Phytoplankton environment DNA sampling device and sampling method

Abstract

The invention discloses a phytoplankton environment DNA sampling device and method.The sampling device comprises a sampling module, a power module, a monitoring module and a collecting module, the sampling module comprises a sampling pipeline, a first-stage filtering unit, a second-stage filtering unit and a third-stage filtering unit, and the first-stage filtering unit, the second-stage filtering unit and the third-stage filtering unit are sequentially connected; the input end of the first-stage filter unit is connected with the output end of the sampling pipeline, and a first filter membrane, a second filter membrane and a third filter membrane of which the pore diameters are sequentially reduced are respectively arranged in the third-stage filter unit; the power module is a peristaltic pump, and the monitoring module is a pressure sensor and is used for monitoring the pressure difference between the two sides of a filter membrane and providing stable water flow; the collecting module comprises a filter membrane collecting bin and a filter membrane box which are respectively connected with the first filter membrane, the second filter membrane and the third filter membrane. The multi-stage dynamic filtering mode is utilized, and the capture rate of phytoplankton environment DNA can be effectively increased through combination of the three-stage filtering unit and the peristaltic pump.

Description

Technical Field

[0001] This invention belongs to the field of environmental monitoring and biotechnology, specifically relating to a phytoplankton environmental DNA sampling device and sampling method, which is applicable to ecological monitoring, environmental assessment, aquaculture and scientific research analysis. Background Technology

[0002] Traditional phytoplankton sampling methods rely on netting or centrifugation, which require repeated manual netting and centrifugation operations. The sample volume processed at one time is limited, resulting in low efficiency. Furthermore, the friction from netting or centrifugation can easily damage cells and disrupt the DNA structure within the cells. Traditional methods expose DNA to room temperature for extended periods, posing a risk of DNA degradation.

[0003] In addition, the single-stage filtration devices used in previous studies have poor adaptability to particle size and are difficult to capture phytoplankton of different sizes, such as diatoms and green algae. At the same time, in water environments containing high-density phytoplankton cells, single-stage filter membranes are prone to rapid saturation, leading to rapid clogging of the filter membrane. Frequent shutdowns to replace the filter membrane also affect the operating frequency.

[0004] When conducting field sampling, traditional phytoplankton sampling equipment and on-site processing equipment, such as water samplers, trawls, centrifuges, and filtration devices, are heavy and not portable enough, making them difficult to adapt to river, stream, tidal flat wetland environments and transportation vehicles such as ships. Traditional sampling methods rely more on manual recording of parameters and lack integrated sensors and central control modules to achieve automated control and real-time monitoring functions.

[0005] Environmental DNA (eDNA) refers to fragments of genetic material released into environmental media such as soil, water, and air through processes like metabolism, shedding, and death. It includes intracellular DNA (such as genomic DNA released from broken cells) and extracellular cell-free DNA, and has become a key research area in the interdisciplinary field of ecology and molecular biology in my country in recent years. Environmental eDNA is easily degraded by environmental factors such as temperature, pH, and enzyme activity; therefore, contamination control during sampling, cryogenic preservation and rapid transportation of samples, and efficient eDNA enrichment are crucial technologies.

[0006] The core of phytoplankton eDNA sampling is "contamination control, rapid enrichment, and sample preservation." Filtration is the most common and reliable method. The sampling volume, filter membrane pore size, and preservation method must be adjusted according to the water type and research objectives to ensure data accuracy. Previous studies used single-stage filtration devices that enrich eDNA using only a single-pore membrane, suitable for clear water with few impurities. However, in high-turbidity water, membrane clogging is common, and it cannot distinguish eDNA particles of different sizes. In practical sampling, multiple single-stage filtrations are often required, but this increases the number of filtrations and time, exposing DNA to room temperature for extended periods, posing a risk of DNA degradation.

[0007] Therefore, there is an urgent need for an integrated, efficient, and graded phytoplankton environmental DNA sampling device and method. Through stepwise screening using multi-pore membranes, it can be adapted to complex water bodies with high turbidity and abundant particulate matter, reducing the probability of membrane clogging. It can also obtain eDNA distribution information related to different particle sizes, facilitating more refined community structure analysis. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a phytoplankton environmental DNA sampling device and sampling method, which can effectively improve the DNA capture rate.

[0009] The technical solution of this invention: A phytoplankton environmental DNA sampling device, comprising a sampling module, a power module, a monitoring module, and a collection module, wherein:

[0010] The sampling module includes a sampling pipe and a first-stage filtration unit, a second-stage filtration unit, and a third-stage filtration unit connected in sequence. The input end of the first-stage filtration unit is connected to the output end of the sampling pipe. The first-stage filtration unit, the second-stage filtration unit, and the third-stage filtration unit are respectively provided with a first filter membrane, a second filter membrane, and a third filter membrane with successively decreasing pore sizes.

[0011] The power module is a peristaltic pump, and the monitoring module is a pressure sensor. The peristaltic pump and the pressure sensor are respectively installed on the connecting pipe between two adjacent filtration units to monitor the pressure difference across the filter membrane and provide a stable water flow.

[0012] The collection module includes a filter membrane collection chamber and a filter membrane box, which are respectively connected to the first filter membrane, the second filter membrane, and the third filter membrane, and are used to replace the filter membranes in real time and collect DNA samples from the phytoplankton environment.

[0013] Furthermore, the pore size of the first filter membrane in the first-stage filtration unit is 20 μm, the pore size of the second filter membrane in the second-stage filtration unit is 5 μm, and the pore size of the third filter membrane in the third-stage filtration unit is 0.45 μm.

[0014] Furthermore, the flow rate of the peristaltic pump can be adjusted within the range of 0.1 L / min to 1 L / min.

[0015] Furthermore, the filter membrane box is a cartridge-type filter membrane box, containing 6 filter membranes with the same pore size.

[0016] Furthermore, the sampling pipe has an anti-clogging barrier at the front end of the inlet, and a coarse filter screen with a pore size of 200μm is placed in the sampling pipe.

[0017] Based on the above-mentioned phytoplankton environmental DNA sampling device, the present invention also provides a sampling method, comprising the following steps:

[0018] Insert the inlet of the sampling pipe of the sampling device into the water body and start sampling;

[0019] After passing through the anti-clogging barrier and coarse filter screen of the sampling pipeline, the water sample enters the first-stage filtration unit, the second-stage filtration unit, and the third-stage filtration unit in sequence. The peristaltic pump of the power module continuously drives the water intake at the initial flow rate, and the water sample passes through the first filter membrane, the second filter membrane, and the third filter membrane in sequence.

[0020] When the sampled water reaches the preset volume, after a single sampling is completed, the filter membrane in the collection module is automatically replaced, and the sampled filter membrane is automatically transferred to the filter membrane collection chamber of the collection module for low-temperature storage.

[0021] Furthermore, the initial flow rate of the peristaltic pump is 0.5 L / min. When the water sample passes through the first filter membrane, the second filter membrane, and the third filter membrane in sequence, if the pressure sensor of the monitoring module detects that the pressure difference across the filter membrane is greater than 10 kPa, the flow rate of the peristaltic pump is controlled to be reduced to 0.3 L / min.

[0022] Furthermore, the maximum sampling volume of the sampling device in a single sampling is 5L.

[0023] The phytoplankton environmental DNA sampling device and sampling method described in this invention utilize a multi-stage dynamic filtration approach. By combining a three-stage filtration unit with a peristaltic pump, the flow rate of the water sample can be effectively regulated, and the flow rate can be automatically adjusted according to the pressure difference across the filter membrane. This balances sampling efficiency with filter membrane lifespan and can effectively improve the capture time of phytoplankton environmental DNA. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the principle of the phytoplankton environmental DNA sampling device described in the embodiment of the present invention.

[0025] Figure 2 yes Figure 1 The diagram shows the principle of the collection module.

[0026] Figure 3 This is a flowchart of the phytoplankton environmental DNA sampling method described in an embodiment of the present invention.

[0027] In the diagram: 11-Anti-clogging fence, 12-Coarse filter screen, 13-Sampling pipe; 21-Monitoring module, 22-Power module, 23-Collection module; 31-First-stage filtration unit, 32-Second-stage filtration unit, 33-Third-stage filtration unit; 41-First filter membrane, 42-Second filter membrane, 43-Third filter membrane. Detailed Implementation

[0028] like Figure 1 As shown, the phytoplankton environmental DNA sampling device of this invention includes a sampling module, a power module, a monitoring module, and a collection module, wherein:

[0029] The sampling module includes a sampling pipe 13 for collecting water samples and a first-stage filtration unit 31, a second-stage filtration unit 32, and a third-stage filtration unit 33 connected in sequence. The sampling pipe 13 is provided with an inlet 131. A wear-resistant anti-clogging barrier 11 is provided at the front end of the sampling pipe 13. The anti-clogging barrier 11 is set with an appropriate spacing between the barriers to prevent the entry of large debris. A coarse filter screen 12 is also placed in the sampling pipe 13. The coarse filter screen has a pore size of 200μm and adopts a detachable structure, which can pre-filter large particles such as branches, leaves, and sand in the water sample.

[0030] Furthermore, the sampling module includes a three-stage filtering unit, namely... Figure 1 The diagram shows a first-stage filtration unit 31, a second-stage filtration unit 32, and a third-stage filtration unit 33. The input end of the first-stage filtration unit 31 is connected to the output end of the sampling pipe 13. Each of the first-stage filtration unit 31, second-stage filtration unit 32, and third-stage filtration unit 33 contains a first filter membrane 41, a second filter membrane 42, and a third filter membrane 43 with progressively decreasing pore sizes. The first filter membrane 41, second filter membrane 42, and third filter membrane 43 are arranged vertically in the order of coarse, medium, and fine pore sizes. Each filtration unit contains a filter membrane. The upper layer is the first filter membrane 41 (coarse pore size filter membrane) with a pore size of 20 μm, primarily trapping large phytoplankton; the middle layer is the second filter membrane 42 (medium pore size filter membrane) with a pore size of 5 μm, primarily trapping medium-sized phytoplankton; and the lower layer is the third filter membrane 43 (fine pore size filter membrane) with a pore size of 0.45 μm, primarily capturing organic debris and viruses containing free biological DNA. The pore size of the above-mentioned filter membranes can also be different graded particle sizes according to the specific conditions of the water body. The above-mentioned filter membranes are arranged in an alternating manner to avoid clogging and superposition.

[0031] Furthermore, the power module 22 is a peristaltic pump, and the monitoring module 21 is a pressure sensor. The peristaltic pump and pressure sensor are respectively installed on the connecting pipe between adjacent two-stage filtration units to monitor the pressure difference across the filter membrane and provide a stable water flow. In this embodiment, the flow rate of the peristaltic pump can be adjusted within the range of 0.1L / min to 1L / min to provide a stable, low-flow water flow, thereby reducing shearing damage to biological DNA during sample collection. The pressure sensor is a piezoresistive sensor, which can monitor the pressure difference across the filter membrane in real time. When the pressure difference exceeds a certain threshold, the peristaltic pump can automatically adjust the water flow rate to decrease, or an audible and visual alarm can be used to remind manual operation to control the flow rate.

[0032] In addition, such as Figure 2 As shown, the collection module 23 includes a filter membrane collection chamber 231, a filter membrane box 232, and a control unit 233, which are connected to the first filter membrane 41, the second filter membrane 42, and the third filter membrane 43, respectively, for real-time replacement of filter membranes and collection of DNA samples from the phytoplankton environment. The filter membrane collection chamber 231 has a built-in -20℃ constant temperature refrigeration unit to continuously preserve the collected samples at low temperatures, delaying the degradation of biological DNA and effectively improving the DNA capture rate. The filter membrane box 232 is a magazine-type filter membrane box with a sealed design. Each filter membrane box can be pre-loaded with 6 filter membranes of the same pore size. The control unit 233 automatically replaces the filter membranes, avoiding cross-contamination caused by manual operation. The control unit 233 is a control system based on an STM32 microcontroller, integrating the monitoring of basic water body physical indicators such as flow rate, water pressure, and water temperature, and automatically generating indicator parameter data and sampling logs.

[0033] The sampling device described in this embodiment can adopt a portable mechanical design structure. The entire sampling device weighs less than 3kg. The outer shell is waterproof and can also support drones or water buoy mounting platforms. Compared with traditional equipment, it greatly shortens the operation time.

[0034] like Figure 3 As shown, based on the above-mentioned phytoplankton environmental DNA sampling device, this embodiment of the invention also provides a sampling method, including the following steps:

[0035] (1) Insert the inlet of the sampling pipe of the sampling device into the water body and start sampling;

[0036] (2) After the water sample passes through the anti-clogging barrier and coarse filter screen of the sampling pipeline, it enters the first-stage filtration unit, the second-stage filtration unit and the third-stage filtration unit in sequence. The peristaltic pump of the power module continuously drives the water to be drawn at the initial flow rate. The water sample passes through the first filter membrane, the second filter membrane and the third filter membrane in sequence.

[0037] (3) When the sampled water reaches the preset volume, after a single sampling is completed, the filter membrane box of the collection module will automatically replace the filter membrane, and the sampled filter membrane will be automatically transferred to the filter membrane collection chamber of the collection module for low-temperature storage.

[0038] In step (2) above, the initial flow rate of the peristaltic pump can be set to 0.5 L / min. When the water sample passes through the first filter membrane, the second filter membrane, and the third filter membrane in sequence, when the pressure sensor of the monitoring module detects that the pressure difference across the filter membrane is greater than 10 kPa, the flow rate of the peristaltic pump is controlled to drop to 0.3 L / min.

[0039] In this embodiment, the first, second, and third filter membranes are made of polycarbonate, a material with high corrosion resistance and low DNA adsorption rate. The sampling device has a maximum single sampling capacity of 5L, making it more suitable for surface water in lakes and rivers. When sampling at greater depths is required, an extension tube needs to be installed.

[0040] In summary, the sampling device and sampling method described in the embodiments of the present invention have the following significant advantages over traditional single-stage sampling and filtering systems:

[0041] 1. The combination of a three-stage filtration structure (coarse, medium, and fine) and a peristaltic pump in this device effectively improves the DNA capture rate by more than 30%.

[0042] 2. This device can automatically replace the filter membrane after a single sampling is completed. The replacement of each filter membrane takes less than 1 minute, which can significantly shorten the manual operation time of field sampling, saving up to 50% of the time. At the same time, it reduces the risk of DNA contamination during the manual replacement of filter membranes.

[0043] 3. This device adopts the concept of collaborative sampling and utilizes an automatic filter membrane switching method to maintain uninterrupted sampling operation, supporting long-term monitoring tasks. It is suitable not only for biological monitoring and scientific research, but also for ecological pollution tracking and ecological status assessment, such as phytoplankton community dynamic tracking, rapid collection of DNA from biomarker species of water pollution, real-time monitoring and early warning of red tides and algal blooms in aquaculture waters, and the development of environmental DNA barcoding monitoring sample collection technology.

[0044] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A phytoplankton environmental DNA sampling device, characterized in that: It includes a sampling module, a power module, a monitoring module, and a collection module, among which: The sampling module includes a sampling pipe and a first-stage filtration unit, a second-stage filtration unit, and a third-stage filtration unit connected in sequence. The input end of the first-stage filtration unit is connected to the output end of the sampling pipe. The first-stage filtration unit, the second-stage filtration unit, and the third-stage filtration unit are respectively provided with a first filter membrane, a second filter membrane, and a third filter membrane with successively decreasing pore sizes. The power module is a peristaltic pump, and the monitoring module is a pressure sensor. The peristaltic pump and the pressure sensor are respectively installed on the connecting pipe between two adjacent filtration units to monitor the pressure difference across the filter membrane and provide a stable water flow. The collection module includes a filter membrane collection chamber and a filter membrane box, which are respectively connected to the first filter membrane, the second filter membrane, and the third filter membrane, and are used to replace the filter membranes in real time and collect DNA samples from the phytoplankton environment.

2. The phytoplankton environmental DNA sampling device according to claim 1, characterized in that: The first filter membrane in the first-stage filtration unit has a pore size of 20 μm, the second filter membrane in the second-stage filtration unit has a pore size of 5 μm, and the third filter membrane in the third-stage filtration unit has a pore size of 0.45 μm.

3. The phytoplankton environmental DNA sampling device according to claim 1, characterized in that: The flow rate of the peristaltic pump can be adjusted within the range of 0.1L / min to 1L / min.

4. The phytoplankton environmental DNA sampling device according to claim 1, characterized in that: The filter cartridge is a magazine-type filter cartridge containing six filter membranes of the same pore size.

5. The phytoplankton environmental DNA sampling device according to claim 1, characterized in that: The sampling pipe is equipped with an anti-clogging barrier at the front end of the inlet, and a coarse filter screen is placed in the sampling pipe.

6. The phytoplankton environmental DNA sampling device according to claim 5, characterized in that: The coarse filter screen has a pore size of 200 μm.

7. The sampling method of the phytoplankton environmental DNA sampling device according to claim 1, characterized in that: Includes the following steps: Insert the inlet of the sampling pipe of the sampling device into the water body and start sampling; After passing through the anti-clogging barrier and coarse filter screen of the sampling pipeline, the water sample enters the first-stage filtration unit, the second-stage filtration unit, and the third-stage filtration unit in sequence. The peristaltic pump of the power module continuously drives the water intake at the initial flow rate, and the water sample passes through the first filter membrane, the second filter membrane, and the third filter membrane in sequence. When the sampled water reaches the preset volume, after a single sampling is completed, the filter membrane in the collection module's filter membrane box is automatically replaced, and the sampled filter membrane is automatically transferred to the filter membrane collection chamber of the collection module for storage.

8. The sampling method according to claim 7, characterized in that: The initial flow rate of the peristaltic pump is 0.5 L / min.

9. The sampling method according to claim 8, characterized in that: When the water sample passes through the first filter membrane, the second filter membrane, and the third filter membrane in sequence, if the pressure sensor of the monitoring module detects that the pressure difference across the filter membrane is greater than 10 kPa, the flow rate of the peristaltic pump is controlled to be reduced to 0.3 L / min.

10. The sampling method according to claim 7, characterized in that: The maximum sampling volume of the sampling device in a single sampling is 5L.

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