switch; the battery is electrically connected to the charging port.

[0020] An environmental DNA sampling method includes the following steps:

[0021] The enrichment device is placed horizontally on the ship, such that the pipes of the first inlet pipe and / or the second inlet pipe extend below the water surface.

[0022] The boat was driven to start traveling along a direction parallel to the riverbank from section one, the enrichment device was turned on, and continuous mobile sampling began. Sampling ended when the boat reached section two.

[0023] Furthermore, the volume of filtered water sample is counted during the sampling process; three parallel biological samples are collected in each route of the mobile sampling.

[0024] Preferably, the sampling method is applicable to large river basin water environments and special species survey and detection environments.

[0025] 3. Beneficial effects

[0026] Compared with existing known technologies, the technical solution provided by this invention has the following significant advantages:

[0027] (1) The present invention provides an environmental DNA filter cartridge, comprising a filter layer and two end caps, wherein the filter layer is located between and connected to the two end caps; the filter layer is folded multiple times to make the cross section of the filter layer wavy, the number of folded layers of the filter layer is 45 to 60, which increases the filtration area, thereby increasing the enrichment volume, thereby increasing the enrichment rate and elution rate of environmental DNA samples, and ultimately increasing the concentration of environmental DNA samples and detecting more species.

[0028] (2) The present invention provides an environmental DNA enrichment device that uses the filter cartridge of the present application and also includes an outer cover, a first port and a second port. The filter cartridge is set inside the outer cover, and the first port and the second port are respectively set on the water inlet side and the water outlet side of the outer cover. The structure is simple and easy to replace, and meets the sampling needs of different filter diameters and enrichment volumes. This tangential flow filtration device effectively increases the filtration volume of the enrichment device and realizes large-capacity rapid enrichment of samples.

[0029] (3) The environmental DNA enrichment device provided by this invention uses a high-flow peristaltic pump as power, has multiple channels with independent channel control, and is easy to rapidly enrich multiple samples simultaneously on-site; combined with a tangential flow enricher with a pleated filter cartridge, it effectively increases the filter volume and achieves large-capacity rapid enrichment of samples; it provides an environmental DNA enrichment device for the investigation and detection of large watershed water environments and special species (such as rare and endangered species, invasive species, etc.), improving the representativeness of samples. The enrichment device of this application has a simple structure, small size, and is easy to carry. By changing the installation position of the enricher, it can achieve multi-functional enrichment under positive and negative pressure to meet the sampling needs of different scenarios. Except for the pump pipe, the collected water sample does not come into contact with any parts of the pump, effectively reducing sample contamination; simple and standard pipeline replacement effectively reduces contamination between samples. The consumables of the enrichment device are selected from commonly used standard parts on the market, reducing consumable costs.

[0030] (4) The present invention provides an environmental DNA sampling method that uses the enrichment device of this application to continuously conduct mobile sampling from section one to section two. The enrichment effect is better than that of sampling at each point separately. For large watershed water environment, special species investigation and detection environment, the mobile sampling enrichment effect is more representative. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the enrichment device structure of the present invention;

[0032] Figure 2 This is a top view of the enrichment device of the present invention;

[0033] Figure 3 This is a sectional view of the enrichment device of the present invention from the DD plane;

[0034] Figure 4 This is a negative pressure enrichment flow path diagram of the enrichment device of the present invention;

[0035] Figure 5 This is a positive pressure enrichment flow path diagram of the enrichment device of the present invention;

[0036] Figure 6 This is a schematic diagram of the enrichment device of the present invention;

[0037] Figure 7 The image shows the analysis of overlapping fish species detected in Example 3 and Comparative Example 1.

[0038] Figure 8 This is a comparison chart of the number of fish detected in different taxonomic ranks for Example 3 and Comparative Example 1.

[0039] Explanation of the labels in the diagram:

[0040] 1. First pump; 11. First water inlet pipe; 12. First drain pipe; 2. Second pump; 21. Second water inlet pipe; 22. Second drain pipe; 3. Main switch; 4. First switch; 5. Second switch; 6. Power display screen; 7. Handle; 8. Charging port; 9. Housing;

[0041] 10. Enrichment unit; 101. Outer casing; 102. Filter element; 103. First port; 104. Second port;

[0042] 13. Battery. Detailed Implementation

[0043] To make the above-mentioned objects, features, and advantages of the invention more apparent and understandable, specific embodiments of the invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0044] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0045] Furthermore, 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 explicitly specified.

[0046] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0047] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0048] It should be noted that when an element is referred to as being "fixed to" or "set on" another element, it can be directly on the other element or there may be an intervening element. When an element is considered to be "connected to" another element, it can be directly connected to the other element or there may be an intervening element. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used herein are for illustrative purposes only and do not represent the only possible implementation.

[0049] Example 1

[0050] An environmental DNA enrichment device according to this embodiment, such as Figure 1-6 As shown, the device includes a housing 9, a first pump 1, a second pump 2, a main switch 3, a first switch 4, a second switch 5, a battery 13, a power display screen 6, and an enrichment unit 10. The main switch 3, the first switch 4, the second switch 5, and the power display screen 6 are located on the surface of the housing 9. The first pump 1 and the second pump 2 are located on the housing 9. The battery 13 is located inside the housing 9 and is used to power the device for normal operation. The main switch 3 is connected to the battery 13, the power display screen 6, the first switch 4, and the second switch 5 by wires. The first switch 4 is connected to the main switch 3 and the first pump 1 by wires. The second switch 5 is connected to the main switch 3 and the second pump 2 by wires. The first pump 1 and the second pump 2 are both peristaltic pumps. Except for the peristaltic pump tubing, the fluid does not come into contact with any part of the peristaltic pump, effectively reducing sample contamination. The peristaltic pump has bidirectional equal flow delivery capability, can generate a high vacuum at the inlet end, and can generate a high positive pressure (0.3 MPa) at the outlet end, which can easily realize multi-functional sampling of negative and positive pressure enrichment of samples, meeting the pressure and flow requirements of different sampling methods. The enrichment device 10 is connected to the first pump 1 and / or the second pump 2. The pipeline of the first pump 1 includes a first inlet pipe 11 and a first drain pipe 12. The first inlet pipe 11 is connected to the inlet end of the first pump 1, and the first drain pipe 12 is connected to the outlet end of the first pump 1. The enrichment device 10 is connected in the middle of the first inlet pipe 11 or the first drain pipe 12. The pipeline of the second pump 2 includes a second inlet pipe 21 and a second drain pipe 22. The second inlet pipe 21 is connected to the inlet end of the second pump 2, and the second drain pipe 22 is connected to the outlet end of the second pump 2. The enrichment device 10 is connected in the middle of the second inlet pipe 21 or the second drain pipe 22.

[0051] It should be noted that the housing 9 serves as protection and support for the equipment, and its material can be low-density, high-strength, corrosion-resistant aluminum alloy and plastic.

[0052] An environmental DNA enrichment device according to this embodiment also includes a charging port 8 and a handle 7. Both the charging port 8 and the handle 7 are disposed on the surface of the housing 9. The charging port 8 is connected to the battery 13 wire for charging through the battery of a dedicated charging device. The handle 7 facilitates carrying the device.

[0053] An environmental DNA enrichment device according to this embodiment, such as Figure 6 As shown, the system includes an outer cover 101, a filter element 102, a first port 103, and a second port 104. The filter element 102 is disposed inside the outer cover 101. The first port 103 and the second port 104 are respectively located on the water inlet side and the water outlet side of the outer cover 101. Water flows from the first port 103 into the second port 104. The filter element 102 includes a filter layer and two end caps. The filter layer is located between and connected to the two end caps. The filter layer is folded multiple times to create a wavy cross-section. The number of folded layers in the filter layer is 40 to 65. In this embodiment, the filter layer is folded multiple times to form a hollow columnar structure. The two ends of the columnar structure are glued to the two end caps. The number of folded layers in the filter layer is 50 to 55, resulting in high enrichment and elution rates. In other embodiments, the number of folded layers in the filter layer is 40 to 45 or 60 to 65.

[0054] Specifically, the enricher 10 is a disposable tangential flow filter, consisting of a complete filter element 102 sealed within a robust disposable polypropylene outer casing 101. The filter element 102 is preferably made of polyethersulfone (PES). The filter element 102 may include a supportless or supported structure. The filter membrane precision (filter diameter) in the filter layer is 0.45 μm to 5 μm. In this embodiment, the filter diameter is 0.45 μm. In other embodiments, filter diameters of 1 μm, 3 μm, or 5 μm are used to meet different enrichment needs. The enricher 10 of this application features a large filtration area, good enrichment effect, high temperature resistance, high pressure resistance, and easy elution. A comparison of the internal structure effects of enrichers 10 with different parameters is shown in Table 1.

[0055] Table 1 Comparison of the effects of the internal structure of enricher 10

[0056]

[0057] Note: Enrichment rate and elution rate are the average values ​​of the three samples; the number of fish species is the total number of fish species detected, meaning that they must be detected in at least two samples simultaneously.

[0058] As shown in Table 1, under the same specifications, materials, and filter membrane precision, the more folds in the filter layer, the larger the filtration area, the larger the sample enrichment volume, and consequently the higher the sample enrichment rate. When the filter element has a supportless structure, with 60-65 folds, the enrichment rate can reach up to 92%, but the corresponding elution rate is only up to 60%. Fewer folds result in a higher elution rate; with 40-45 folds, the elution rate can reach up to 95%, but the corresponding enrichment rate is only up to 80%. For environmental DNA sample detection, with 50-55 folds, the enrichment rate is up to 90%, and the corresponding elution rate is up to 95%, both showing high enrichment and elution rates, resulting in better monitoring performance and the detection of up to 45 fish species. Compared to common filter structures on the market, the supportless design effectively improves the elution efficiency and detection effect of the enrichment device 10. In subsequent embodiments, the filter element 102 of the enrichment device 10 is selected to be without a support plate, with a filter membrane accuracy of PES 0.45μm, a height of 1.5 inches, a diameter of 60mm, and 50 folds in the filter layer.

[0059] Example 2

[0060] An environmental DNA enrichment device according to this embodiment can meet the sampling needs of both negative pressure enrichment flow path and positive pressure enrichment flow path.

[0061] The negative pressure enrichment flow path steps of the environmental DNA enrichment device are as follows:

[0062] Place the device near a water source. First, install the enricher 10 in the middle of the first inlet pipe 11 according to the water inlet and outlet directions. Connect one end of the first inlet pipe 11 to the inlet port of the peristaltic pump (first pump 1) and place the other end at the water source. Connect the first drain pipe 12 to the outlet of the peristaltic pump (first pump 1) to form a closed pipeline. Turn on the main switch 3, and the power display screen 6 can display the power and voltage of the battery 13 in real time. Turn on the corresponding channel switch (first switch 4), and the corresponding peristaltic pump (first pump 1) will start running. When the peristaltic pump is running continuously, the sample is sucked from the water source into the inlet pipe, passes through the enricher 10 and the peristaltic pump, and is discharged from the drain pipe. The environmental DNA sample in the enricher 10 is enriched by the vacuum negative pressure of the peristaltic pump. Based on the negative pressure enrichment flow path, cross-contamination between samples can be avoided by replacing the enricher 10, cleaning or replacing the inlet pipe each time.

[0063] In some other embodiments, the enricher 10 can be installed in the middle of the second inlet pipe 21 in the direction of water inlet and outlet to form a single-channel negative pressure enrichment flow path for the enrichment device; or the enricher 10 can be installed in the middle of the first inlet pipe 11 and the second inlet pipe 21 in the direction of water inlet and outlet to form a dual-channel negative pressure enrichment flow path for the enrichment device.

[0064] The positive pressure enrichment flow path steps of the environmental DNA enrichment device are as follows:

[0065] Place the device near a water source. First, connect one end of the first inlet pipe 11 to the inlet port of the peristaltic pump (first pump 1), and place the other end at the water source. Install the enricher 10 in the middle of the first drain pipe 12 according to the water inlet and outlet directions. Connect the first drain pipe 12 to the outlet of the peristaltic pump (first pump 1) to form a closed pipeline. Turn on the main switch 3, and the power display screen 6 can display the power and voltage of the battery 13 in real time. Turn on the corresponding channel switch (first switch 4), and the corresponding peristaltic pump (first pump 1) will start running. When the peristaltic pump is running continuously, the sample is sucked from the water source into the inlet pipe, passes through the peristaltic pump and the enricher 10, and is discharged from the drain pipe. The environmental DNA sample in the enricher 10 is enriched by the high pressure at the outlet of the peristaltic pump. Based on the positive pressure enrichment flow path, cross-contamination between samples can be avoided by replacing the enricher 10, replacing or cleaning the inlet pipe, peristaltic pump pipe and drain pipe each time.

[0066] In some other embodiments, the enricher 10 can be installed in the middle of the second drain pipe 22 in the direction of water inlet and outlet to form a single-channel positive pressure enrichment flow path for the enrichment device; or the enricher 10 can be installed in the middle of the first drain pipe 12 and the second drain pipe 22 in the direction of water inlet and outlet to form a dual-channel positive pressure enrichment flow path for the enrichment device.

[0067] Example 3

[0068] This embodiment of an environmental DNA sampling method, tangential flow filtration mobile sampling, includes the following steps:

[0069] S1. Place the enrichment device horizontally at a suitable sampling location;

[0070] S2, such as Figure 4 Piping setup: Install the enrichment device 10 between the first inlet pipe 11 and the second inlet pipe 21 according to the water inlet and outlet directions; connect one end of the first inlet pipe 11 to the inlet port of the peristaltic pump (first pump 1) and place the other end at the water source; connect the first drain pipe 12 to the outlet of the peristaltic pump (first pump 1) to form a closed pipeline; connect one end of the second inlet pipe 21 to the inlet port of the peristaltic pump (second pump 2) and place the other end at the water source; connect the second drain pipe 22 to the outlet of the peristaltic pump (second pump 2) to form a closed pipeline.

[0071] The peristaltic pump tubing connecting the enrichment unit 10 and the peristaltic pump needs to be replaced for each sample, while the peristaltic pump tubing at the drain end can be reused.

[0072] S3. Turn on the main switch 3 and check the voltage and power level through the power display (if the power is insufficient, it needs to be charged before operation, and the power level must be ≥50%).

[0073] S4. Connect a peristaltic pump tube of appropriate length to the inlet port of the enricher 10 and place the peristaltic pump tube below the water surface;

[0074] S5. When a boat traveling parallel to the riverbank starts from section YCS16 of the Yangtze River (section one, i.e., point A), the first switch 4 and the second switch 5 are turned on to enrich and sample the surface water through two channels. In this embodiment, three parallel biological samples are collected, so two enrichment devices are used. The discharged water is collected in a container, and the volume of the filtered water sample is counted.

[0075] At the start of filtration, a syringe can be used to time and measure the filtration rate, and the filtration time can be estimated based on the pre-filtered water volume. At half the filtration time, the syringe can be used again to time and measure the filtration rate. If the flow rate slows down significantly, the filtration time can be extended.

[0076] S6. When the ship reaches the YCS17 section of the Yangtze River (section two, i.e. point D), the filtration ends, the peristaltic pump tube is lifted out of the water and the relevant channel pumps continue to run to empty the water sample in the large-capacity enrichment device 10.

[0077] S7. Close the first switch 4 and the second switch 5, record the filtered water volume as 20L / sample, and collect three biological parallel samples (i.e., 3 biological replicates) for each route, for a total of 3 samples (60L of water samples in total).

[0078] S8. Remove the large-capacity enrichment device 10, remove the Luer inner spiral plug, open the vent, and use a 50ml syringe to inject 50ml of DNA later storage buffer into the connector.

[0079] S9. After adding the storage buffer, tighten the Luer inner screw plug and vent, then seal with a sealing film, shake well, mark the sample site number with a marker, and finally put the sample into a small sealed bag for storage.

[0080] In this embodiment, DNA extraction was performed using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germany). The extracted DNA was amplified by PCR, and the resulting products were subjected to next-generation sequencing. Sequencing data underwent quality control, effective sequence assembly, OTU clustering, and OTU species annotation. Species abundance analysis was performed based on the OTU species annotations. For detailed results, please refer to [link to relevant documentation]. Figure 7 Species diversity analysis, results are detailed in [link to relevant documentation]. Figure 8 The concentration of DNA extracted from the product was detected using a NanoDrop micro-spectrophotometer (as shown in Table 2), and the number of fish species detected was counted (as shown in Table 3).

[0081] Remark: Figure 7 and Figure 8 The data in the table represents the total number of fish species detected, meaning that at least two samples must be detected simultaneously.

[0082] Comparative Example 1

[0083] This comparative example describes an environmental DNA sampling method, using a single-point sampling method via membrane filtration, comprising the following steps:

[0084] Two cross-sections were selected in the middle and lower reaches of the Yangtze River estuary. Sampling points were set up at distances of 150m, 300m, 300m and 1500m from the left bank, respectively, at the two cross-sections. Sampling was conducted at the surface, middle and bottom layers, for a total of 24 sampling points to ensure full coverage of the entire cross-sectional profile during sampling.

[0085] 4.5L of water sample was collected at each location using a water sampler;

[0086] Using a vacuum pump as the power source, each 4.5L water sample was filtered through a 0.45μm polyethersulfone membrane, meaning each membrane filtered 1.5L of water sample. There were 3 biological replicates at each site, for a total of 72 samples (108L of water sample).

[0087] The filter membranes enriched with environmental DNA were placed into 5 ml centrifuge tubes and stored at -20°C until DNA extraction was performed.

[0088] The DNA extraction method used in this comparative example employed the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germany). The extracted DNA was amplified by PCR, and the resulting products were subjected to next-generation sequencing. Sequencing data underwent quality control, effective sequence assembly, OTU clustering, and OTU species annotation. Species abundance analysis was performed based on the OTU species annotations. The results are as follows: Figure 7 As shown; Species diversity analysis results are as follows. Figure 8 As shown in Table 2, the concentration of DNA extracted from the product was detected using a NanoDrop micro-spectrophotometer, and the number of fish species detected was counted (as shown in Table 3).

[0089] Remark: Figure 7 and Figure 8 The data in the table represents the total number of fish species detected, meaning that at least two samples must be detected simultaneously.

[0090] Table 2. Results of DNA concentration detection in products extracted by tangential flow filtration and membrane filtration methods.

[0091]

[0092] The DNA concentration extracted from samples obtained by different sampling methods was analyzed against the extraction effect of the same kit. The results showed a significant difference in DNA concentration between the two sampling methods (p<0.0001). The DNA concentration extracted by the tangential flow filtration method in Example 3 was significantly higher than that of the membrane filtration method in Comparative Example 1. The average DNA concentration of the tangential flow filtration sample (35.64 ng / μl) was 3.4 times that of the membrane filtration sample (10.40 ng / μl), and the DNA concentration was more stable compared to the membrane filtration method.

[0093] Table 3. Results of the number of fish species detected by a single filter in tangential flow filtration and membrane filtration methods.

[0094]

[0095] Note: The number of fish species detected is the average number of fish species detected per sample.

[0096] Analysis of the number of fish species detected by a single filter under the two sampling methods revealed a significant difference (p<0.0001). The number of fish species detected by the tangential flow filtration method in Example 3 was significantly higher than that by the membrane filtration method in Comparative Example 1. The average number of fish species detected by the tangential flow filtration method (31 species) was 3.0 times that of the average number of fish species detected by the membrane filtration method (9.71≈10 species), and the results were more stable than those of the membrane filtration method.

[0097] Comparative Example 2

[0098] This comparative example describes an environmental DNA sampling method that employs single-point sampling, essentially the same as in Example 3. The difference lies in replacing the walk-through sampling from point A to point D with single-point sampling at points A, B, C, and D. Each point has three biological replicates, resulting in a total of 12 samples (240L of water sample).

[0099] The DNA extraction method for the samples obtained in this comparative example was the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germany), and the number of fish species detected was counted (as shown in Table 4).

[0100] Table 4. Results of fish species counts from single-point and mobile monitoring using tangential flow filtration method.

[0101]

[0102] Note: The data in Table 4 represents the total number of fish species detected, meaning that at least two samples must be detected simultaneously.

[0103] As can be seen from Example 3 and Comparative Example 2, the wide-area environmental DNA sample enrichment effect is better than that of sampling at each location separately, and the enrichment effect of the wide-area sampling is more representative.

[0104] The present invention and its embodiments have been described above illustratively. This description is not restrictive, and the figures shown are only one embodiment of the present invention; the actual structure is not limited thereto. Therefore, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, such designs should fall within the protection scope of the present invention.

Claims

1. An environmental DNA enrichment device, characterized in that: The device includes a housing (9), a first pump (1), a second pump (2), a main switch (3), a first switch (4), a second switch (5), and an enricher (10). The main switch (3), the first switch (4), and the second switch (5) are disposed on the surface of the housing (9). The first pump (1) and the second pump (2) are disposed on the housing (9). The first pump (1) and the second pump (2) are both peristaltic pumps. The first switch (4) is connected to the main switch (3) and the first pump (1) by wires. The second switch (5) is connected to the main switch (3) and the second pump (2) by wires. The enrichment device (10) is connected to the first pump (1) and / or the second pump (2). The pipeline of the first pump (1) includes a first inlet pipe (11) and a first outlet pipe (12). The pipeline of the second pump (2) includes a second inlet pipe (21) and a second outlet pipe (22). The enrichment device (10) is connected in the middle of the first inlet pipe (11) and / or the second inlet pipe (21). The enrichment device (10) includes an outer cover (101) and a filter element (102), the filter element (102) being disposed inside the outer cover (101); the filter element (102) includes a filter layer and end caps at both ends, the filter layer being located between and connected to the end caps at both ends; the filter layer is folded multiple times to make the cross-section of the filter layer wavy, the number of folds of the filter layer is 50 to 55, the filtered water sample volume is ≥20 L, and the filter membrane precision in the filter layer is 0.45 μm; the filter element (102) includes a structure without a support plate; the enrichment rate of the filter element (102) is ≥90%, and the elution rate of the filter element (102) is ≥95%; The filter membrane is made of polyethersulfone.

2. The environmental DNA enrichment device according to claim 1, characterized in that: The enrichment device (10) also includes a first port (103) and a second port (104), which are respectively located on the water inlet side and the water outlet side of the outer casing (101).

3. The environmental DNA enrichment device according to claim 2, characterized in that: The first water inlet pipe (11) is connected to the inlet end of the first pump (1), and the first drain pipe (12) is connected to the outlet end of the first pump (1); the second water inlet pipe (21) is connected to the inlet end of the second pump (2), and the second drain pipe (22) is connected to the outlet end of the second pump (2).

4. The environmental DNA enrichment device according to claim 3, characterized in that: It also includes a battery (13), a power display screen (6) and a charging port (8); the battery (13) is disposed inside the housing (9), and the power display screen (6) and the charging port (8) are disposed on the surface of the housing (9); the main switch (3) is electrically connected to the battery (13), the power display screen (6), the first switch (4) and the second switch (5); the battery (13) is electrically connected to the charging port (8).

5. An environmental DNA sampling method, characterized in that: Includes the following steps: The enrichment device of claim 3 or 4 is placed horizontally on the ship, such that the pipes of the first inlet pipe (11) and / or the second inlet pipe (21) extend below the water surface; Drive the boat to start traveling from section one along a direction parallel to the riverbank, turn on the enrichment device, and start continuous mobile sampling. When the boat reaches section two, the sampling ends and the water sample in the enrichment device (10) is emptied. Remove the enrichment device (10), add the preservation buffer, and seal. The volume of filtered water sample is counted during the sampling process.

6. The environmental DNA sampling method according to claim 5, characterized in that: Three parallel biological samples were collected along each route of the mobile sampling.

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