Detection device and rapid detection method for enteromorpha microscopic propagules

Abstract

The invention belongs to the technical field of detection devices, and discloses a detection device and a rapid detection method for enteromorpha microscopic propagules. According to the detection device, a connector and a sample feeding pipe and a connecting pipe at the bottom and the top of a filter are limited through a first protection assembly, a second protection assembly and a limiting assembly respectively, and meanwhile, protection blocks are used for protecting the outer sides of the first protection assembly, the second protection assembly and the filter, so that the filter is prevented from being easily loosened due to vibration during operation of the filter; the rapid detection method comprises the following steps: 1, filtering a collected water sample through a filter membrane in a filter instrument, and extracting high-purity DNA (deoxyribonucleic acid); 2, cloning and purifying the enteromorpha specific gene, determining the DNA concentration of the target gene, and establishing a standard curve; and 3, detecting the copy number of the enteromorpha microcosmic propagule in the water body through qPCR, and calculating the cell abundance according to the copy number of the target gene in a single cell to realize rapid identification and quantitative detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection devices, and particularly relates to a detection device for Enteromorpha micro-propagules and a rapid detection method. BACKGROUND

[0002] The Enteromorpha green tide in the Yellow Sea is one of the most serious ecological disasters in the coastal waters of China. Since 2007, the large-scale Enteromorpha green tide has been ravaging the coastal waters of the Yellow Sea in China year after year, seriously affecting the marine and coastal ecological environment, threatening the marine landscape, tourism and mariculture, and causing huge economic losses and serious social impacts.

[0003] Micro-propagules are the "seed" sources of the green tide, and refer to diploid cell groups, detached tissue blocks, single cells and other green algae fragments that only undergo vegetative reproduction. Micro-propagules are widely distributed in the coastal waters, and therefore, the spatiotemporal distribution and population dynamics of the Enteromorpha micro-propagules are important contents of the operational monitoring of the Enteromorpha green tide in the Yellow Sea. The abundance of the micro-propagules of the green algae of the genus Ulva is an important biological index of the green tide investigation, and the related quantitative methods are widely used in the current green tide investigation, providing important biological data for predicting the potential green tide biomass in seawater and predicting the scale and source of the green tide.

[0004] At present, in order to rapidly detect the Enteromorpha micro-propagules or suspended algal filament fragments in water, it is necessary to use a water environment DNA filter to carry out environmental DNA sampling, to filter seawater on a filter membrane in layers, and to detect the DNA sample on the filter membrane. However, in the process of field or marine investigation, the filter is prone to loosening due to vibration when the filter is running in the filter instrument, thereby affecting the filtering effect, and in severe cases, the filter may fall off, so that the water sample cannot be normally filtered, and the final detection effect is affected.

[0005] Therefore, it is necessary to invent a detection device for Enteromorpha micro-propagules and a rapid detection method to solve the above problems according to the actual needs of the investigation and monitoring of the Enteromorpha micro-propagules. SUMMARY

[0006] In view of the problems in the prior art, the application provides the following technical scheme:

[0007] The detection device for Enteromorpha micro-propagules comprises a filter instrument body, a connecting head fixedly connected to the top of the filter instrument body, a filter arranged at the top of the connecting head, and a connecting protection assembly arranged on the outer side of the connecting head.

[0008] The connection protection assembly includes a protective block, a protective cover, a first protection component, and a second protection component. The bottom of the protective block is fixedly installed on the top of the filter body. The protective block is located outside the connector and the filter. The protective cover is located on the top of the protective block. The first protection component is located outside the connector and the filter. The second protection component is located outside the filter.

[0009] As a preferred embodiment of the above technical solution, there are multiple sets of filters, protective blocks, and protective covers, which are arranged in a linear array on the top of the filter body. The filter contains multiple layers of filter membranes with filter holes that gradually increase in diameter. Sample inlet tubes are fixedly installed on both the top and bottom sides of the filter. The sample inlet tube at the bottom of the filter is connected to the connector. The first protective component and the second protective component are located on both sides of the connector and the filter, and are arranged symmetrically with respect to the center of the filter.

[0010] As a preferred embodiment of the above technical solution, the first protective component includes a limiting block, a pushing block, a first rack, a gear, a rotating rod, a second rack, and a moving block;

[0011] The limiting block is slidably connected to the top of the filter body. A pushing block is fixedly installed on one side of the limiting block, and a first rack is fixedly installed on one side of the pushing block. A gear meshes with one side of the first rack, and a rotating rod is fixedly installed in the middle of the gear. The bottom of the rotating rod is rotatably connected to the top of the filter body. A second rack meshes with one side of the gear, and a moving block is fixedly connected to one side of the second rack.

[0012] As a preferred embodiment of the above technical solution, the limiting block is located outside the connector, and an arc-shaped groove is provided on the side of the limiting block near the connector. The bottom of the filter is located at the top of the limiting block, and the second protective component is located at the top of the moving block. The moving block, the pushing block, the first rack and the second rack are all slidably connected to the top of the filter body. The first magnetic block is fixedly installed on both sides of the moving block, and the second magnetic block is fixedly installed on the inner wall of the protective block. The first magnetic block and the second magnetic block are magnetically connected.

[0013] As a preferred embodiment of the above technical solution, the second protective component includes a fixed block, a movable block, a slider, and a limiting plate; the fixed block and the movable block are fixedly installed at the top, the slider is fixedly installed at the bottom of the movable block, the slider and the fixed block are slidably connected to the inner wall of the fixed block, one end of the limiting plate is hinged to the movable block, and a limiting groove is provided on the side of the limiting plate away from the movable block.

[0014] As a preferred embodiment of the above technical solution, one end of the limiting plate is located inside the fixing block, and the other end is located on the top of the fixing block. A movable groove is provided on the top of the limiting plate located behind the filter. A limiting rod is provided in the movable groove. The limiting rod is L-shaped and slides in the movable groove through a fixing spring. A slot is provided on the top of the limiting plate located in front of the filter. The limiting rod and the slot are located on both sides of the limiting groove. The limiting rod is inserted into the limiting groove. A fixing hole is provided on the side of the limiting plate near the inner wall of the fixing block.

[0015] As a preferred embodiment of the above technical solution, the filter is provided with limiting components on both the left and right sides. There are four sets of limiting components, which are symmetrically arranged with respect to the center of the filter. The limiting components include a mounting block, a fixing plate, and a first telescopic rod. The mounting block and the fixing plate are fixedly installed to the inner wall of the protective block. One end of the first telescopic rod is hinged to the mounting block, and the other end is located on the top of the fixing plate. The first telescopic rod is inserted into the fixing hole.

[0016] As a preferred embodiment of the above technical solution, a movable component is provided between the filter and the limiting block. The number of movable components is the same as the number of protective blocks. The movable components are connected by connecting blocks. The movable component includes a movable plate, a compression spring, and a second telescopic rod. A groove is provided on the top of the limiting block, and the movable plate is located inside the groove. A compression spring is fixedly installed at the bottom of the movable plate. One end of the compression spring is fixedly installed to the inner wall of the bottom of the groove. An opening is provided on the outer wall of the protective block, and a fixing groove is provided on the inner wall of the bottom of the opening. The second telescopic rod is fixedly installed inside the fixing groove, and the top of the second telescopic rod is fixedly installed to the bottom of the movable plate. One end of the movable plate in the protective blocks located on both sides of the filter body is slidably connected to the inner wall of the protective block, and the other end is fixedly installed to the connecting block. The movable plate in the protective block located in the middle of the filter body is slidably connected to the inner wall of the opening on both sides, and is fixedly connected to the connecting block.

[0017] This invention also provides a rapid detection method using a detection device for microscopic propagules of Ulva prolifera, comprising the following steps:

[0018] Step 1: Use a water sampler to collect water samples from the top and bottom layers. After filtering, place the water sample into a sample container. Then, install the filter on the filter body and limit the filter using the first and second limiting components. Connect the sample container to the inlet tube of the filter body through the connecting tube, and connect the collection tube to the outlet of the filter body. Start the filter body to filter the water sample. After filtration, move the first and second protective components to remove the limiting components from the filter. Remove the filter by moving the components and place the filter membrane into a 2.0ml sterile centrifuge tube for freezing and storage.

[0019] Step 2: Add 500 μL of lysis buffer and 20 μL of proteinase K to a centrifuge tube containing a cryofilter membrane; take 500 mg of soil sample and add 500 μL of lysis buffer and 20 μL of proteinase K to a 2.0 ml centrifuge tube; incubate at 65℃ for 4 h (with inverting and shaking to mix); centrifuge at 10000 x g / min at room temperature for 10 min, and collect the supernatant; add 1.5 volumes of chloroform:isoamyl alcohol (24:1), centrifuge at 10000 x g / min for 10 min, and collect the supernatant; add 1 volume of isopropanol, and precipitate overnight at 4℃; centrifuge at 4℃ for 10 min to collect the DNA precipitate; wash with 70% ethanol and dry; dissolve the precipitate in 100 μL of LTE buffer (pH=8) for later use to obtain high-purity DNA;

[0020] Step 3: Establishing the standard curve. Select a *Ulva prolifera*-specific gene fragment and design primers. Use a T-vector PCR product cloning kit to clone and purify the amplified product of the specific gene fragment, extract plasmid DNA, determine the plasmid DNA concentration, and dilute the plasmid DNA. Calculate the standard curve using the logarithm of the plasmid DNA concentration. 10 C v The x-axis represents the critical cycle value (C) of the qPCR reaction, and the y-axis represents the critical cycle value (C) of the qPCR reaction. t Plot the qPCR standard curve with y=0.05 as the ordinate.

[0021] Step 4: Calculate the copy number of *Ulva prolifera* micropropagules in the sample by comparing the sample DNA amplification with the standard curve; calculate the copy number of the target gene in a single cell based on the DNA concentration and gamete cell concentration of the target gene; calculate the corresponding micropropagule cell abundance based on the copy number abundance of *Ulva prolifera* micropropagules obtained by qPCR, thereby achieving rapid identification and quantitative detection of *Ulva prolifera* micropropagules.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) The present invention limits the connector and the sample inlet tube at the bottom of the filter by the first protective component, and limits the sample inlet tube and the connecting tube at the top of the filter by the second protective component and the limiting component, so that the connection at both ends of the filter is limited, thereby improving the stability of the connection. At the same time, the protective block protects the first protective component, the second protective component and the outside of the filter, preventing the first protective component and the second protective component from being collided, which would cause the connection at both ends of the filter to loosen. This would make the filter easy to loosen due to vibration when the filter body is running, thereby affecting the filtration and detection effect.

[0024] (2) By moving the moving component, the sample tubes at the bottom of the three filters are separated from the connectors, and the tops of the three filters can be moved to the outside of the protective block, which makes it easier to remove the three filters without disassembling them one by one, thus improving the efficiency of filter removal and thus improving the efficiency of detection.

[0025] (3) The present invention can sequentially intercept and filter substances of different particle sizes through the filter membranes on multiple filters. Through the layer-by-layer classification, various unwanted components in the sample can be removed more precisely and comprehensively, greatly improving the filtration accuracy and overall effect, making the final filtered product purer and meeting the needs of subsequent analysis and detection, thereby improving the filtration efficiency and facilitating detection. Attached Figure Description

[0026] Figure 1 The diagram shown is a schematic representation of the overall structure of the embodiment;

[0027] Figure 2 The diagram shown is a structural diagram of the filter body, protective block, and filter of an embodiment.

[0028] Figure 3 The diagram shown is an internal structural diagram of the protective block in the embodiment;

[0029] Figure 4 The diagram shown is a cross-sectional view of the protective block, the first protective component, the second protective component, and the filter in the embodiment.

[0030] Figure 5 The diagram shown is a structural diagram of the protective block, the first protective component, and the second protective component of the embodiment;

[0031] Figure 6 The diagram shown is a structural diagram of the first protective component and the second protective component in the embodiment;

[0032] Figure 7 The diagram shown is a structural diagram of the second protective component of the embodiment;

[0033] Figure 8 The diagram shown is a structural diagram of the protective block and the moving component of an embodiment;

[0034] Figure 9 The diagram shown is a schematic of the SYBRGREEN dye method in the example.

[0035] In the diagram: 1. Filter body; 2. Connector; 3. Filter; 4. Protective block; 5. Protective cover; 6. Sample inlet tube; 7. Limiting block; 8. Pushing block; 9. First rack; 10. Gear; 11. Second rack; 12. Moving block; 13. First magnetic block; 14. Second magnetic block; 15. Fixed block; 16. Movable block; 17. Limiting plate; 18. Limiting rod; 19. Slot; 20. Mounting block; 21. Fixed plate; 22. First telescopic rod; 23. Connecting block; 24. Moving plate; 25. Compression spring; 26. Second telescopic rod; 27. Slider. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments.

[0037] Example 1

[0038] This invention provides a device for detecting microscopic propagules of *Ulva prolifera*, such as... Figures 1 to 4 As shown, the filter includes a filter body 1, a connector 2 fixedly connected to the top of the filter body 1, a filter 3 disposed on the top of the connector 2, and a connection protection assembly disposed on the outside of the connector 2. The connection protection assembly includes a protective block 4, a protective cover 5, a first protection assembly, and a second protection assembly. The bottom of the protective block 4 is fixedly installed on the top of the filter body 1, and the protective block 4 is located outside the connector 2 and the filter 3. The protective cover 5 is located on the top of the protective block 4. The first protection assembly is located outside the connector 2 and the filter 3, and the second protection assembly is disposed outside the filter 3. There are multiple sets of filters 3, protective blocks 4, and protective covers 5, and they are arranged in a linear array on the top of the filter body 1. The filter contains multiple layers of filter membranes with filter pores, and the diameter of the filter pores gradually increases. Sample inlet tubes 6 are fixedly installed on both the top and bottom sides of the filter 3. The sample inlet tube 6 located at the bottom of the filter 3 is inserted into the connector 2. The first protection assembly and the second protection assembly are located on both sides of the connector 2 and the filter 3, and they are symmetrically arranged with respect to the center of the filter 3.

[0039] Connector 2 facilitates connection to the sample inlet tube 6 at the bottom of filter 3, allowing filter 3 to be installed on filter body 1. When filter body 1 is not in use, protective cover 5 and protective block 4 protect connector 2 from damage, preventing it from affecting the connection with filter 3. Open protective cover 5, place filter 3 inside protective block 4, and connect its bottom sample inlet tube 6 to connector 2. Then connect the connecting tube containing the sample container to the sample inlet tube 6 at the top of filter body 1. Moving the first protective component restricts the outer side of connector 2, ensuring a secure connection between the sample inlet tube 6 at the bottom of filter 3 and connector 2. Moving the second restricting component restricts the sample inlet tube 6 at the top of filter 3 and connecting tube, ensuring stable connections at both ends of the filter 3. 100 mL of water is filtered through filter body 1 using a 0.22 μm filter membrane. After filtration through a 200 μm sieve, the water sample is then... The 0.22μm filter membrane filters 100mL of water sample. Multiple samples can be filtered simultaneously through three filters 3, improving the filtration effect. Through the filter membranes on multiple sets of filters, substances of different particle sizes can be intercepted and filtered sequentially. Smaller diameter filter pores intercept smaller substances first. As the water continues to flow through the filter membrane, slightly larger diameter filter pores then filter the larger particles. Through this layer-by-layer grading, various unwanted components in the sample can be removed more finely and comprehensively, greatly improving the filtration accuracy and overall effect. The final filtered product is purer and meets the requirements of subsequent analysis and testing, thereby improving filtration efficiency. The protective block 4 can limit and protect the outer sides of the filter 3 and connector 2, as well as the first and second protective components, preventing the filter 3 and connector 2 from being impacted and affecting the limiting effect of the first and second protective components, thus affecting the filtration and testing results.

[0040] like Figures 4 to 6 As shown, the first protective assembly includes a limiting block 7, a pushing block 8, a first rack 9, a gear 10, a rotating rod, a second rack 11, and a moving block 12. The limiting block 7 is slidably connected to the top of the filter body 1. The pushing block 8 is fixedly installed on one side of the limiting block 7, and the first rack 9 is fixedly installed on one side of the pushing block 8. The gear 10 meshes with one side of the first rack 9. A rotating rod is fixedly installed in the middle of the gear 10. The bottom of the rotating rod is rotatably connected to the top of the filter body 1. The second rack 11 meshes with one side of the gear 10. A movable block 12 is fixedly connected to one side, a limiting block 7 is located outside the connector 2, and an arc-shaped groove is opened on the side of the limiting block 7 near the connector 2. The bottom of the filter 3 is located at the top of the limiting block 7, and the second protective component is located at the top of the movable block 12. The movable block 12, the pushing block 8, the first rack 9 and the second rack 11 are all slidably connected to the top of the filter body 1. A first magnetic block 13 is fixedly installed on both sides of the movable block 12, and a second magnetic block 14 is fixedly installed on the inner wall of the protective block 4. The first magnetic block 13 and the second magnetic block 14 are magnetically connected.

[0041] After the filter 3 is placed inside the protective block 4, the sample inlet tube 6 at the bottom of the filter 3 is inserted into the connector 2. Then, the fixed block 15 moves the moving block 12 towards the inner wall of the protective block 4. When the moving block 12 moves, it drives the second rack 11 to move, so that the gear 10 drives the rotating rod to rotate along the top of the filter body 1. This causes the first rack 9 to drive the pushing block 8 and the limiting block 7 to move closer to the connector 2. When the moving block 12 moves to the second magnetic block 14, the position of the moving block 12 is fixed by the mutual attraction between the first magnetic block 13 and the second magnetic block 14. At the same time, the pushing block 8 pushes the limiting block 7 to the outside of the connector 2. The arc groove fits against the outside of the connector 2, protecting and limiting the outside of the connector 2. This keeps the connection between the connector 2 and the sample inlet tube 6 stable and improves the stability of the filter 3 connection.

[0042] like Figures 4 to 7 As shown, the second protective assembly includes a fixed block 15, a movable block 16, a slider 27, and a limiting plate 17. The fixed block 15 and the top of the movable block 12 are fixedly installed, and the slider 27 is fixedly installed on the top of the movable block 16. Both the slider 27 and the movable block 16 are slidably connected to the inner wall of the fixed block 15. One end of the limiting plate 17 is hinged to the movable block 16. A limiting groove is formed on the side of the limiting plate 17 away from the movable block 16. One end of the limiting plate 17 is located inside the fixed block 15, and the other end is located on the top of the fixed block 15. The limiting plate is located behind the filter 3. The top of plate 17 has a movable groove, and a limiting rod 18 is installed in the movable groove. The limiting rod 18 is L-shaped and is slidably installed in the movable groove by a fixing spring. One end of the limiting rod 18 is located on the top of the limiting plate 17, and the other end is located on the side of the limiting plate 17 near the inner wall of the protective block 4. The top of the limiting plate 17 located in front of the filter 3 has a slot 19. The limiting rod 18 and the slot 19 are located on both sides of the limiting groove. The limiting rod 18 is inserted into the limiting groove. The side of the limiting plate 17 near the inner wall of the fixing block 15 has a fixing hole.

[0043] After the position of the movable block 12 is limited by the first magnetic block 13 and the second magnetic block 14, the front and rear limiting plates 17 of the filter 3 are moved upward, so that the movable block 16 moves upward along the inner wall of the fixed block 15. When the slider 27 moves to the top of the fixed block 15, the movable block 16 and the limiting plate 17 move completely to the outside of the fixed block 15, pushing the limiting rod 18 to move towards the limiting plate 17 and squeezing the fixing spring. Then, the two limiting plates 17 are rotated. When the limiting plate 17 is rotated to a state that is level with the top of the filter 3, the limiting rod 18 is released. Under the action of the fixing spring, the limiting rod 18 is inserted into the slot 19, so that the inner wall of the limiting groove on one side of the two limiting plates 17 is closed. The sample inlet tube 6 at the top of the filter 3 is tightly fixed to the connection between the sample inlet tube 6 and the connecting tube. The limiting plate 17 then limits the connection between the sample inlet tube 6 and the connecting tube, improving stability and facilitating sample filtration. When the limiting rod 18 is moved away from the slot, the fixing spring is compressed, so that the limiting rod 18 is no longer inserted with 19. Then, the two limiting plates 17 are rotated to the vertical position, so that the limiting plates 17 are away from the sample inlet tube 6 at the top of the filter 3. The limiting plate 17 is moved downward, which drives the movable block 16 and the slider 17 to move into the interior of the fixed block 15. Then, the first limiting component is moved away from the connector 3 and the sample inlet tube 6, so that the filter 3 can be easily removed from the protective block 4.

[0044] like Figures 3 to 5 As shown, there are four sets of limiting components on both the left and right sides of the filter 3, and they are arranged symmetrically with respect to the center of the filter 3. The limiting components include a mounting block 20, a fixing plate 21, and a first telescopic rod 22. The mounting block 20 and the fixing plate 21 are fixedly installed on the inner wall of the protective block 4. One end of the first telescopic rod 22 is hinged to the mounting block 20, and the other end is located on the top of the fixing plate 21. The first telescopic rod 22 is inserted into the fixing hole.

[0045] The mounting block 20 facilitates the installation of the first telescopic rod 22 on the inner wall of the protective block 4, and the fixing plate 21 facilitates the support of the first telescopic rod 22. When the first telescopic rod 22 is in the retracted state, and the limiting plate 17 is located outside the sample inlet tube 6 at the top of the filter 3, the first telescopic rod 22 is rotated 90 degrees so that the first telescopic rod 22 rotates to the top of the filter 3 and keeps it horizontal. Then the first telescopic rod 22 is extended so that one end of the first telescopic rod 22 is inserted into the fixing hole on one side of the limiting plate 17 to limit the limiting plate 17, thereby improving the stability of the limiting plate 17 in limiting the connection between the sample inlet tube 6 and the connecting tube, preventing the limiting plate 17 from moving after being hit, which would cause the connection between the connecting tube and the sample inlet tube 6 to shake and make it inconvenient to perform testing.

[0046] like Figures 5 to 8As shown, a movable assembly is provided between the filter 3 and the limiting block 7. The number of movable assemblies is the same as the number of protective blocks 4. The movable assemblies are connected by a connecting block 23. The movable assembly includes a movable plate 24, a compression spring 25, and a second telescopic rod 26. A groove is provided on the top of the limiting block 7. The movable plate 24 is located inside the groove. The compression spring 25 is fixedly installed at the bottom of the movable plate 24. One end of the compression spring 25 is fixedly installed to the inner wall of the bottom of the groove. A movable opening is provided on the outer wall of the protective block 4. A fixing groove is provided on the inner wall of the bottom of the movable opening. The second telescopic rod 26 is fixedly installed inside the fixing groove. The top of the second telescopic rod 26 is fixedly installed to the bottom of the movable plate 24. One end of the movable plate 24 in the protective blocks 4 located on both sides of the filter body 1 is slidably connected to the inner wall of the protective block 4, and the other end is fixedly installed to the connecting block 23. The movable plate 24 in the protective block 4 located in the middle of the filter body 1 is slidably connected to the inner wall of the movable opening on both sides, and is fixedly connected to the connecting block 23.

[0047] Three moving components are connected by connecting block 23. When connecting block 23 is moved upward, one end of the moving plate 24 in the protective block 4 on both sides of the filter body 1 moves upward along the inner wall of the protective block 4, and the other end moves upward along the inner wall of the movable port. This causes the compression spring 25 to stretch and return to its original state, and at the same time, it drives the second telescopic rod 26 to move upward and extend. Both ends of the moving plate 24 in the middle protective block 4 move upward along the inner wall of the movable port. The compression spring 25 stretches and returns to its original state, and it drives the second telescopic rod 26 to move upward and extend. This causes the moving plate 24 to push the filter 3 upward, and it drives the sample inlet tube 6 at the bottom of the filter 3 to move upward. This causes the top of the filter 3 to move to the outside of the protective block 4, and then the filter 3 can be taken out from the protective block 4. This makes it easier to improve the efficiency of removal and improve the detection effect.

[0048] The present invention also provides a rapid detection method using the above-mentioned detection device for Ulva prolifera microscopic propagules, comprising the following steps:

[0049] Step 1: Use a water sampler to collect water samples from the top and bottom layers. After filtering, place the water sample into a sample container. Then, install filter 3 on the filter body 1. Filter 3 is confined by the first and second limiting components. Connect the sample container to the inlet tube 6 of the filter body 1 through the connecting tube. At the same time, connect the collection tube to the outlet of the filter. Start the filter body 1 to filter the water sample. After filtration, move the first and second protective components to remove the confining effect on filter 3. Remove filter 3 by moving the components and place the filter membrane into a 2.0ml sterile centrifuge tube for freezing and storage.

[0050] Step 2: Add 500 μL of lysis buffer and 20 μL of proteinase K to a centrifuge tube containing a cryofilter membrane; take 500 mg of soil sample and add 500 μL of lysis buffer and 20 μL of proteinase K to a 2.0 ml centrifuge tube; incubate at 65℃ for 4 h (with inverting and shaking to mix); centrifuge at 10000 x g / min at room temperature for 10 min, and collect the supernatant; add 1.5 volumes of chloroform:isoamyl alcohol (24:1), centrifuge at 10000 x g / min for 10 min, and collect the supernatant; add 1 volume of isopropanol, and precipitate overnight at 4℃; centrifuge at 4℃ for 10 min to collect the DNA precipitate; wash with 70% ethanol and dry; dissolve the precipitate in 100 μL of LTE buffer (pH=8) for later use to obtain high-purity DNA;

[0051] Step 3: Establishing the standard curve. Select a *Ulva prolifera*-specific gene fragment and design primers. Use a T-vector PCR product cloning kit to clone and purify the amplified product of the specific gene fragment, extract plasmid DNA, determine the plasmid DNA concentration, and dilute the plasmid DNA. Calculate the standard curve using the logarithm of the plasmid DNA concentration. 10 C v The x-axis represents the critical cycle value (C) of the qPCR reaction, and the y-axis represents the critical cycle value (C) of the qPCR reaction. t Plot the qPCR standard curve with y=0.05 as the ordinate.

[0052] Step 4: Compare the sample DNA amplification results with the standard curve to calculate the copy number of Ulva prolifera in the sample; calculate the copy number of the target gene in a single cell based on the DNA concentration and gamete cell concentration of the target gene; calculate the corresponding micropropagule cell abundance based on the copy number abundance of Ulva prolifera obtained by qPCR, thereby achieving rapid identification and quantitative detection of Ulva prolifera micropropagules.

[0053] Working principle: During use, the water sampler collects water samples from both the surface and bottom layers. After filtering through a 200μm sieve, the water sample is placed into the sample container. Then, the protective cover 5 is opened, and the filter 3 is placed inside the protective block 4, with its bottom inlet tube 6 connected to the connector 2. The moving fixed block 15 drives the moving block 12 to move towards the inner wall of the protective block 4. When the moving block 12 moves, it drives the second rack 11 to move, causing the gear 10 to drive the rotating rod to rotate along the top of the filter body 1. This causes the first rack 9 to drive the pushing block 8 and the limiting block 7 to move closer together. As the connector 2 moves, when the moving block 12 moves to the second magnetic block 14, the position of the moving block 12 is fixed by the mutual attraction between the first magnetic block 13 and the second magnetic block 14. Simultaneously, the pushing block 8 pushes the limiting block 7 to the outside of the connector 2, where it fits against the outside of the connector 2 through the arc-shaped groove, thus protecting and limiting the outside of the connector 2. Then, the limiting plates 17 on both sides of the filter 3 move upwards, causing the movable block 16 to move upwards along the inner wall of the fixed block 15. When the movable block 16 reaches the top of the fixed block 15, the limiting block... The fixed plate 17 is moved completely to the outside of the fixed block 15, pushing the limiting rod 18 to move towards the limiting plate 17 and squeezing the fixing spring. Then, the two limiting plates 17 are rotated. When the limiting plate 17 is rotated to a state where it is level with the top of the filter 3, the limiting rod 18 is released. Under the action of the fixing spring, the limiting rod 18 is inserted into the slot 19, so that the inner wall of the limiting groove on one side of the two limiting plates 17 is tightly fixed to the connection between the sample inlet tube 6 and the connecting tube at the top of the filter 3. Thus, the connection between the sample inlet tube 6 and the connecting tube is limited by the action of the limiting plate 17. Then, the first telescopic rod 22 is rotated ninety degrees, so that the first telescopic rod 22 is rotated to the top of the filter 3 and keeps it level with it. Then, the first telescopic rod 22 is extended, so that one end of the first telescopic rod 22 is inserted into the fixing hole on one side of the limiting plate 17, limiting the limiting plate 17. Thus, the filter 3 is fixed on the filter body 1. The sample container is connected to the sample inlet tube 6 of the filter body 1 through the connecting tube. At the same time, the collection tube is connected to the sample outlet of the filter. The filter body 1 is started to filter.

[0054] After filtration is completed, the first protective component, the second protective component, and the limiting component are moved to no longer limit the filter 3. Then, the connecting block 23 is moved upward, so that one end of the moving plate 24 in the protective blocks 4 on both sides of the filter body 1 moves upward along the inner wall of the protective block 4, and the other end moves upward along the inner wall of the movable port. This causes the compression spring 25 to stretch and return to its original state, and at the same time, it drives the second telescopic rod 26 to move upward and extend. The moving plate 24 in the middle protective block 4 moves upward along the inner wall of the movable port at both ends. The compression spring 25 stretches and returns to its original state, and it drives the second telescopic rod 26 to move upward and extend. This causes the moving plate 24 to push the filter 3 upward, and it drives the sample inlet tube 6 at the bottom of the filter 3 to move upward, so that the top of the filter 3 moves to the outside of the protective block 4. Then, the filter 3 is taken out from the protective block 4.

[0055] Place the filter membrane into a 2.0 ml sterile centrifuge tube and freeze it. Add 500 μL of lysis buffer and 20 μL of proteinase K to the centrifuge tube containing the frozen filter membrane.

[0056] Take 500 mg of soil sample and add 500 μL of lysis buffer and 20 μL of proteinase K to a 2.0 ml centrifuge tube; incubate at 65 °C for 4 h (with inverting and shaking to mix); centrifuge at 10000 x g / min at room temperature for 10 min, and collect the supernatant; add 1.5 volumes of chloroform:isoamyl alcohol (24:1), centrifuge at 10000 x g / min for 10 min, and collect the supernatant; add 1 volume of isopropanol, and precipitate overnight at 4 °C; centrifuge at 4 °C for 10 min to collect the DNA precipitate; wash with 70% ethanol and dry; dissolve the precipitate in 100 μL of LTE buffer (pH=8) for later use to obtain high-purity DNA; use a vector kit to amplify the target gene product (environmental DNA extracted in step two)... NA) cloning and purification: Following the product instructions, the plasmid was introduced into competent cells. The cells were plated on LB agar plates containing 20 μL IPTG (100 mM) and 100 μL X-gal (20 mg / mL) ampicillin and incubated overnight at 37°C. White bacteria were picked and quantitatively amplified using PFX-F / R primers to confirm the inserted fragment and successful cloning. Cells containing the inserted fragment were collected by centrifugation, and plasmid DNA was extracted using a plasmid extraction kit. A standard curve was obtained by amplifying the plasmid DNA. The number of *Ulva prolifera* microscopic reproductive cells in the sample was calculated by comparing the sample DNA amplification with the standard curve, enabling rapid identification and quantitative detection of *Ulva prolifera* microscopic reproductive cells.

[0057] Example 2

[0058] The research team previously developed a quantitative real-time PCR (qPCR) method for detecting microscopic propagules of *Ulva prolifera* targeting specific DNA fragments. This method was then used to detect water samples from the shallow waters of northern Jiangsu, and the results were compared with traditional culture methods. The detection efficiency and distribution patterns of the two methods for *Ulva prolifera* microscopic propagules were basically consistent. However, compared to traditional culture methods, qPCR is more sensitive and efficient, making it more suitable for on-site monitoring of microscopic propagules or suspended algal filaments.

[0059] Construction of Real-Time Quantitative PCR Detection Method

[0060] (1) Primer screening and development. Specific primers that have undergone preliminary application verification studies or specific primers designed for the 18S rDNA of Ulva prolifera were selected as primers for real-time quantitative PCR detection. The specificity of the primers was verified using the NCBI database.

[0061] (2) DNA extraction. Take 1g of *Ulva prolifera* thallus and extract genomic DNA using a plant genomic DNA extraction kit.

[0062] (3) Primer verification. The genomic DNA of *Ulva prolifera* was amplified by conventional PCR using the screened and developed primers. The effectiveness of the amplification primers was verified by agarose gel electrophoresis, and the amplification products were sequenced and compared with the primer design sequence.

[0063] (4) Determination of real-time quantitative PCR reaction conditions. Primers were synthesized, and the sample was diluted with Ulva prolifera genomic DNA to prepare the reaction system. Real-time quantitative PCR amplification was performed, and the reaction conditions were adjusted according to the melting curve.

[0064] Establishing a standard curve

[0065] The quantitatively amplified fragment was cloned and purified using a T-vector PCR product cloning kit. Following the product instructions, the PCR amplification product was inserted into the pUCm-T vector and ligated overnight at 16°C. The plasmid was then introduced into *E. coli* TOP10 competent cells using a heat shock method (42°C water bath for 90 s). The cells were then plated on LB agar plates containing ampicillin and incubated overnight at 37°C with inverted incubation. White colonies were picked and PCR amplified using quantitative primers to confirm the inserted fragment. Positive colonies were picked and incubated overnight at 37°C with shaking in liquid medium containing ampicillin. In the fifth step, cells containing the inserted fragment were collected by centrifugation, and plasmid DNA was extracted using a plasmid extraction kit. The plasmid DNA concentration (P, ng / μL) was determined using a NanoVue (USA) analyzer, and the copy number concentration of the plasmid DNA was calculated based on the plasmid molecular weight, etc.

[0066] In the formula: Cv is the plasmid copy number concentration (copies / μL); P is the measured mass concentration of plasmid DNA extract (ng / μL); Lv and Lf are the number of bases (bp) of the plasmid vector and the target fragment, respectively. In this study, Lv = 2773bp and Lf = 297bp; 6.02 × 1023 is Avogadro's constant (i.e., number of moles); 660 is the average molecular weight of double-stranded DNA bases (g / mol); 109 is the conversion factor between ng and g.

[0067] Plasmid DNA was serially diluted (0, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, and a blank control group), with each concentration set up in triplicate. Quantitative amplification was performed using a real-time PCR instrument to obtain the Ct values ​​(critical cycle values ​​for qPCR) of samples with different DNA concentrations.

[0068] A standard curve was plotted with the logarithm of plasmid DNA concentration on the x-axis and Ct value on the y-axis.

[0069] Establishment of correspondence

[0070] Dry weights of 1 mg, 10 mg, 100 mg, 1 g, and 10 g of *Ulva prolifera* algae were taken and disrupted using an ultrasonic disruptor. The disrupted algae were then placed in a 5 L artificially prepared marine environment. Free DNA was collected by filtration using an environmental DNA sampler. After 10-fold serial dilutions, each sample was processed using the SYBR Green dye method (e.g., ...). Figure 9 As shown, real-time quantitative PCR was performed to achieve rapid quantitative detection by observing changes in fluorescence signal intensity. The Ct value of the sample was obtained and converted into DNA copy number using a standard curve. The correspondence between DNA copy number and the dry weight of *Ulva prolifera* was established, i.e., the range of DNA copy number of 1g of floating *Ulva prolifera* at a fixed dilution factor.

[0071] Field Application

[0072] (1) Large-scale sampling. Based on the on-site survey stations of marine seaweed green tide population and environmental factors, environmental DNA sampling was carried out using a water environment DNA filter. 5L of seawater was collected in layers, filtered onto the filter membrane, and environmental DNA protection solution was added. The samples were then stored in a -20℃ refrigerator or liquid nitrogen for laboratory testing and analysis after being brought ashore.

[0073] (2) Fixed-point continuous sampling. Relying on the fixed-point continuous station at sea, the environmental DNA online automatic collection instrument (automatic control, which can automatically enrich 9 environmental DNA samples through processes such as water sample washing, rinsing, sampling, emptying, and adding fixative) is used to carry out automatic environmental DNA sampling. Every six hours, 5L of seawater is collected in layers, filtered onto the filter membrane, and environmental DNA protection solution is added. The samples are then stored in a -20℃ refrigerator or liquid nitrogen for laboratory testing and analysis after being brought ashore.

[0074] (3) Environmental DNA Sample Detection. The filter membrane sample was broken up by agitation (speed, time, temperature) using grinding beads and lysis buffer. DNA contained in the environmental medium was fully released using physical and chemical methods, and impurities such as proteins, lipids, polysaccharides, and RNA were removed from the sample. DNA was purified using conventional molecular biology techniques such as centrifugation column method and magnetic bead method, with the procedure described in GB / T40226. The sample DNA concentration should not be less than 1 ng / μL, with an optimal range of 10-100 ng / μL. The absorbance ratio at 260 nm and 280 nm (OD260nm / OD280nm) should be within the range of 1.7-2.0, and the OD260nm / OD230nm ratio should be greater than 2.0. After parallel aliquoting, the DNA was stored at -20℃ or below, avoiding repeated freeze-thaw cycles. Quantitative PCR amplification was performed according to the real-time quantitative PCR reaction conditions established in this study. Based on the sample Ct values, the standard curve, and the corresponding relationship, the approximate biomass of the *Ulva prolifera* population at the survey station was calculated.

[0075] Results Analysis

[0076] This study compares the differences between environmental DNA monitoring technology and the traditional trawl "sampling-culture-identification" monitoring method in terms of the detection rate, relative biomass, spatial distribution, and temporal variation of *Ulva prolifera*, assessing the feasibility of operational application of environmental DNA monitoring technology. Spatial distribution analysis was conducted on environmental DNA detection data obtained from large-scale survey stations, and distribution maps were drawn. Temporal variation analysis was conducted on environmental DNA detection data obtained from fixed-point continuous stations, and temporal variation maps were drawn. Subsequent analysis, combined with environmental factor survey results, will further investigate the relationship between the spatiotemporal variation characteristics of *Ulva prolifera* green tide populations and environmental factor regulation.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it.

Claims

1. A device for detecting microscopic propagules of *Ulva prolifera*, characterized in that, The filter includes a filter body (1), a connector (2) is fixedly connected to the top of the filter body (1), a filter (3) is provided on the top of the connector (2), and a connection protection component is provided on the outside of the connector (2); the connection protection component includes a protection block (4), a protection cover (5), a first protection component and a second protection component, the bottom of the protection block (4) is fixedly installed on the top of the filter body (1), the protection block (4) is located on the outside of the connector (2) and the filter (3), the protection cover (5) is located on the top of the protection block (4), the first protection component is provided on the outside of the connector (2) and the filter (3), and the second protection component is provided on the outside of the filter (3).

2. The detection device for microscopic propagules of *Ulva prolifera* according to claim 1, characterized in that, There are multiple sets of the filter (3), protective block (4) and protective cover (5), and they are arranged in a linear array on the top of the filter (3) body. The filter (3) is provided with multiple layers of filter membranes, and filter pores are opened on the filter membranes. The diameter of the filter pores gradually increases. The sample inlet tubes (6) are fixedly installed on the upper and lower sides of the filter (3). The sample inlet tube (6) located at the bottom of the filter (3) is inserted into the connector (2). The first protective component and the second protective component are located on both sides of the connector (2) and the filter (3), and they are arranged symmetrically with respect to the center of the filter (3).

3. The detection device for microscopic propagules of *Ulva prolifera* according to claim 2, characterized in that, The first protective component includes a limiting block (7), a pushing block (8), a first rack (9), a gear (10), a rotating rod, a second rack (11), and a moving block (12). The limiting block (7) is slidably connected to the top of the filter body (1). The pushing block (8) is fixedly installed on one side of the limiting block (7). The first rack (9) is fixedly installed on one side of the pushing block (8). The gear (10) meshes on one side of the first rack (9). The rotating rod is fixedly installed in the middle of the gear (10). The bottom of the rotating rod is rotatably connected to the top of the filter body (1). The second rack (11) meshes on one side of the gear (10). The moving block (12) is fixedly connected to one side of the second rack (11).

4. The detection device for microscopic propagules of *Ulva prolifera* according to claim 3, characterized in that, The limiting block (7) is located outside the connector (2), and the limiting block (7) has an arc-shaped groove on the side near the connector (2). The bottom of the filter (3) is located at the top of the limiting block (7). The second protective component is located at the top of the moving block (12). The moving block (12), the pushing block (8), the first rack (9) and the second rack (11) are all slidably connected to the top of the filter body (1). The first magnetic block (13) is fixedly installed on both sides of the moving block (12). The second magnetic block (14) is fixedly installed on the inner wall of the protective block (4). The first magnetic block (13) and the second magnetic block (14) are magnetically connected.

5. The detection device for microscopic propagules of *Ulva prolifera* according to claim 4, characterized in that, The second protective component includes a fixed block (15), a movable block (16), a slider (27), and a limiting plate (17); the fixed block (15) and the movable block (12) are fixedly installed at the top, and the slider (27) is fixedly installed at the bottom of the movable block (16). The slider (27) and the movable block (16) are slidably connected to the inner wall of the fixed block (15). One end of the limiting plate (17) is hinged to the movable block (16), and a limiting groove is provided on the side of the limiting plate (17) away from the movable block (16).

6. The rapid detection device for microscopic propagules of *Ulva prolifera* according to claim 5, characterized in that, One end of the limiting plate (17) is located inside the fixing block (15), and the other end is located on the top of the fixing block (15). A movable groove is provided on the top of the limiting plate (17) located behind the filter (3). A limiting rod (18) is provided in the movable groove. The limiting rod (18) is L-shaped and is slidably set in the movable groove by a fixing spring. A slot (19) is provided on the top of the limiting plate (17) located in front of the filter (3). The limiting rod (18) and the slot (19) are both located on both sides of the limiting groove. The limiting rod (18) is inserted into the limiting groove. A fixing hole is provided on the side of the limiting plate (17) near the inner wall of the fixing block (15).

7. The detection device for microscopic propagules of *Ulva prolifera* according to claim 6, characterized in that, The filter (3) is provided with limiting components on both the left and right sides. There are four sets of limiting components, which are symmetrically arranged with respect to the center of the filter (3). The limiting components include a mounting block (20), a fixing plate (21), and a first telescopic rod (22). The mounting block (20) and the fixing plate (21) are fixedly installed on the inner wall of the protective block (4). One end of the first telescopic rod (22) is hinged to the mounting block (20), and the other end is located on the top of the fixing plate (21). The first telescopic rod (22) is inserted into the fixing hole.

8. The detection device for microscopic propagules of *Ulva prolifera* according to claim 7, characterized in that, A movable assembly is provided between the filter (3) and the limiting block (7). The number of movable assemblies is the same as the number of protective blocks (4). The movable assemblies are connected by a connecting block (23). The movable assembly includes a movable plate (24), a compression spring (25), and a second telescopic rod (26). A groove is provided on the top of the limiting block (7). The movable plate (24) is located inside the groove. A compression spring (25) is fixedly installed at the bottom of the movable plate (24). One end of the compression spring (25) is fixedly installed to the inner wall of the bottom of the groove. The protective block (4) is located outside the groove. The wall has an opening for movement, and the bottom inner wall of the opening has a fixed groove. The second telescopic rod (26) is fixedly installed inside the fixed groove. The top of the second telescopic rod (26) is fixedly installed to the bottom of the moving plate (24). One end of the moving plate (24) located in the protective block (4) on both sides of the filter body (1) is slidably connected to the inner wall of the protective block (4), and the other end is fixedly installed to the connecting block (23). The moving plate (24) located in the protective block (4) in the middle of the filter body (1) has sliding connections to the inner walls of the openings on both sides, and is fixedly connected to the connecting block (23).

9. A rapid detection method for microscopic propagules of *Ulva prolifera*, characterized in that, The detection device for the microscopic propagation of *Ulva prolifera* as described in claim 8 includes the following steps: Step 1: Use a water sampler to collect water samples from the top and bottom layers. After filtering the water samples, place them in a sample container. Then install the filter (3) on the filter body (1). The filter (3) is limited by the first limiting component and the second limiting component. The sample container is connected to the inlet tube (6) of the filter body (1) through the connecting tube. At the same time, the collection tube is connected to the outlet of the filter. Start the filter body (1) to filter the water sample. After filtration, move the first protection component and the second protection component to no longer limit the filter (3). Remove the filter (3) through the moving component and put the filter membrane into a 2.0ml sterile centrifuge tube for freezing and storage. Step 2: DNA extraction. Add 500 μL of lysis buffer and 20 μL of proteinase K to a centrifuge tube with a cryofilter membrane; incubate at 65°C for 4 hours, inverting and shaking to mix; centrifuge at 10000 x g / min at room temperature for 10 minutes, and collect the supernatant; Add 1.5 volumes of chloroform:isoamyl alcohol (24:1), centrifuge at 10000xg / min for 10 min, and collect the supernatant; add 1 volume of isopropanol, and precipitate overnight at 4°C; centrifuge at 4°C for 10 min to collect the DNA precipitate. Wash and dry with 70% ethanol; dissolve the precipitate in 100 μL of LTE buffer (pH=8) for later use to obtain high-purity DNA; Step 3: Establishment of the standard curve. Select a specific gene fragment from *Ulva prolifera* and design primers. Use a T-vector PCR product cloning kit to clone and purify the amplified product of the specific gene fragment, extract plasmid DNA, determine the plasmid DNA concentration, and dilute the plasmid DNA. Plot a qPCR standard curve with the logarithm of the plasmid DNA concentration (log10Cv) on the x-axis and the critical cycle value (Ct) of the qPCR reaction on the y-axis. Step 4: Calculate the copy number of *Ulva prolifera* micropropagules in the sample by comparing the sample DNA amplification with the standard curve; calculate the copy number of the target gene in a single cell based on the DNA concentration and gamete cell concentration of the target gene; calculate the corresponding micropropagule cell abundance based on the copy number abundance of *Ulva prolifera* micropropagules obtained by qPCR, thereby achieving rapid identification and quantitative detection of *Ulva prolifera* micropropagules.

Images