A high-efficiency collection system and method based on nematode negative phototaxis, oxygenotaxis and hydro taxis

Abstract

The application discloses a high-efficiency collection system and method based on nematode negative phototaxis, oxygenotaxis and hydro taxis, relates to the technical field of biological sample collection and detection, and comprises a modified Bellman funnel module, a modified shallow tray module, a comprehensive regulation and control module and a mild auxiliary agent configuration unit. The module integrates a precise light gradient and a simple oxygenation assembly, and sterile water is replaced by a mild permeation wetting auxiliary agent extraction solution. The method covers the following steps: configuring a corresponding concentration extraction solution, pretreating a sample, building a module and constructing a water-seeking and directional light gradient environment, oxygenating to maintain dissolved oxygen, collecting and centrifuging the sample after constant temperature standing. The auxiliary agent can be selected from Tween-20, organic silicon and potassium cocoyl glycinate, and is single or complex, and different samples correspond to different concentrations; oxygenation can be intermittently performed by a syringe or a micro air pump. The system and method can be adapted to various plant parasitic nematodes such as pine wood nematodes and various samples, are efficient in collection, and the collection liquid can be directly used for subsequent experiments such as microscopy, culture and PCR.

Description

Technical Field

[0001] This invention relates to the field of biological sample collection and detection technology, specifically to a highly efficient collection system and method based on the negative phototaxis, oxygen tropism, and hydrotropism of nematodes. Background Technology

[0002] In plant pathology and soil animal ecology research, the efficient collection of nematodes, especially plant parasitic nematodes, has always been a key focus and challenge in basic research. Plant parasitic nematodes, such as pine wood nematodes, root-knot nematodes, and stem nematodes, often hide inside plant tissues or in the soil microenvironment. They are small in size, have limited mobility, and are significantly affected by environmental factors. Traditional collection methods often suffer from low efficiency, significant sample damage, and numerous interferences in subsequent experiments, thus hindering the in-depth development of related research.

[0003] Early nematode collection relied heavily on natural separation or simple water immersion. These methods depended on the passive aggregation of nematodes through their own movement, which was not only time-consuming but also lacked the ability to actively regulate environmental factors, making it difficult to improve collection efficiency based on the biological characteristics of nematodes. For example, the Bellman funnel method, a classic collection technique, utilizes the principle of hydrotaxis to guide nematodes into the collection solution. However, in routine operations, the collection solution is often sterile water, which lacks effective wetting of the nematode surface and sample matrix. This causes nematodes to easily become trapped in the gaps between dry samples and unable to escape. At the same time, natural light in the collection solution area can easily induce negative phototaxis stress in some nematodes, inhibiting their downward movement and further reducing the collection rate. While shallow dish-type devices are simple to operate, they generally suffer from problems such as the inability to precisely control the light gradient and unstable dissolved oxygen environment. Insufficient dissolved oxygen can slow down the metabolism of nematodes or even cause them to enter a dormant state, while excessive oxygen may accelerate microbial reproduction, contaminate the sample, or destroy the activity of nematodes.

[0004] Regarding the regulation of environmental factors, traditional methods for utilizing oxygen attraction are mostly limited to static water bodies, failing to consider the dynamic dissolved oxygen requirements of nematodes. The application of phototaxis is often limited to single-mode shading or full-sunlight treatment, failing to establish a directional light gradient between the sample area and the collection liquid area, making it difficult to guide nematodes to migrate along specific paths. Furthermore, the sample pretreatment process lacks standardized procedures; for example, cutting pine wood into excessively large pieces can create internal dry zones, and insufficient soil sieving can leave impurities that hinder nematode release. These details all affect the final collection results.

[0005] In terms of the use of auxiliary agents, conventional collection solutions are mostly pure water or simply supplemented with chemical reagents. Although these can dissolve some of the waxy coating on the nematode's surface, the highly irritating components can easily cause damage to the nematode's body wall, reduce its viability, lead to morphological distortion during subsequent microscopic examination, low survival rate during culture, and susceptibility to interference from inhibitors during PCR amplification. For example, while some chlorine-containing auxiliary agents can enhance wettability, they can damage the integrity of nematode DNA; high concentrations of surfactants may alter the osmotic pressure of the collection solution, causing nematode cell rupture. Therefore, developing a wetting system that is both mild and highly efficient is crucial for improving collection quality.

[0006] In recent years, although some studies have attempted to optimize single environmental factors (such as improving the funnel's light-shielding design and adding ventilation devices), a systematic approach to multi-factor synergistic regulation has not yet been developed, especially lacking the integrated application of the linkage mechanism among the negative phototaxis, oxygen tropism, and hydrotropism of nematodes. Existing devices generally suffer from problems such as dispersed components, uncontrollable parameters, and poor adaptability, making it difficult to meet the differentiated collection needs of different sample types (such as dry, resinous pine wood, high-organic-matter soil, and hard root nodules). For example, the collection of pine wood nematodes needs to take into account the hydrophobicity problem caused by the high resin content of pine wood, while the collection of root-knot nematode nodules needs to address the problem of slow nematode release within dense tissue. Traditional general-purpose devices are insufficient in their ability to respond to such specific needs.

[0007] In summary, existing nematode collection technologies still have significant shortcomings in terms of precise control of environmental factors, development of mild adjuvants, sample compatibility, and standardization of collection procedures. There is a need to construct a highly efficient collection system that can integrate negative phototaxis guidance, oxygen tropism maintenance, and hydrotaxis enhancement, and is compatible with different sample characteristics, in order to improve collection efficiency and sample quality, and provide reliable material support for nematode classification and identification, pathogenic mechanism research, and biological control. Summary of the Invention

[0008] The purpose of this invention is to provide a highly efficient collection system and method based on the negative phototaxis, oxygen tropism, and hydrotropism of nematodes. By improving modules, precisely controlling light and oxygen, and using mild additives, a highly efficient collection system and method adaptable to multiple samples is constructed, thereby improving collection efficiency. The obtained samples can be directly used for subsequent experiments.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a highly efficient collection system and method based on the negative phototaxis, oxytaxis, and hydrotaxis of nematodes, comprising a modified Bellman funnel module, a modified shallow dish module, a comprehensive control module, and a mild adjuvant preparation unit; both the modified Bellman funnel module and the modified shallow dish module integrate a precise light gradient component and a simple oxygenation component, and use a mild penetrating wetting adjuvant extract instead of conventional sterile water to achieve synergistic guidance of the negative phototaxis, oxytaxis, and hydrotaxis of nematodes; The simplified oxygenation assembly includes an inflation tube, a sterile syringe and / or a micro air pump, and a 0.22μm sterile filter. The end of the inflation tube can reach the bottom of the collection liquid, and the end is smoothed to avoid scratching the container or disturbing the collection liquid. The bottom of the shallow dish of the modified Bellman funnel module is coated with black on the outer wall. The outer wall of the collection area, the outer wall of the funnel, and the outer wall of the collection tube of the modified Bellman funnel module are all covered or coated with black light-absorbing material to keep the illuminance of the collection liquid area stable at <5 lux and the illuminance of the sample area controlled at 50-200 lux, forming a clear light gradient between light and dark zones. The modified Bellman funnel module retains the main structure of the funnel, and its collection area is physically isolated from the sample carrying area. The collection area is located below the sample carrying area and does not contact the sample. The modified shallow dish module is an open-top shallow container, which is divided into a sample placement area and a collection liquid area. The sample placement area and the collection liquid area are set apart, and the volume of the collection liquid area meets the sedimentation requirements of nematodes.

[0010] Furthermore, the mild penetrating wetting agent is one or a combination of two of the following: Tween-20, organosilicon (Silwet L-77), and potassium cocoyl glycinate; The mild penetrating and wetting agent has low surface tension, which can enhance the wetting and penetrating ability of the collected liquid on the nematodes in the sample matrix, while avoiding damage to the nematode's surface structure and physiological activity.

[0011] Further, the concentration of Tween-20 used is 0.003%-0.005% (v / v), the concentration of the organosilicon used is 0.001%-0.002% (v / v), and the concentration of potassium cocoyl glycinate used is 0.002%-0.004% (v / v); the compound additive is 50% of the concentration of each individual additive. When two additives are used in combination, weigh out 50% of the recommended concentration of each additive and mix them in sterile water. Stir well to form a compound extract to balance the wetting performance requirements of different sample matrices and reduce the potential impact of a single additive.

[0012] Furthermore, the simplified oxygenation component draws 5-20 mL of sterile air using a sterile syringe, or intermittently injects air into the bottom of the collection liquid by setting parameters using a micro air pump, maintaining the dissolved oxygen concentration of the collection liquid at 5-8 mg / L; for larger nematode collection scenarios such as large (over 20 cm in diameter) nematode separation equipment used in customs timber quarantine and large wood block quarantine, small to medium-sized air replenishment devices such as aquarium air pumps or small oxygen generators are connected to air filling tubes for oxygenation; Intermittent oxygenation refers to non-continuous ventilation, which controls the duration of ventilation and the duration of cessation of ventilation by controlling the interval time, so as to avoid excessive fluctuations in dissolved oxygen due to continuous agitation of the collected liquid or the escape of nematodes due to mechanical stimulation.

[0013] Furthermore, the end of the aerated capillary is 0.5-1 cm from the bottom of the collected liquid, and the end of the aerated capillary needs to be smoothed. The inflatable tube is made of medical-grade polyethylene or silicone, with an inner diameter ≤1mm, uniform wall thickness, and the end is heat-melted or polished to form a blunt round shape, ensuring that no sharp edges are generated during the oxygenation process to scratch the inner wall of the collection container or puncture the filter membrane.

[0014] Furthermore, the integrated control module includes a portable dissolved oxygen meter and an illuminometer, used to monitor the dissolved oxygen concentration and light gradient parameters of the collected liquid in real time, ensuring that the parameters are accurate and controllable; The portable dissolved oxygen meter probe can be inserted into the collection liquid area to measure the dissolved oxygen concentration. During measurement, avoid touching the bottom or side wall of the container. The illuminance meter probe is placed at the center of the sample area and 1 cm below the liquid surface in the collection liquid area to read the illuminance values ​​respectively. The measurement results are used to adjust the light source intensity and oxygenation frequency.

[0015] Furthermore, the mild adjuvant extract is prepared with sterile water as a base, is freshly prepared and used immediately, and can be directly used as a nematode collection solution without subsequent washing, and does not interfere with subsequent experiments such as microscopic examination, culture, and PCR. The pH value of the mild adjuvant extract is controlled at 6.5-7.5, which is close to the osmotic pressure of the nematode's living environment. After preparation, it should be sealed and stored and used within 2 hours to avoid degradation of the adjuvant or microbial growth due to prolonged storage.

[0016] Furthermore, it includes the following core steps: S1: Prepare a mild adjuvant extract with the corresponding concentration according to the sample type. The adjuvant is one or a combination of two of the following: Tween-20, organosilicon, and potassium cocoyl glycinate. S2: Sample pretreatment: Pine wood is cut into 0.5-5cm sawdust, soil is sieved through a 2mm sieve to remove stones and impurities, root nodules are cleaned and cut open with a sterile blade to expose the internal tissue, and the sample is spread in a thickness of 1-2cm to ensure sufficient hydrotactic interaction with the collection liquid area. S3: Construct a modified Bellman funnel module or a modified shallow dish module, physically shield the collection liquid area from light, add mild adjuvant extract to 1 / 3-1 / 2 of the volume of the collection liquid area, and construct a hydrotropic environment in which the sample area is kept moist by matrix capillary action, the collection liquid area is stably stored, and the sample does not directly contact the collection liquid, so as to promote the nematodes to migrate to the collection liquid area along the humidity gradient. S4: Construct a directional light gradient. The liquid collection area is completely wrapped with a black light shield or placed in a dark box to achieve an illuminance of <5 lux. The sample area is illuminated from the side and above by a bright blue LED cold light source at a distance of 30-60cm. The light source power is ≤5W and equipped with a heat dissipation device to ensure that the temperature of the illuminated area is ≤1℃ higher than the ambient temperature. The illuminance is controlled at 50-200 lux. S5: Insert the air-filled capillary tube to the bottom of the collection liquid, connect the front end to a 0.22μm sterile filter via a Luer connector, and slowly inject 5-20mL of sterile air using a sterile syringe, with an injection time of ≥10 seconds. The oxygenation frequency is once every 6 hours; or control the micro air pump through a timer switch and a gas flow meter to intermittently introduce sterile air into the collection liquid to maintain the dissolved oxygen concentration of the collection liquid at 5-8mg / L. S6: Keep at a constant temperature of 20-25℃, or use the modified Bellman funnel method to keep for 3-10 minutes, avoiding vibration during the process; or use the modified shallow dish method to keep for 6-24 hours, with ambient temperature fluctuations ≤±1℃. S7: Collect the collected liquid, centrifuge at a low speed of 800-1000 rpm for 5-10 minutes, discard the supernatant and take the precipitate to obtain the nematode sample, which can be used directly for subsequent experiments.

[0017] Furthermore, in step S1, the dried resinous pine wood is treated with a 0.001%-0.002% organosilicon extract, utilizing the strong permeability of organosilicon to the resinous matrix to promote nematode release; the root-knot nematode nodules are treated with a 0.003%-0.005% Tween-20 extract, utilizing the mild dispersion effect of Tween-20 on plant tissues to expose the nematodes inside the nodules; the soil samples are treated with a 0.002%-0.004% potassium cocoyl glycinate extract, relying on its zwitterionic properties to adapt to the complex ionic environment of the soil; the mixed samples are treated with a combination of organosilicon + potassium cocoyl glycinate or Tween-20 + potassium cocoyl glycinate extract, taking into account the permeability and dispersion requirements of different matrices.

[0018] Further, in step S5, the oxygenation parameters of the micro air pump are a flow rate of 5-12 mL / min, operating for 5 minutes and stopping for 25 minutes, forming a 30-minute cycle of charging and stopping. The method is applicable to the collection of plant parasitic nematodes such as pine wood nematode, root-knot nematode, and stem nematode, and is suitable for sample types such as dry resinous pine wood, soil, root-knot nematode nodules, and plant stem slices (thickness ≤3 mm). During the collection process, the sample does not have direct contact with the collection liquid area, and the nematodes are guided to migrate autonomously into the collection liquid by hydrotropism.

[0019] This invention provides a highly efficient collection system and method based on the negative phototaxis, oxytaxis, and hydrotaxis of nematodes, which has the following beneficial effects: 1. This system utilizes the synergistic effects of nematodes' negative phototaxis, oxytaxis, and hydrotaxis. A precise light gradient (<5 lux in the collection liquid area, protected from light; 50-200 lux in the sample area, blue light) guides the nematodes' directional migration. Combined with an oxygenation component maintaining dissolved oxygen at 5-8 mg / L, it simulates the nematodes' suitable microenvironment, stimulating migration. Mild additives (Tween-20, organosilicon, etc.) reduce the surface tension of the liquid, enhancing wetting and penetration onto the nematode surface, reducing migration resistance, and avoiding interference from the "weakness" of conventional sterile water. Experiments show that the modified Bellman funnel method allows for effective collection within 3-10 minutes of settling, shortening the time and improving the nematode integrity rate compared to traditional methods. It significantly increases the collection volume, especially for difficult-to-process samples such as dried, resinous pine, providing sufficient high-activity samples for subsequent testing.

[0020] The system is designed to cover various sample types, including pine wood, soil, root nodules, and stems. Through differentiated adjuvant configurations (e.g., low-concentration organosilicon for dried pine wood, Tween-20 for root nodules, and potassium cocoyl glycinate for soil) and standardized sample pretreatment (0.5-5cm sawdust, soil sieved through a 2mm sieve), it specifically optimizes the release and migration conditions of nematodes in different substrates. The modular structure (with optional Bellman funnel / shallow dish modules) supports both small-volume precision separation and expansion to large-scale applications (e.g., customs timber quarantine). Simple oxygenation components (syringe / micro-pump) meet different scale requirements. It can be set up without complex equipment, allowing for rapid application in both basic laboratories and field operations, significantly improving the adaptability and ease of use for nematode collection.

[0021] Traditional methods using sterile water can easily lead to residual moisture on the nematode surface, morphological deformation, or require additional washing steps that may affect activity. This system uses a mild adjuvant extract instead of sterile water. The adjuvant concentration is optimized (e.g., Tween-20 at only 0.003%-0.005% v / v), possessing both wetting and low-irritant properties, and can be used directly as the collection solution, avoiding nematode inactivation due to sudden changes in osmotic pressure or mechanical damage. The collected nematodes, after low-speed centrifugation, can be used for microscopic examination, culture, or PCR, eliminating the washing step. This preserves the natural morphology and physiological state of the nematodes and prevents adjuvant residues from inhibiting enzyme activity or interfering with molecular detection, providing highly reliable samples for nematode identification and pathogenicity analysis in scientific research and production.

[0022] The integrated control module is equipped with a portable dissolved oxygen meter and illuminance meter, which can monitor and provide feedback on the dissolved oxygen concentration and light gradient parameters of the collected solution in real time. Combined with timed oxygenation (once every 6 hours, injection ≥10 seconds) and a fixed illumination range (50-200 lux in the sample area), it eliminates environmental fluctuations caused by human error. A physical light-shielding design (blackened funnel outer wall, illumination <5 lux in the collection area) avoids stray light interference. The smooth end of the aeration capillary, positioned 0.5-1 cm from the bottom of the liquid, ensures uniform oxygen diffusion without dead zones. This precise control significantly improves the consistency of collection results across different batches and operators, making it particularly suitable for quarantine or research scenarios requiring quantitative analysis, providing technical support for data comparability and the accuracy of conclusions.

[0023] The system uses mild additives (such as potassium cocoyl glycinate as a natural surfactant), which are highly biodegradable and leave no toxic residues, avoiding the environmental pollution and potential hazards to operators associated with traditional chemical reagents. The oxygenation components are connected via a 0.22μm sterile filter to prevent external microbial contamination of the collection solution; the freshly prepared extract reduces storage losses and the risk of spoilage. The modular design reduces consumable costs (e.g., reusable funnels and shallow trays), and the miniaturized equipment (syringes, aquarium air pumps) has low energy consumption, making it suitable for long-term field use or high-frequency testing. The overall solution balances efficient collection with environmental safety, meeting the requirements of modern biological detection technology for both environmental friendliness and biosafety. Attached Figure Description

[0024] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0025] Figure 1 This is a diagram showing the overall structure of the system of the present invention; Figure 2 This is a flowchart of the improved Bellman funnel module of the present invention; Figure 3 This is a flowchart of the improved shallow dish module of the present invention; Figure 4 This is a flowchart of the integrated control module of the present invention; Figure 5 This is a flowchart of the efficient nematode collection method of the present invention. Detailed Implementation

[0026] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of apparatuses consistent with some aspects of this disclosure as detailed in the appended claims.

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0028] How to use: First, preliminary preparations are carried out. Mild adjuvant extracts are prepared according to the sample type, using sterile water as a base and prepared immediately for use as the collection solution. For dried, resinous pine wood, an organosilicon extract is used; for root-knot nematode nodules, Tween-20 extract is used; for soil samples, potassium cocoyl glycinate extract is used; and for mixed samples, a corresponding compound extract is used. Simultaneously, a modified Bellman funnel or shallow dish module, a comprehensive control module, and a simple oxygenation assembly are prepared. The ends of the aeration tubes are checked for smoothness, and all components are ensured to be clean and sterile.

[0029] In the sample pretreatment stage, samples are processed to a suitable state according to their type: pine wood is cut into fine wood chips, soil is sieved, root nodules are cleaned and cut open, and the thickness of the treated sample is controlled at 1-2cm to ensure that the sample is uniform and loose.

[0030] The acquisition module was set up, and after selecting the appropriate module, the collection liquid area was physically shielded from light. A prepared mild auxiliary extract was added to create a hydrotropic environment, ensuring that the sample remained moist but did not come into direct contact with the collection liquid. A directional light environment was constructed using the module's built-in precise light gradient component, completely shielding the collection liquid area from light. The sample area used a bright blue cold LED light source, and the distance to the light source was controlled to avoid temperature rise, thus forming a reasonable light gradient.

[0031] Initiate oxygenation control by inserting the air-filled capillary tube with the series sterile filters to the bottom of the collection liquid, maintaining an appropriate distance between the end and the bottom. Sterile air can be slowly injected using a sterile syringe, controlling the injection time and oxygenating at fixed intervals; alternatively, a miniature air pump can be controlled via a timer switch and gas flow meter to intermittently introduce sterile air. Real-time monitoring using a portable instrument within the integrated control module maintains the dissolved oxygen concentration in the collection liquid within a suitable range. For large collection equipment, appropriate small to medium-sized air supply devices should be selected.

[0032] Constant temperature static incubation involves placing the entire collection system in a suitable room temperature environment. The modified Bellman funnel method requires a short static incubation period, while the modified shallow dish method extends the incubation time. During this period, environmental stability must be maintained to avoid light and temperature fluctuations affecting nematode migration.

[0033] Sample collection and processing: After standing, collect the collected liquid, centrifuge at low speed and take the precipitate to obtain nematode samples. No additional washing steps are required, and it can be directly used for subsequent experimental operations such as microscopic examination, culture, and PCR.

[0034] Precautions: All components must be sterile, and the inflatable capillary tubes must be cleaned promptly after use; the extract should be prepared and used immediately to avoid ineffectiveness; parameters should be monitored throughout the process to ensure accurate and controllable collection conditions, thereby improving the efficiency and purity of nematode collection.

[0035] Example: Example 1: Collection of pine wood nematodes from dried resinous pine wood using a modified Bellman funnel module This embodiment uses a modified Bellman funnel module, a simple oxygenation component, a comprehensive control module, and a mild additive preparation unit to construct a collection system for collecting pine wood nematodes from dried, resinous pine wood samples. The entire process relies on the nematodes' negative phototaxis, oxygen attraction, and hydrotaxis to achieve efficient separation without interfering with subsequent experiments.

[0036] First, a mild adjuvant extract was prepared using sterile water as a base, along with a freshly prepared 0.0015% (v / v) organosilicon (Silwet L-77) extract. This concentration is suitable for the wetting needs of dry, resinous pine wood while avoiding damage to pine nematodes. Next, the condition of each component was checked to ensure that the outer wall of the modified Bellman funnel module was blackened, and that the collection area, funnel, and collection tube were all black, achieving a light intensity of <5 lux in the collection area and a controllable light intensity in the sample area within the range of 50-200 lux. The end of the aeration tube of the simple oxygenation component was smoothed, and a 0.22μm sterile filter was connected in series at the front end to ensure sterile aeration and prevent sharp structures from scratching the nematodes.

[0037] In the sample pretreatment stage, the dried resinous pine samples were cut into wood chips of 1-3 cm in size to ensure uniform size and avoid large samples from affecting nematode migration. The treated wood chips were then evenly spread on the sample area of ​​the modified Bellman funnel with a thickness of 1.2 cm to ensure that the sample layer is loose and breathable, and to facilitate the subsequent construction of a hydrotactic environment.

[0038] A data collection system was constructed by adding a prepared organosilicon extract to the collection area of ​​a modified Bellman funnel, ensuring that the extract served as the collection liquid. This created a hydrotropic environment where the sample area was moist, the collection liquid area remained liquid, and the sample did not directly contact the collection liquid. A directional light environment was constructed using a precise light gradient component. The collection liquid area was completely shielded from light by its blackened outer wall. The sample area used a bright blue cold LED light source, and the distance between the light source and the sample area was controlled to ensure no temperature rise in the illuminated area. The illuminance in the sample area was stabilized at 80 lux in real time using a lux meter.

[0039] Initiate the oxygenation process by inserting the air-filled tube to the bottom of the collection solution, with the end 0.8 cm from the bottom. Use a sterile syringe to draw 12 mL of sterile air and slowly inject it into the collection solution over 12 seconds. Set the oxygenation frequency to once every 6 hours. Monitor the dissolved oxygen concentration of the collection solution in real time using a portable dissolved oxygen meter with the integrated control module to ensure it is stably maintained at approximately 6 mg / L.

[0040] The entire collection system was placed in a constant temperature environment of 22℃ and allowed to stand for 6 minutes using a modified Bellman funnel method. During this time, the environment was kept sealed to avoid interference from light and temperature fluctuations that could affect nematode migration. After standing, the collected liquid was collected and centrifuged at low speed. The precipitate after centrifugation was used to obtain the pine wood nematode sample. This sample did not require further washing and could be directly used for microscopic examination and PCR detection experiments.

[0041] Example 2: Collection of stem nematodes from soil using an improved shallow tray module This embodiment uses a modified shallow tray module, a simple oxygenation component, a comprehensive control module, and a mild additive preparation unit to construct a collection system for collecting stem nematodes from soil samples. It fully utilizes the advantages of the system's light gradient control, oxygenation control, and mild extraction to improve collection efficiency and sample purity.

[0042] Mild adjuvant extract preparation: Using sterile water as a base, prepare a 0.003% (v / v) potassium cocoyl glycinate extract. This adjuvant effectively wets soil particles, promotes stem nematode detachment from the soil matrix, and does not affect the activity of stem nematodes. It can also be used directly as a collection solution. Inspect the improved shallow tray module to ensure that its integrated precision light gradient component and simple oxygenation component are functioning properly, the end of the aeration capillary is smooth and free of burrs, and the 0.22μm sterile filter is undamaged.

[0043] Sample pretreatment: The soil sample to be collected is sieved through a 2mm sieve to remove impurities such as stones and plant debris, to avoid clogging the oxygenation components or interfering with nematode collection. The sieved soil is then evenly spread in the sample area of ​​the modified shallow dish, with a thickness controlled at 1.5cm, ensuring a flat soil layer to facilitate nematode migration to the collection liquid area.

[0044] Module Construction and Environmental Control: A prepared potassium cocoyl glycinate extract was added to the collection area of ​​the modified shallow dish to ensure the soil in the sample area remained moist but did not directly contact the collection solution, thus creating a stable hydrotropic environment. The lighting environment was set using a precise light gradient component. The collection area employed physical light shielding combined with blackening the module's outer wall to ensure an illuminance of <5 lux. The sample area used a bright blue LED cold light source, and the light source distance was controlled to maintain a stable illuminance of 150 lux in the sample area, with no temperature rise observed in the illuminated area. Parameters were calibrated in real-time using a lux meter.

[0045] Oxygenation control: An air-filled capillary tube with a 0.22μm sterile filter connected in series was inserted to the bottom of the collection liquid, with the end 0.6cm from the bottom. A miniature air pump was used as the oxygenation device. The pump parameters were controlled by a timer switch and a gas flow meter, with a flow rate set to 8mL / min, operating for 5 minutes and stopping for 25 minutes in an intermittent oxygenation mode. The dissolved oxygen concentration in the collection liquid was monitored in real time using a portable dissolved oxygen meter and maintained at around 7mg / L to provide a suitable dissolved oxygen environment for stem nematodes and promote their migration to the collection liquid area.

[0046] Constant temperature settling: The data acquisition system was placed in a constant temperature environment of 23℃ and settling for 18 hours using a modified shallow tray method. During this period, the system was kept away from contact to prevent soil loosening or fluctuations in light gradient and dissolved oxygen concentration. Parameters were checked periodically through the integrated control module during the settling process to ensure the stability of all indicators.

[0047] Sample collection and processing: After settling, slowly collect the collected liquid and put it into a centrifuge tube for low-speed centrifugation. Take the precipitate to obtain the stem nematode sample. The sample does not need to be washed and can be directly used for culture and microscopic analysis. The experimental results are not significantly interfered with.

[0048] Example 3: Collection of root-knot nematodes from root nodules using a modified Bellman funnel module This embodiment uses a modified Bellman funnel module, a simple oxygenation component, a comprehensive control module, and a mild adjuvant preparation unit to collect root-knot nematodes from root nodule samples. By optimizing the light gradient, dissolved oxygen, and extraction conditions, it achieves efficient separation and preservation of the activity of root-knot nematodes.

[0049] Preparation of mild adjuvant extract: Using sterile water as a base, a 0.004% (v / v) Tween-20 extract was prepared. Tween-20 effectively wets the root nodule tissue, promotes the detachment of root-knot nematodes from the nodules, and is mild and non-irritating, maintaining the activity of root-knot nematodes and meeting the needs of subsequent experiments. The modified Bellman funnel module was inspected, confirming that its outer wall blackening treatment was satisfactory, the collection area could achieve complete light protection, and the precision light gradient component could properly adjust the light intensity.

[0050] Sample pretreatment: Select root nodule samples containing root-knot nematodes, rinse the surface soil and impurities with sterile water, and then cut the nodules with a sterile knife to expose the internal tissue, facilitating the detachment of root-knot nematodes. Evenly spread the treated nodules in the sample area of ​​the modified Bellman funnel, controlling the thickness to 1 cm to avoid nodule stacking that could hinder nematode migration.

[0051] System setup and parameter control: Prepared Tween-20 extract was added to the collection zone of the modified Bellman funnel to create a hydrotropic environment, ensuring the nodule sample was moist but not in direct contact with the collection solution. A light gradient environment was set up, with the illuminance in the collection zone <5 lux, and a bright blue cold LED light source used in the sample zone. The distance of the light source was adjusted to stabilize the illuminance in the sample zone at 100 lux, and real-time monitoring was conducted using a lux meter to ensure no temperature rise occurred.

[0052] Oxygenation procedure: Insert an air-filled capillary tube with a 0.22μm sterile filter connected in series at the front end to the bottom of the collection solution, with the end 0.7cm from the bottom. Use a sterile syringe to draw 15mL of sterile air and slowly inject it into the collection solution over 15 seconds. Oxygenation should be performed every 6 hours. Monitor the dissolved oxygen concentration in the collection solution using a portable dissolved oxygen meter, maintaining it at approximately 6.5mg / L to ensure the activity and migration of root-knot nematodes.

[0053] Isothermal settling and sample collection: The system was placed in a constant temperature environment of 24℃ and allowed to stand for 8 minutes using a modified Bellman funnel method. After settling, the collected liquid was collected, centrifuged at low speed, and the precipitate was taken to obtain root-knot nematode samples. These samples can be directly used for microscopic examination, PCR detection, and subsequent culture experiments without interference from the extract.

[0054] Example 4: Collection of plant parasitic nematodes from mixed samples using a modified shallow dish module This embodiment uses a modified shallow tray module, a simple oxygenation component, a comprehensive control module, and a mild adjuvant preparation unit to collect mixed populations of pine wood nematodes and stem nematodes from mixed samples containing soil and plant stem slices. By compounding mild adjuvants and precise environmental control, the simultaneous and efficient collection of multiple nematodes is achieved.

[0055] Preparation of mild adjuvant extract: Using sterile water as a base, a mild adjuvant is prepared by combining organosilicon and potassium cocoyl glycinate. The concentration of organosilicon is 0.0008% (v / v), and the concentration of potassium cocoyl glycinate is 0.0015% (v / v), both 50% of the concentration used for a single adjuvant. The compound extract is prepared and used immediately, and can simultaneously meet the wetting requirements of different matrices in mixed samples. It is non-damaging to various nematodes and can be used directly as a collection solution.

[0056] Sample pretreatment: Cut the plant stems in the mixed sample into 1-2cm pieces, and sieve the soil through a 2mm sieve to remove impurities. Then, mix the treated stem pieces with the sieved soil evenly and spread them on the sample area of ​​the modified shallow dish. The spreading thickness is controlled to 1.8cm to ensure that the mixed sample is evenly distributed and facilitates the migration of nematodes to the collection liquid area.

[0057] Module Construction and Environmental Control: The prepared compound extraction solution was added to the modified shallow tray collection area to ensure the mixed sample remained moist but did not directly contact the collection solution, thus creating a stable hydrotropic environment. The precise light gradient component was adjusted; the collection area achieved an illuminance of <5 lux through blackening the outer wall and physical light shielding. The sample area used a bright blue LED cold light source, controlling the illuminance at 180 lux, and was calibrated in real-time using a lux meter to prevent temperature increases caused by light exposure.

[0058] Oxygenation control: A miniature air pump was used as the oxygenation device. A 0.22μm sterile filter was connected in series at the front end of the air-filling tube, extending to the bottom of the collection liquid, with the end 0.9cm from the bottom. The air pump parameters were set via a timer switch and a gas flow meter, with a flow rate of 10mL / min. Intermittent oxygenation was carried out for 5 minutes on and 25 minutes off. The dissolved oxygen concentration in the collection liquid was monitored using a portable dissolved oxygen meter and maintained at around 7.5mg / L to provide a suitable dissolved oxygen environment for different nematodes.

[0059] Isothermal settling and sample collection: The acquisition system was placed in a constant temperature environment of 21℃ and settling for 22 hours using a modified shallow dish method, maintaining environmental stability and avoiding external interference. After settling, the collected liquid was collected, and the precipitate was removed by low-speed centrifugation to obtain a mixed sample containing pine wood nematodes and stem nematodes. This sample could be used directly for subsequent classification, detection, and culture experiments without washing.

[0060] Example 5: Collection of pine wilt nematodes from customs timber quarantine based on a large-scale modified Bellman funnel module This embodiment is designed for customs timber quarantine scenarios. It uses a large modified Bellman funnel module with a diameter of 25cm, a small and medium-sized air supply device (air pump for aquariums), a comprehensive control module, and a mild adjuvant preparation unit to collect pine wood nematodes from large wood blocks. This meets the needs of batch and efficient collection in quarantine work and strictly complies with aseptic and experimental compatibility requirements.

[0061] Mild additive extraction solution preparation: Based on sterile water, a 0.002% (v / v) organosilicon extraction solution is prepared to meet the wetting requirements of large wood blocks while ensuring the activity of pine wood nematodes. The extraction solution can be used directly as the collection solution without further treatment. The large modified Bellman funnel module was inspected to ensure proper blackening of its outer wall. The collection area, funnel, and outer wall of the collection tube are all black, achieving an illuminance of <5 lux in the collection area. The precise light gradient component covers the sample area, ensuring uniform illumination.

[0062] Sample pretreatment: Cut the large wood blocks to be quarantined into 2-5cm sawdust, remove surface contaminants, and spread them evenly in the sample area of ​​the large modified Bellman funnel. The thickness of the sawdust layer should be controlled at 2cm to ensure that the sawdust layer is loose, which facilitates the migration of nematodes and avoids clogging the collection channel.

[0063] System Setup and Environmental Control: A prepared organosilicon extract was added to the collection area of ​​a large modified Bellman funnel to create a hydrotropic environment, ensuring the sawdust samples were moist but not in direct contact with the collection liquid. A light gradient environment was set up, with the collection liquid area completely protected from light. The sample area used a bright blue cold LED light source, and the light source distance was adjusted to maintain a stable illuminance of 200 lux in the sample area. Real-time monitoring with a lux meter ensured no temperature rise in the illuminated area, meeting the requirements for a stable environment during quarantine work.

[0064] Oxygenation control: An aquarium air pump was used as the air supply device. A 0.22μm sterile filter was connected in series at the front end of the air supply tube, extending to the bottom of the collection liquid, with the end 1cm away from the bottom to ensure uniform aeration. The air pump parameters were controlled by a timer switch and a gas flow meter, with a flow rate of 12mL / min and intermittent oxygenation for 5 minutes on and 25 minutes off. The dissolved oxygen concentration in the collection liquid was monitored in real time using a portable dissolved oxygen meter, maintaining it at around 8mg / L to ensure the activity and migration efficiency of the nematodes in batches.

[0065] Isothermal settling and sample collection: The system was placed in a constant temperature environment of 25℃ and allowed to stand for 10 minutes using a large modified Bellman funnel method. After settling, the collected liquid was slowly collected and centrifuged in batches at low speed. The precipitates were then combined to obtain pine wood nematode samples. These samples can be directly used for microscopic examination, PCR testing, and other experiments required for quarantine, ensuring accurate and reliable quarantine results without interference from the extract.

[0066] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A highly efficient collection system based on the negative phototaxis, oxytaxis, and hydrotaxis of nematodes, characterized in that, Includes an improved Bellman funnel module, an improved shallow dish module, a comprehensive control module, and a mild additive preparation unit; Both the modified Bellman funnel module and the modified shallow dish module integrate a precision light gradient component and a simple oxygenation component, and use a mild penetrating wetting agent extract instead of conventional sterile water; the simple oxygenation component includes an air-filled capillary tube, a sterile syringe and / or a micro air pump, and a 0.22μm sterile filter; The end of the inflatable capillary can reach the bottom of the collection liquid; the bottom of the shallow dish of the modified Bellman funnel module, or the outer wall, is blackened; the outer wall of the collection area of ​​the modified Bellman funnel module, the outer wall of the funnel, and the outer wall of the collection tube are black, so as to achieve an illuminance of <5 lux in the collection liquid area and an illuminance of 50-200 lux in the sample area.

2. The data acquisition system according to claim 1, characterized in that, The mild penetrating and wetting aid is one or a combination of two of the following: Tween-20, organosilicon (Silwet L-77), and potassium cocoyl glycinate.

3. The data acquisition system according to claim 2, characterized in that, The concentration of Tween-20 used is 0.003%-0.005% (v / v), the concentration of the organosilicon used is 0.001%-0.002% (v / v), and the concentration of the potassium cocoyl glycinate used is 0.002%-0.004% (v / v); the compound additives are 50% of the concentration of each individual additive.

4. The data acquisition system according to claim 1, characterized in that, The simple oxygenation component draws 5-20 mL of sterile air with a sterile syringe, or intermittently injects air into the bottom of the collection liquid by setting parameters through a micro air pump, maintaining the dissolved oxygen concentration of the collection liquid at 5-8 mg / L. For larger nematode collection scenarios, such as the large (over 20 cm in diameter) nematode separation equipment used in customs timber quarantine and large wood block quarantine, small to medium-sized air supply equipment such as aquarium air pumps or small oxygen generators are required.

5. The data acquisition system according to claim 1, characterized in that, The end of the aerated capillary tube should be 0.5-1 cm from the bottom of the collected liquid, and the end of the aerated capillary tube should be smoothed.

6. The data acquisition system according to claim 1, characterized in that, The integrated control module includes a portable dissolved oxygen meter and an illuminometer, which are used to monitor the dissolved oxygen concentration and light gradient parameters of the collected liquid in real time to ensure that the parameters are accurate and controllable.

7. The data acquisition system according to claim 1, characterized in that, The mild adjuvant extract is prepared with sterile water as a base, and can be used immediately after preparation. It can be used directly as a nematode collection solution without subsequent washing and does not interfere with subsequent experiments such as microscopic examination, culture, and PCR.

8. A method for efficient nematode collection using the system described in any one of claims 1-7, characterized in that, The core steps include the following: S1: Prepare a mild adjuvant extract with the corresponding concentration according to the sample type. The adjuvant is one or a combination of two of the following: Tween-20, organosilicon, and potassium cocoyl glycinate. S2: Sample pretreatment: pine wood is cut into 0.5-5cm sawdust, soil is sieved through a 2mm sieve, root nodules are cleaned and cut open, and the sample thickness is 1-2cm. S3: Construct a modified Bellman funnel module or a modified shallow dish module, physically shield the collection liquid area from light, add a mild auxiliary extract, and create a hydrotropic environment where the sample area is moist, the collection liquid area is liquid-containing, and the sample does not come into direct contact with the collection liquid. S4: Construct a directional light gradient, ensure the illuminance in the collection liquid area is <5 lux and completely shield it from light, and use a bright blue cold LED light source at a relatively far distance in the sample area (to prevent the temperature in the illuminated area from rising), with an illuminance of 50-200 lux; S5: Insert the air-filled capillary tube to the bottom of the collection liquid, connect a 0.22μm sterile filter in series at the front end, and slowly inject 5-20mL of sterile air using a sterile syringe, with an injection time of ≥10 seconds. The oxygenation frequency is once every 6 hours; or control the micro air pump through a timer switch and a gas flow meter to intermittently introduce sterile air into the collection liquid to maintain the dissolved oxygen concentration of the collection liquid at 5-8mg / L. S6: Let stand at a constant temperature of 20-25℃, or for 3-10 minutes using the modified Bellman funnel method, or for 6-24 hours using the modified shallow dish method. S7: Collect the collected liquid, centrifuge at low speed, and take the precipitate to obtain the nematode sample, which can be used directly for subsequent experiments.

9. The data acquisition method according to claim 8, characterized in that, In step S1, dried resinous pine wood is treated with 0.001%-0.002% organosilicon extract, root-knot nematode nodules are treated with 0.003%-0.005% Tween-20 extract, soil samples are treated with 0.002%-0.004% potassium cocoyl glycinate extract, and mixed samples are treated with a combination of organosilicon + potassium cocoyl glycinate or Tween-20 + potassium cocoyl glycinate extract.

10. The data acquisition method according to claim 8, characterized in that, In step S5, the oxygenation parameters of the micro air pump are a flow rate of 5-12 mL / min, working for 5 minutes and stopping for 25 minutes; the method is applicable to the collection of plant parasitic nematodes such as pine wood nematode, root-knot nematode, and stem nematode, and is suitable for sample types such as dried resinous pine wood, soil, root-knot nematode nodules, and plant stem slices.

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