A method and system for determining the number of parasite eggs in pet feces

Abstract

This invention relates to the field of parasite egg quantity detection technology, specifically to a method and system for determining the number of parasite eggs in pet feces. The method involves pre-treating a fecal sample by mixing and shaking it with a compound releasing agent; collecting the egg-enriched solution through multi-stage filtration and backwashing; purifying the solution using a double-layer density gradient medium at specific speeds and times to form an egg-enriched layer; adding fluorescein diacetate and propidium iodide for active staining; collecting the stained egg layer at a flow rate of 0.5 ml / min; injecting the test suspension into a counting chamber; acquiring color images under a fluorescence microscope; and using a computer for color separation, target identification, deduplication of overlapping areas, and automatic counting to obtain the number of live and dead eggs and calculate the number of eggs per gram of feces. This method improves the egg recovery rate, simplifies the operation, enhances resistance to interference from impurities, and accurately distinguishes between live and dead eggs, making it suitable for the precise quantitative detection of parasite eggs in pet feces.

Description

Technical Field

[0001] This invention relates to the field of insect egg quantity detection technology, and in particular to a method and system for determining the number of insect eggs in pet feces. Background Technology

[0002] Intestinal parasite infections in pets (such as roundworms, hookworms, and whipworms) are a common public health problem. Accurately measuring the number of eggs in feces (EPG, eggs per gram of feces) is crucial for assessing the severity of infection, developing a deworming plan, and evaluating treatment effectiveness. Currently, the McMaster method is widely used in the industry. This method involves weighing a measured amount of feces, mixing it with a saturated saline solution or zinc sulfate solution, filtering it, and then injecting the filtrate into a specially designed McMaster counting chamber. The number of eggs in two grid areas of the chamber is observed and counted under a microscope, and finally, the EPG value is calculated using a formula.

[0003] However, existing technologies have the following shortcomings: First, traditional flotation solutions (such as saturated saline) have limited density, resulting in low flotation efficiency for heavier eggs (such as trematode and whipworm eggs), easily leading to missed detections and underestimating the count results. Second, the process requires manual stirring, filtering, and transfer, which is cumbersome and demands high operator skill, leading to significant differences in results among different operators. Third, impurities, fats, and air bubbles in fecal samples often resemble the morphology of eggs, interfering with microscopic identification and causing false positives or counting errors. Fourth, conventional methods cannot effectively distinguish between live and dead eggs, and only live eggs are infectious, which is crucial for developing precise treatment plans.

[0004] To address the problems of existing methods for quantitative detection of parasite eggs in pet feces, such as low egg recovery rate (especially for eggs with higher specific gravity), cumbersome operation steps and poor result repeatability, severe interference from impurities leading to low identification accuracy, and inability to distinguish the viability of eggs, this paper proposes a simple, high-recovery, interference-resistant method that can identify the viability of eggs. Summary of the Invention

[0005] The purpose of this invention is to provide a method and system for determining the number of parasite eggs in pet feces, which improves the egg recovery rate, simplifies the operation, enhances the ability to resist interference from impurities, and can accurately distinguish between live and dead eggs, making it suitable for the precise quantitative detection of parasite eggs in pet feces.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for determining the number of parasite eggs in pet feces, comprising the following steps: Pet feces samples were mixed with a compound release agent and shaken to obtain a homogenized suspension. The homogenized suspension is subjected to multi-stage filtration to remove impurities and collect the insect egg enrichment solution. The insect egg enrichment solution was centrifuged at 1800 rpm for 10 minutes to enrich the insect eggs at the medium interface, forming an insect egg enrichment layer. Add insect egg activity staining solution to the egg enrichment layer for staining, and then collect the egg layer at a flow rate of 0.5 ml per minute to obtain the test egg suspension. The suspension of the insect eggs to be tested was injected into a quantitative counting plate, and a color fluorescence image was acquired under a fluorescence microscope. Then, color separation, target identification, deduplication of overlapping areas, and automatic counting were performed to obtain the number of live insect eggs and the number of dead insect eggs. The number of live eggs and the total number of eggs per gram of feces are calculated based on the number of live eggs and the number of dead eggs.

[0007] The composite release agent consists of phosphate buffer, 0.5% by mass / volume of Tween-80 surfactant, and 200 units of pepsin per milliliter, and is preheated to 37 degrees Celsius before use; the oscillation treatment is performed by oscillating at 2500 revolutions per minute for 5 minutes using a vortex oscillator.

[0008] The multi-stage filtration system includes, from top to bottom, a coarse filter with a pore size of 500 micrometers, an impurity interception screen with a pore size of 300 micrometers, and an insect egg interception screen with a pore size of 40 micrometers. The specific method for collecting the insect egg enrichment solution is as follows: after gravity filtration is completed, a compound release agent is used to rinse the insect egg trap from the bottom to the top, and the rinsing solution is collected as the insect egg enrichment solution.

[0009] During the centrifugation of the insect egg enrichment solution at a speed of 1800 rpm, the pre-filled double-layer density gradient medium in the centrifuge tube consists of: a lower layer of 5 ml of sucrose-sodium iodide mixture with a density of 1.28 g / cm³, and an upper layer of 5 ml of sucrose solution with a density of 1.18 g / cm³; after centrifugation, the insect egg enrichment layer is located at the top of the upper medium and at the interface between the two mediums.

[0010] The staining solution for the live insect eggs is a phosphate buffer containing fluorescein diacetate at a final concentration of 10 micrograms per milliliter and propidium iodide at a final concentration of 5 micrograms per milliliter. The staining process is as follows: 2 milliliters of staining solution are added to the egg enrichment layer, mixed well, and incubated at room temperature in the dark for 5 minutes. Live eggs fluoresce green and dead eggs fluoresce red.

[0011] The egg layer was collected at a constant flow rate of 0.5 ml per minute, with a collection volume of 3 ml, to obtain the egg suspension to be tested.

[0012] The process involves injecting the suspension of the insect eggs to be tested into a quantitative counting plate, acquiring color fluorescence images under a fluorescence microscope, and then performing color separation, target identification, deduplication of overlapping areas, and automatic counting to obtain the number of live and dead insect eggs, including: 20 μL of the test insect egg suspension was injected into a quantitative counting chamber with a depth of 0.2 mm and an area of ​​100 square millimeters. Panoramic color fluorescence images were captured using a fluorescence microscope equipped with a color cooled digital camera under 488 nm blue excitation light. The acquired panoramic color fluorescence image was decomposed into green and red channels, and adaptive threshold segmentation and connected component analysis were performed on them respectively. Based on area and roundness, candidate regions for live eggs and candidate regions for dead eggs were selected.

[0013] The method further includes: When a panoramic color fluorescence image is stitched together from multiple adjacent fields of view, the overlapping areas of adjacent fields of view are identified, and candidate targets with a centroid distance less than a preset threshold are merged into the same insect egg, and duplicate records are deleted. Then, the number of targets retained in the green channel and the red channel are counted respectively as the number of live insect eggs and the number of dead insect eggs.

[0014] The calculation of the number of live worm eggs and the total number of worm eggs per gram of feces, based on the number of live worm eggs and the number of dead worm eggs, includes: The average number of live eggs measured by two independent counting chambers is taken as N_live, and the average number of dead eggs is taken as N_dead. The total volume of the egg layer is divided by the volume of the counting chamber to obtain the dilution factor, which is then divided by the weight of the fecal sample. The number of live eggs per gram of feces and the number of dead eggs per gram of feces are calculated separately. The two are added together to obtain the total number of eggs per gram of feces.

[0015] In a second aspect, the present invention provides a system for determining the number of parasite eggs in pet feces, applied to a method for determining the number of parasite eggs in pet feces as provided in the first aspect, comprising: The sample pretreatment module is used to mix pet feces samples with a compound release agent and shake them to obtain a homogenized suspension; the homogenized suspension is then subjected to multi-stage filtration to remove impurities and collect the egg-enriched solution; the egg-enriched solution is centrifuged at 1800 rpm for 10 minutes to enrich the eggs at the medium interface, forming an egg-enriched layer; an egg activity staining solution is added to the egg-enriched layer for staining, and then the egg layer is collected at a flow rate of 0.5 ml per minute to obtain the egg suspension to be tested; The egg count module is used to acquire a color fluorescence image of the egg suspension to be tested on a quantitative counting plate, and then perform color separation, target recognition, deduplication of overlapping areas and automatic counting to obtain the number of live eggs and the number of dead eggs; based on the number of live eggs and the number of dead eggs, the number of live eggs and the total number of eggs per gram of feces are calculated.

[0016] This invention discloses a method and system for determining the number of parasite eggs in pet feces. The method involves pre-treating the fecal sample by mixing and shaking it with a compound releasing agent; collecting the egg-enriched solution through multi-stage filtration and backwashing; purifying the solution using a double-layer density gradient medium at specific speeds and times to form an egg-enriched layer; adding fluorescein diacetate and propidium iodide for active staining; collecting the stained egg layer at a flow rate of 0.5 ml / min; injecting the test suspension into a counting chamber; acquiring color images under a fluorescence microscope; and using a computer for color separation, target identification, deduplication of overlapping areas, and automatic counting to obtain the number of live and dead eggs and calculate the number of eggs per gram of feces. This method improves the egg recovery rate, simplifies the operation, enhances resistance to interference from impurities, and accurately distinguishes between live and dead eggs, making it suitable for the precise quantitative detection of parasite eggs in pet feces. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0018] Figure 1 This is a schematic diagram illustrating the steps of a method for determining the number of parasite eggs in pet feces according to the first embodiment of the present invention.

[0019] Figure 2 This is a flowchart illustrating a method for determining the number of parasite eggs in pet feces provided by the present invention. Detailed Implementation

[0020] 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 numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application.

[0021] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0022] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0023] The first embodiment of this application is as follows: Please see Figures 1-2 This invention provides a method for determining the number of parasite eggs in pet feces, comprising the following steps: S1. Mix the pet feces sample with the compound release agent and shake to obtain a homogenized suspension.

[0024] Specifically, first, collect pet feces samples. Using a disposable feces collection scoop, select the central portion of fresh feces from the pet being tested, away from the ground or litter box, to avoid contamination by environmental pollutants. Collect at least 5 grams, place it in a sealed sample collection cup, and deliver it to the testing laboratory within 2 hours. If immediate testing is not possible, store the sample at 4 degrees Celsius for no more than 24 hours.

[0025] Before starting the formal processing, remove the sample from the refrigerated environment and allow it to warm to room temperature (approximately 20 to 25 degrees Celsius). Prepare a clean sample pretreatment tube, which is a transparent plastic tube with a conical bottom and a sealing screw cap. The side of the tube is marked with graduations for 10 ml, 15 ml, and 20 ml. Use the matching quantitative sampling spoon, which is calibrated so that one level spoonful of wet feces weighs exactly 2.0 grams (with an error of no more than ±0.05 grams). Use the sampling spoon to scoop out one level spoonful of the warmed fecal sample, gently scraping off any excess feces from the back of the spoon until the feces are level with the rim. Transfer this spoonful of fecal sample directly to the bottom of the sample pretreatment tube, taking care to avoid feces adhering to the inner wall of the tube opening.

[0026] Next, add the compound release agent to the sample pretreatment tube. The compound release agent is a pre-prepared working solution dispensed into reagent bottles, consisting of phosphate buffer (pH 7.4) as the solvent, 0.5% (w / v) of Tween-80 surfactant, and a final concentration of 200 units per milliliter of pepsin. Before use, preheat the compound release agent in a 37°C water bath for 15 minutes. Accurately measure 10 ml of the preheated compound release agent using a wide-mouth pipette and slowly inject it along the inner wall of the sample pretreatment tube, allowing the liquid to directly flush the fecal sample at the bottom of the tube. After injection, the fecal sample should be completely submerged in the compound release agent.

[0027] Tighten the screw caps on the sample pretreatment tubes to ensure a tight seal. Mount the pretreatment tubes onto the rack of a vortex mixer (also known as a vortex oscillator) and adjust the oscillator speed to 2500 rpm. Start the oscillator and continue oscillating for 5 minutes. During oscillation, the liquid inside the tubes experiences strong turbulence and shear force. Combined with the emulsifying and dispersing effects of Tween-80 on the lipid components of feces, and the decomposition of mucoproteins and fibrin in feces by pepsin, the fecal matrix rapidly disintegrates into a homogenized suspension. The originally clumps of feces are broken down into individual suspended particles, while the parasite eggs (including roundworm eggs, hookworm eggs, whipworm eggs, etc.) attached to the fecal residue detach from the residue due to the reduction of interfacial tension by surfactants and are uniformly dispersed in the liquid phase. After 5 minutes of oscillation, the liquid inside the tubes appears as a uniform light brown suspension without obvious clumps or stratification. At this point, the homogenization and pretreatment of the sample are complete.

[0028] Finally, remove the sample pretreatment tube containing the homogenized suspension from the vortex mixer and let it stand upright for about 15 seconds to allow the liquid adhering to the tube wall to flow back to the bottom of the tube. After confirming that there are no obvious undisintegrated large pieces of feces in the tube, the subsequent multi-stage filtration and impurity removal steps can be performed. Throughout the entire pretreatment process, the sample pretreatment tube must be kept sealed to prevent external impurities from entering or liquid from splashing.

[0029] S2. The homogenized suspension is subjected to multi-stage filtration to remove impurities and collect the insect egg enrichment solution.

[0030] Specifically, the homogenized suspension is filtered through a multi-stage filtration system. First, the suspension comes into contact with a first 500-micron coarse filter screen from top to bottom. This screen intercepts large impurities such as hair, grass clippings, and unbroken feed particles. The filtered liquid then drips into the second layer. The second layer is a 300-micron impurity interception screen, used to trap medium-sized plant fibers and grit. After two layers of filtration, the liquid still contains a large amount of fine residue and target insect eggs. Most of these fine residues are less than 100 microns in diameter. However, to ensure that no insect eggs are missed, a third layer uses an egg-retaining screen with a 40-micron pore size. Because common parasite eggs in pet feces (such as Toxocara canis eggs, which are approximately 75 micrometers in diameter, hookworm eggs, and whipworm eggs, which are approximately 55 micrometers in diameter) are all larger than 40 micrometers, all the eggs are trapped on the upper surface of the third filter. Most of the tiny impurities smaller than 40 micrometers (such as bacterial clumps, starch granules, and pigment particles) pass through the filter with the liquid and flow into the waste collection cup below. The entire filtration process relies on gravity to proceed naturally, without applying any external force to avoid damaging the eggs.

[0031] When all the liquid in the sample pretreatment tube has drained and no more droplets are dripping from the filter screen, filtration is complete. At this point, remove the waste collection cup and place a clean 15 ml centrifuge tube in its original position as the egg enrichment receiving tube. Then, detach the multi-stage filtration assembly from the sample pretreatment tube and invert it so that the egg trap (third layer) is on top. Using a syringe (without the needle) containing 10 ml of compound release agent, slowly push the compound release agent upwards from below the egg trap (i.e., the side from which the liquid originally flowed out), causing the liquid to flow backwards through the mesh of the egg trap. Under the impact of the reverse water flow, the eggs adhering to the surface of the filter screen are washed away and flow out from above the filter screen with the rinsing solution. Collect this rinsing solution directly into the 15 ml centrifuge tube below. To ensure complete collection, repeat the reverse rinsing twice, using 5 ml of compound release agent each time, collecting a total of approximately 15 ml of liquid. This liquid is an egg enrichment solution, which contains the vast majority of eggs separated from feces, and the total volume is controlled (15 ml) to facilitate subsequent density gradient centrifugation.

[0032] First, it should be noted that the composite release agent used in this invention is not a single commercially available product, but a specially formulated working solution according to the requirements of this method. Its specific components and functions are as follows: Basic solvent: phosphate buffer, concentration 0.01 mol / L, pH adjusted to 7.4. This buffer maintains the stability of the acid-base environment of the entire treatment system, preventing protein denaturation or loss of enzyme activity in the eggs due to pH fluctuations.

[0033] Surfactant: Tween-80 (chemical name: polysorbate 80), added at a concentration of 0.5% by mass-volume ratio. Tween-80 is a nonionic surfactant that can reduce the surface tension of liquids, effectively emulsify the lipid components in feces, disperse fat particles, and help worm eggs detach from the surface of fecal particles.

[0034] Biological enzyme: pepsin, with a final activity concentration of 200 units per milliliter. Pepsin can hydrolyze adhesive mucin and undigested fibrin in feces, breaking down the sticky matrix encapsulating worm eggs into soluble small molecules, thereby releasing the worm eggs.

[0035] The above components should be mixed thoroughly before use and preheated in a 37°C water bath for 15 minutes to ensure optimal enzyme activity. This compound release agent does not contain organic solvents or strong acids or bases and does not damage the morphology and activity of insect eggs.

[0036] During multi-stage filtration, the target insect eggs are trapped on the upper surface of the bottom-level egg-retaining mesh. If the traditional "forward rinsing" method is used, that is, rinsing from the top down from the retaining mesh, with the rinsing liquid flowing in the same direction as the liquid during filtration, two problems are likely to occur: First, the insect eggs may be further pressed into the mesh or embedded in the fiber gaps, making them difficult to wash away; second, some fine residues may re-cover the surface of the insect eggs, reducing the efficiency of subsequent purification.

[0037] This invention employs a "reverse collection of rinsing liquid" method. Specifically, the rinsing liquid is sprayed from below and above the egg-retaining net, i.e., against the original filtration direction. The advantages of this method are: Highly efficient stripping: The reverse water flow can "push" out the insect eggs embedded in the mesh without squeezing them.

[0038] Reduced losses: After being backflushed, the insect eggs are suspended in the rinsing solution and will not re-adhere to the mesh surface.

[0039] Volume controllable: Most of the insect eggs can be collected with a small amount of rinsing solution, resulting in a high concentration of insect egg enrichment solution.

[0040] Therefore, backwashing is one of the key technical means to ensure a high egg recovery rate.

[0041] S3. Centrifuge the egg enrichment solution at 1800 rpm for 10 minutes to enrich the eggs at the medium interface and form an egg enrichment layer.

[0042] Specifically, this invention employs a double-layer density gradient medium, which is pre-filled into dedicated density gradient centrifuge tubes before use. The density gradient centrifuge tubes are made of transparent polypropylene with a conical bottom, and are marked with 5 ml, 10 ml, and 15 ml graduations. The tube caps are leak-proof screw caps.

[0043] The specific preparation method for the bilayer density gradient medium is as follows (prepared in advance and provided as a component of the detection kit): Lower layer medium: Weigh sucrose and sodium iodide, dissolve them in distilled water and adjust the density to 1.28 grams per cubic centimeter, while adding 0.1% sodium azide as a preservative. The volume of this layer is 5 ml. The high-density (1.28) sucrose-sodium iodide mixture can effectively float heavier insect eggs (such as whipworm eggs and fluke eggs, with a specific gravity of about 1.20-1.25) and prevent most fecal residue (specific gravity usually greater than 1.30) from settling into the egg layer.

[0044] Upper medium: Weigh sucrose, dissolve it in distilled water, and adjust the density to 1.18 grams per cubic centimeter. Add 0.1% sodium azide. The volume of this medium is also 5 ml. A sucrose solution with a density of 1.18 is suitable for flotating insect eggs with a low specific gravity (such as roundworm eggs, with a specific gravity of approximately 1.10-1.15) and forms a buffer layer to prevent impurities in the sample from directly entering the lower layer.

[0045] In a centrifuge tube, slowly pour the lower layer medium (5 ml) along the tube wall, then carefully pour the upper layer medium (5 ml) on top. A clearly visible interface will form between the two layers due to their density difference. Avoid mixing the two layers during the process; a long needle or separator can be used. The prepared double-layer gradient centrifuge tubes can be stored at 4 degrees Celsius for up to one week.

[0046] Slowly add the approximately 15 ml of egg enrichment solution collected in step two along the inner wall of the density gradient centrifuge tube using a pipette, ensuring it covers the top layer of the two-layer medium. Avoid disturbing the liquid surface, maintaining the three-layer structure (top layer: egg enrichment solution; middle layer: sucrose solution with a density of 1.18; bottom layer: sucrose-sodium iodide mixture with a density of 1.28). After addition, the total volume in the centrifuge tube should be approximately 25 ml (15 ml enrichment solution + 5 ml upper layer medium + 5 ml lower layer medium). At this point, the liquid level in the centrifuge tube should be about 1 cm from the opening, leaving sufficient space to prevent overflow during centrifugation.

[0047] Density gradient centrifuge tubes containing samples are symmetrically placed in a horizontal rotor centrifuge. The characteristic of a horizontal rotor is that the basket can swing freely during centrifugation, ensuring that the centrifuge tubes remain parallel to the direction of centrifugal force, which is beneficial for forming a flat density gradient interface.

[0048] The selection of centrifugation speed is based on the following considerations: This method selects 1800 revolutions per minute (approximately 400 × g relative centrifugal force). If the rotation speed is too low (e.g., below 1000 rpm), the centrifugal force is insufficient, and the insect eggs cannot effectively migrate to the density gradient interface, resulting in incomplete separation of impurities and insect eggs. If the rotation speed is too high (e.g., exceeding 3000 rpm), the large centrifugal force will cause some of the heavier insect eggs (such as whipworm eggs) to pass through the density gradient layer and settle to the bottom of the tube, which may also cause the insect eggs to deform or break.

[0049] After preliminary experimental optimization, a rotation speed of 1800 rpm can ensure that the vast majority of insect eggs (specific gravity range 1.10-1.25) migrate to the medium interface with a density that matches their density under the action of centrifugal force, while preventing fine particles in the impurities (specific gravity usually greater than 1.30) from floating up and interfering with the egg layer.

[0050] The selection of centrifugation time is based on the following considerations: This method sets the centrifugation time at 10 minutes. If the time is too short (e.g., less than 5 minutes), the eggs have not yet completed the migration from the sample layer to the gradient interface, resulting in a low recovery rate. If the time is too long (e.g., more than 20 minutes), the eggs may be excessively piled up at the density interface and squeezed against each other. Furthermore, prolonged centrifugation may lead to a decrease in egg viability (although this method involves subsequent viability staining, physical damage to live eggs should still be minimized).

[0051] Ten minutes is a balance point, allowing the insect eggs to fully complete the flotation migration while maintaining their morphology and good activity. Under the conditions of 1800 rpm and 10 minutes, the recovery rate of insect eggs can reach over 95%, and the impurity removal rate exceeds 90%.

[0052] Centrifugation process: Tightly close the centrifuge lid, set the speed to 1800 rpm, and the time to 10 minutes. Set both acceleration and deceleration to medium (avoid sudden acceleration or deceleration to prevent disturbance of the gradient interface). Start the centrifuge and allow it to run smoothly. When the time is up, allow the centrifuge to decelerate and stop naturally. Do not use emergency braking to prevent liquid surface fluctuations from causing mixing of the gradient layer.

[0053] After centrifugation, carefully open the centrifuge lid, remove the centrifuge tubes, and place them vertically on the tube rack, avoiding shaking. At this point, four clear bands can be observed from the bottom of the tube to the liquid surface: Bottom layer (lowest layer): A small amount of dense, dark brown to black precipitate, mainly consisting of coarse, heavy impurities with a specific gravity exceeding 1.30, such as undigested mineral particles and metal fragments. This layer does not contain target insect eggs.

[0054] At the interface between the lower and upper media: located between the 5 ml and 10 ml scales, it appears as a thin, milky-white band. This band is rich in heavier insect eggs, such as whipworm eggs and fluke eggs, which cluster at the boundary between densities of 1.28 and 1.18, because their specific gravity falls precisely between the two layers.

[0055] Top layer of upper medium: Located between 10 ml and 15 ml, i.e., the top area of ​​the upper medium, a relatively wide milky white suspended layer is visible. This layer is rich in less dense insect eggs, such as roundworm eggs and hookworm eggs, which float on top of the medium with a density of 1.18.

[0056] The uppermost liquid (protozoan egg enrichment layer): has become almost clear or light yellow, in which most of the original fine impurities have settled or floated to the surface and been removed.

[0057] The second zone (at the interface) and the third zone (at the top of the upper medium) together constitute the egg enrichment layer, with a volume of approximately 2 to 3 ml. The eggs in these two zones have been separated from most of the minute impurities, resulting in significantly improved purity, and can be directly used for subsequent viable staining.

[0058] Before proceeding to the next step (step four, active staining), carefully aspirate the top layer of clear liquid (approximately 15 ml) using a long-needle syringe or micropipette, taking care not to disturb the underlying egg layer. Leave approximately 10 ml of liquid (including the egg layer at the top and interface of the upper medium). At this point, density gradient centrifugation purification is complete, and the centrifuge tube contains only the medium system with high-purity eggs, ready for the active staining step.

[0059] S4. Add insect egg activity staining solution to the egg enrichment layer for staining, and then collect the egg layer at a flow rate of 0.5 ml per minute to obtain the egg suspension to be tested.

[0060] Specifically, after completing the third step of density gradient centrifugation purification, a clear egg-rich layer formed in the centrifuge tube. This layer was located at the top of the upper medium and at the interface between the upper and lower medium layers, with a total volume of approximately 2 to 3 ml. Although the eggs at this stage had been separated from most impurities, they still contained a small amount of density gradient medium components (sucrose solution) and had not yet been activity-labeled. To ensure accurate identification and differentiation of live and dead eggs during subsequent counting, activity staining was necessary. Furthermore, to remove the egg-rich layer from the centrifuge tube without damage for counting, a controlled collection technique was required, with flow rate control being crucial.

[0061] This invention employs a dual fluorescence staining method, utilizing the differences in metabolic activity and cell membrane integrity between live and dead eggs to distinguish between them.

[0062] The staining solution for the insect eggs is a freshly prepared working solution, with phosphate buffer as the solvent and a pH adjusted to 7.4. The staining solution contains two fluorescent dyes: Fibroside diacetate (FDA): The final concentration is 10 micrograms per milliliter. FDA itself is non-fluorescent, but it can freely pass through intact cell membranes into the interior of live insect eggs. Under the action of esterases in the live insect eggs, FDA is hydrolyzed into fluorescein, which has a strong green fluorescence. Because fluorescein carries a charge, it can no longer pass through intact cell membranes and is therefore retained inside the live insect eggs, causing the live insect eggs to emit green fluorescence under blue excitation light (wavelength approximately 488 nanometers).

[0063] Propidium iodide (PI): The final concentration is 5 micrograms per milliliter. PI is a red fluorescent nucleic acid dye that cannot penetrate intact cell membranes. PI can only enter the egg when the cell membrane is damaged (i.e., a dead or dying egg), where it binds to DNA and emits red fluorescence (excitation wavelength approximately 535 nanometers, emission wavelength approximately 617 nanometers).

[0064] When the two dyes are used in combination, live eggs fluoresce only in green, while dead eggs fluoresce only in red. Eggs with damaged cell membranes but still containing residual esterase activity may emit both green and red light (appearing yellow or orange), but in practice, they are still classified as dead or low-viability eggs. This dual-staining method can clearly distinguish between live and dead eggs in the same field of view and is not affected by the fluorescence of fecal residues.

[0065] After the third step of centrifugation is completed, do not disturb the layered structure inside the centrifuge tube. First, use a long-handled pipette or a vacuum aspirator connected to a tubing to carefully remove the top layer of clear liquid above the egg-rich layer (i.e., the clarified part of the protozoan egg-rich liquid layer), leaving the liquid level about 1 cm above the egg-rich layer to prevent the eggs from being aspirated.

[0066] Next, take the pre-prepared and light-protected staining solution for the eggs and use a micropipette to draw 2 ml of the staining solution. Gently insert the tip of the pipette into the centrifuge tube below the liquid surface, close to the area above the egg-rich layer, and slowly inject the staining solution, avoiding direct impact on the egg layer. After injection, the total liquid volume in the centrifuge tube should be approximately 4 to 5 ml (the original 2 to 3 ml of egg-rich solution plus 2 ml of staining solution).

[0067] Next, using a thin, sterile glass rod or a special stirring needle, gently insert it into the bottom of the centrifuge tube and slowly rotate it at a speed of about 10 revolutions per minute, lifting it up and down twice to thoroughly mix the staining solution with the egg-rich layer. Be careful to be gentle to avoid creating air bubbles or damaging the egg structure. Then, cap the centrifuge tube and place it horizontally on a shaker. Incubate at 30 revolutions per minute at room temperature in the dark for 5 minutes. Shaking incubation ensures that the dye evenly contacts the eggs, guaranteeing thorough and consistent staining.

[0068] Five minutes later, remove the centrifuge tube. At this point, green fluorescein has accumulated inside the live eggs, and the nuclei of dead eggs are stained red. The stained sample should be immediately used to collect the egg layer to avoid prolonged storage, which could lead to fluorescence quenching or changes in the viability of live eggs.

[0069] After staining, the egg-rich layer (i.e., the liquid layer containing stained eggs) located in the middle of the centrifuge tube needs to be collected as the egg suspension to be tested for subsequent counting. Because the egg-rich layer is sandwiched between two liquid layers and has a small volume (approximately 2 to 3 ml), direct pouring or rough aspiration can easily lead to contamination by upper impurities or omission of lower eggs. Therefore, this invention uses a micro-injection pump with a long needle for precise collection and strictly controls the collection flow rate.

[0070] Take a sterile 5 ml syringe and attach a 15 cm long, 0.7 mm (21 G) flat-tipped needle. Secure the syringe to the advance seat of the microinfusion pump and adjust the pump parameters to "draw mode" (i.e., the pump pulls back the syringe piston, drawing liquid into the syringe). Attach a 2 cm long piece of soft silicone tubing to the needle tip as a cushioning protection to prevent the needle tip from damaging the eggs.

[0071] The present invention sets the collection flow rate at 0.5 ml per minute, and this value is determined based on the following considerations: Preventing egg breakage: The shells of insect eggs (especially live eggs) have some elasticity but limited shear resistance. If the flow rate is too high (e.g., exceeding 2 ml per minute), the liquid will generate high shear force and turbulence at the needle tip inlet, which can easily cause the egg shells to rupture or the contents to leak out, affecting the accuracy of subsequent counting. A flow rate of 0.5 ml per minute is in a laminar flow state, and the mechanical stress of the fluid on the insect eggs is minimal.

[0072] To avoid mixing between layers: the liquids above and below the egg-rich layer have different densities and compositions (the upper layer is a sucrose solution with a density of 1.18, and the lower layer is a sucrose-sodium iodide mixture with a density of 1.28). If the collection speed is too fast, eddies will form, drawing in either the clear liquid above or the precipitate below, resulting in excessive impurities or media mixed into the suspension to be tested, interfering with the counting. Slow collection allows the needle tip to remain in the middle of the egg layer, utilizing the viscosity of the liquid itself for stable aspiration.

[0073] To ensure complete collection: The total volume of the egg-rich layer is approximately 2 to 3 ml. Collecting at a rate of 0.5 ml per minute takes 4 to 6 minutes to complete. While this time is slightly longer, it ensures that the eggs are fully and completely absorbed, and allows the operator to observe the needle tip position in real time and make adjustments as needed. Collecting too quickly may be completed in a few seconds, but this easily leads to the loss of eggs attached to the tube wall or interface.

[0074] Set the injection pump speed to 0.5 ml per minute and the total target collection volume to 3 ml (slightly larger than the estimated volume of the egg layer). Start the injection pump and slowly retract the syringe plunger to create negative pressure at the needle tip.

[0075] The operator holds the centrifuge tube at approximately a 30-degree angle and slowly inserts the tip of a long needle through the centrifuge tube opening downwards until the needle tip is precisely in the center of the egg-rich layer (visible as a milky white band to the naked eye). Under microscope assistance or good lighting, the milky white egg solution can be seen gradually drawn into the syringe. During collection, the operator needs to slowly pull the needle upwards according to the drop in liquid level, keeping the needle tip in the center of the egg-rich layer. When the liquid collected in the syringe reaches 3 ml (i.e., the set volume of the micro-injection pump is reached), the extraction automatically stops. At this point, the syringe contains 3 ml of milky white suspension of the tested eggs, which contains stained live and dead eggs and is essentially free of lower medium or upper impurities.

[0076] Remove the syringe from the microinjection pump, carefully remove the long needle, and replace it with a regular disposable needle (or use a needleless syringe tip). Gently push the suspension of eggs to be tested into a clean 1.5 ml centrifuge tube (3 ml can be divided into two portions, or a larger volume tube can be used). Gently invert twice to mix, ensuring the eggs are evenly distributed in the suspension, and then immediately proceed to step five for quantitative counting. If a short storage period (no more than 30 minutes) is required before counting, the centrifuge tube should be stored in an ice bath away from light to prevent fluorescence quenching and alteration of egg activity.

[0077] Through the above-described meticulous staining and collection operations, this invention successfully obtained an egg suspension that was both labeled with its active state and maintained high purity and intact morphology, laying a solid foundation for subsequent accurate counting.

[0078] S5. Inject the suspension of the insect eggs to be tested into a quantitative counting plate, place it under a fluorescence microscope to collect a color fluorescence image, and then perform color separation, target identification, deduplication of overlapping areas and automatic counting to obtain the number of live insect eggs and the number of dead insect eggs.

[0079] Specifically, after completing the fourth step of viable staining and egg layer collection, 3 ml of egg suspension was obtained. The live eggs in this suspension accumulated green luciferin, emitting green fluorescence under specific wavelength light excitation; the nuclei of dead eggs were stained with propidium iodide, emitting red fluorescence under the same excitation light. To accurately and efficiently count the number of live and dead eggs, this invention employs fluorescence microscopy combined with a computer-automated identification and counting system, replacing manual visual counting.

[0080] The quantitative counting device used in this step consists of the following components: Quantitative counting plate: Consistent with the aforementioned scheme, it is a detachable dedicated counting plate. A counting chamber with a fixed depth of 0.2 mm is located in the center of the counting plate. The bottom of the counting chamber is etched with a 1 mm × 1 mm standard grid, arranged in 10 rows × 10 columns, for a total counting area of ​​100 square millimeters. Sample inlets and vents are located at both ends of the counting chamber to facilitate uniform filling of the liquid.

[0081] Fluorescence microscope: Equipped with a 10x objective lens and a blue excitation system (center wavelength 488 nm). This microscope emits uniformly intense excitation light, causing the insect eggs in the counting chamber to fluoresce. The microscope is also equipped with a trinocular observation head, one of which connects to a color cooled digital camera (image acquisition device).

[0082] Color cooled digital camera: With a resolution of at least 5 megapixels, it features high sensitivity and low noise, capable of capturing fluorescence signals in the green spectrum (center wavelength approximately 520 nm) and red spectrum (center wavelength approximately 617 nm) respectively. The camera connects to a computer via a data cable.

[0083] Computer: Equipped with a fluorescence image acquisition card and automatic counting and analysis software. This software includes built-in color segmentation algorithms, particle counting algorithms, and a data management module.

[0084] Before counting, wipe the quantitative counting plate clean with anhydrous ethanol and allow it to air dry. Place the counting plate horizontally on the stage of the fluorescence microscope and secure it with the slide clips. Adjust the microscope focus to make the grid lines at the bottom of the counting plate clearly visible. Start the camera and computer software, set the exposure time to 100 milliseconds, and the gain to medium, ensuring that the image brightness is moderate and there is no overexposure.

[0085] Use a micropipette to draw 20 μL of the suspension of insect eggs to be tested (before use, gently invert the container several times to ensure even distribution of the eggs), and inject it evenly through the sample dispensing port of the quantitative counting plate. Due to capillary action, the liquid will automatically fill the entire counting chamber, and excess liquid will flow out through the vent. Let the counting plate stand for 2 minutes to allow the eggs to settle naturally onto the grid plane at the bottom of the counting chamber, facilitating subsequent focusing and imaging.

[0086] Start the computer's automatic counting software and execute the following image acquisition process: The software sends a command to control the excitation source of the fluorescence microscope to turn on, and the blue excitation light (488 nm) illuminates the counting chamber.

[0087] Under software control, a color-cooled digital camera automatically captures a full-field color fluorescence image. This image contains fluorescence information of all insect eggs in the counting chamber, with live eggs appearing as green dots or ellipses, and dead eggs appearing as red dots or ellipses. The background (bottom of the counting chamber and the liquid) is dark or has weak fluorescence.

[0088] To ensure image quality, the software automatically performs white balance adjustment and background subtraction to remove brightness unevenness caused by the optical system.

[0089] Since the total area of ​​the counting chamber is 100 square millimeters, a single field of view cannot cover the entire chamber under a 10x objective lens. Therefore, the software drives the automatic stage (if the microscope is equipped with one) or prompts the operator to manually move the stage to acquire images of 9 to 12 adjacent fields of view sequentially in a zigzag pattern, with a 10% overlap between each field of view. All the acquired images are automatically stitched together to form a complete panoramic color fluorescence image of the counting chamber.

[0090] After a panoramic color fluorescence image of the counting chamber is acquired by a color-cooled digital camera, the image is transmitted to automatic counting and analysis software on a computer. The software sequentially performs color separation, candidate egg identification, duplicate removal of overlapping areas, and quantity counting according to a preset algorithm. Each step is described in detail below.

[0091] A panoramic color fluorescence image is essentially a two-dimensional matrix composed of pixels, with each pixel containing brightness values ​​for three color channels: red, green, and blue. Since live eggs emit green fluorescence and dead eggs emit red fluorescence, while the background (the dark field at the bottom of the counting chamber and the unstained liquid) has very low brightness in all channels, the two fluorescence signals can be decoupled through color channel separation. The specific operation is as follows: The software first converts the panoramic image from its display format to a three-channel array in memory. Then, it extracts the green channel array and the red channel array separately, forming two independent grayscale images. Green channel grayscale image: In this image, the areas that originally fluoresce green (where live eggs are located) show a higher grayscale value (brighter), while the areas that fluoresce red (where dead eggs are located) have a lower grayscale value in this channel, and the background is close to black.

[0092] Red channel grayscale image: In this image, the area that originally emitted red fluorescence (the location of dead insect eggs) shows a higher grayscale value, while the location of live insect eggs shows a lower grayscale value in this channel.

[0093] To further eliminate weak crosstalk between the two channels (e.g., the green fluorescence of live eggs might be slightly recorded by the red channel), the software applies a linear transformation to correct each channel image: using pre-acquired pure green fluorescence and pure red fluorescence standards, the crosstalk coefficient is calculated, and the contribution from the other channel is subtracted from the target channel. This results in two pure monochromatic fluorescence images: a green fluorescence image (primarily showing live eggs) and a red fluorescence image (primarily showing dead eggs).

[0094] The purpose of target recognition is to distinguish real insect eggs from background noise and fluorescent impurities from the two monochrome fluorescence images mentioned above, and to determine the location and boundary of each insect egg.

[0095] (1) The software first preprocesses the green fluorescence image and the red fluorescence image respectively, including: Median filtering: Using a 3×3 pixel filter window, each pixel in the image is traversed, and the original pixel value is replaced with the median gray value of the neighboring pixels. This operation can effectively remove isolated salt-and-pepper noise (such as tiny fluorescent dust particles) while preserving the clarity of the egg edges.

[0096] Background homogenization: Since the lighting source may have unevenness with bright centers and dark edges, the software uses an estimated background image (obtained by applying a large-kernel Gaussian blur to the entire image) to subtract it pixel by pixel from the original image, so that the gray values ​​of the entire background tend to be uniform.

[0097] (2) After preprocessing, the software uses an adaptive thresholding algorithm for each image to divide the pixels in the image into "foreground" (candidate regions for insect eggs) and "background". Unlike the fixed threshold, the adaptive threshold dynamically calculates the segmentation threshold of each pixel based on the mean and standard deviation of the gray values ​​of its local neighborhood. Specifically, for each pixel in the image, the software takes a neighborhood window of a fixed size (e.g., 31×31 pixels) around the pixel, calculates the mean and standard deviation of the gray values ​​of all pixels within the window, and then sets the threshold to "mean value minus an empirical constant multiplied by the standard deviation". Pixels with gray values ​​higher than this threshold are marked as foreground, otherwise as background.

[0098] This method can adapt to subtle changes in brightness in different areas of the image, avoiding the omission of some insect eggs or misjudgment of the background due to uneven lighting.

[0099] (3) After thresholding, the foreground pixels in the image form several independent white connected regions (each region corresponds to a potential insect egg). The software uses an eight-neighbor connectivity algorithm (i.e., each pixel is considered connected to its eight neighboring pixels in the upper, lower, left, right, and four diagonal directions) to scan the entire image, assigning a unique number to each connected region and recording the set of pixels contained in that region. Simultaneously, the software calculates the following characteristics of each connected region: Area: The total number of pixels within the area.

[0100] Perimeter: The pixel length of the region boundary.

[0101] Roundness: Calculated from area and perimeter (no formula required; can be described as "the degree to which the shape of the area is close to a circle").

[0102] Centroid coordinates: the average row and column coordinates of all pixels in the region.

[0103] (4) Not all connected regions are eggs. Some tiny fluorescent impurities (such as dye crystals) or non-specifically stained particles can also form connected regions. The software filters each candidate region based on pre-defined morphological parameters of the eggs: First, the software removes areas that are too small (less than 80% of the minimum projected area of ​​a typical insect egg) or too large (more than 120% of the maximum projected area of ​​a typical insect egg), which usually correspond to impurities or two eggs stuck together.

[0104] Secondly, the software evaluates the roundness of the area: insect eggs are usually round or oval after natural settling, with a high roundness value; while slender impurity particles (such as fiber fragments) have a low roundness value. The software retains areas with roundness within a preset range (roughly corresponding to an aspect ratio of no more than 1.5).

[0105] Finally, for candidate regions in the green fluorescence image, the software also checks their internal grayscale uniformity: live eggs are uniformly stained because they are filled with fluorescein; while false positive regions caused by autofluorescence often appear as irregular spots. The software eliminates regions with excessively high coefficients of variation by calculating the coefficient of variation (the ratio of standard deviation to mean) of pixel grayscale values ​​within the region.

[0106] After the above screening, the connected regions retained in the green fluorescent image were officially labeled as "live egg targets," and the connected regions in the red fluorescent image were labeled as "dead egg targets." The centroid coordinates and pixel area of ​​each target were recorded.

[0107] Since panoramic images are stitched together from multiple adjacent fields of view, there is approximately a 10% overlap between them. During the stitching process, the same insect egg located within the overlapping area may be captured by two separate fields of view, resulting in duplicates in the merged panoramic image, appearing as two very close targets. To avoid duplicate counting, the software performs overlapping area deduplication.

[0108] (1) Determine the overlap boundary. During the image acquisition stage, the software records the stage coordinates of each field of view. During panoramic stitching, the software knows the overlap pixel width between adjacent fields of view. The software generates a global coordinate system, maps the image of each field of view to this coordinate system, and marks the pixel range of all areas belonging to the overlap region.

[0109] (2) Merge neighboring targets. The software traverses all identified live insect egg targets (dead insect eggs are treated in the same way) and checks whether the centroid distance between any two targets is less than a threshold (this threshold is set to 1.2 times the diameter of a typical insect egg, for example, the number of pixels corresponding to 100 micrometers). If the centroid distance between two targets is less than this threshold, and they are located in the overlapping areas of two different fields of view, then the two targets are considered to correspond to the same insect egg.

[0110] For multiple targets identified as the same insect egg, the software performs a merging operation: retaining one target (e.g., the one with the largest area), deleting the remaining duplicate targets, and updating the centroid of the final target to the average of the centroids of all deleted targets, so as to more accurately reflect the actual location of the insect egg.

[0111] (3) Edge processing: For insect eggs located in non-overlapping areas, the software does not perform any merging operation. In addition, the software also checks those insect eggs whose center of gravity is close to the edge of the panoramic image to ensure that they are not partially truncated (if part of the insect egg is outside the image boundary, its area will be significantly smaller than the normal value. Such targets have been eliminated in the morphology screening stage and therefore will not be included in the count).

[0112] After removing duplicates from overlapping areas, each insect egg corresponds to only one target record in the panoramic image.

[0113] After deduplication of overlapping areas, the software counts the total number of live insect eggs retained in the green fluorescent image and the total number of dead insect eggs retained in the red fluorescent image. These two integers represent the total number of live insect eggs (N_live_primitive) and the total number of dead insect eggs (N_dead_primitive) detected in the entire counting chamber.

[0114] Since two independent counting chambers are needed for each sample, the software repeats the entire process described above to process the image of the second counting chamber, obtaining the second set of data. The software automatically calculates the average of the two counting chambers: Nlive = (Nlive original from the first chamber + Nlive original from the second chamber) / 2, Ndead = (Ndead original from the first chamber + Ndead original from the second chamber) / 2. The calculation results are rounded to one decimal place (rounded to the integer in actual EPG calculations) or stored directly as floating-point numbers.

[0115] Finally, the software displays the following information on the computer screen: First counting board: number of live eggs, number of dead eggs; Second counting board: number of live eggs, number of dead eggs; Average: Number of live eggs, number of dead eggs.

[0116] If the relative deviation between the two counts (the ratio of the difference between the two live egg counts to the mean) exceeds 10%, the software will output a warning message: "The count deviation is too large. It is recommended to repeat the experiment."

[0117] The software also saves the above results in a designated log file, along with a panoramic color fluorescence image file and a pseudo-color overlay marker image (with green circles outlining identified live eggs and red circles outlining dead eggs), for subsequent manual review or as the original record.

[0118] At this point, the data processing and automatic counting process is complete, and the obtained Nlive and Nl are automatically transferred to the result calculation module in step six (or manually entered by the operator). The core calculation of the entire process involves image processing, morphological analysis, and statistical judgment, falling under the category of G06F (electro-digital data processing), and achieving efficient and accurate identification and counting of the fluorescent color of insect eggs.

[0119] S6. Calculate the number of live eggs and the total number of eggs per gram of feces based on the number of live eggs and the number of dead eggs.

[0120] Specifically, the number of live eggs (EPG live) and the total number of eggs (EPG total) per gram of feces are calculated using the following formulas: Where Vtotal is the total collection volume of the egg enrichment layer in the third step (3 ml), Vcalculated is the total volume of the counting chamber (the area of ​​the counting chamber is 100 square millimeters multiplied by the depth of 0.2 millimeters, which equals 20 cubic millimeters, or 0.02 ml), and W is the weight of the fecal sample taken in the first step (2 grams). Therefore: Live eggs (EPG live) = (N live × V total / V total) / W = (N live × 3 ml / 0.02 ml) / 2 g = N live × 75.

[0121] Number of dead eggs (EPG dead) = (N dead × V total / V total) / W = N dead × 75.

[0122] Total number of eggs (total EPG) = live EPG + dead EPG.

[0123] The final report shows the number of live and dead roundworm eggs per gram of feces, for example, "1200 EPG of live roundworm eggs and 300 EPG of dead roundworm eggs were detected." This method, through standardized pretreatment, efficient purification, viability staining, and accurate counting, completely solves the technical problems of missed detection of roundworm eggs, operational errors, interference from impurities, and inability to distinguish viability in existing technologies.

[0124] The second embodiment of this application is as follows: This invention provides a system for determining the number of parasite eggs in pet feces, applied to a method for determining the number of parasite eggs in pet feces as provided in the first embodiment, comprising: The sample pretreatment module is used to mix pet feces samples with a compound release agent and shake them to obtain a homogenized suspension; the homogenized suspension is then subjected to multi-stage filtration to remove impurities and collect the egg-enriched solution; the egg-enriched solution is centrifuged at 1800 rpm for 10 minutes to enrich the eggs at the medium interface, forming an egg-enriched layer; an egg activity staining solution is added to the egg-enriched layer for staining, and then the egg layer is collected at a flow rate of 0.5 ml per minute to obtain the egg suspension to be tested; The egg count module is used to acquire a color fluorescence image of the egg suspension to be tested on a quantitative counting plate, and then perform color separation, target recognition, deduplication of overlapping areas and automatic counting to obtain the number of live eggs and the number of dead eggs; based on the number of live eggs and the number of dead eggs, the number of live eggs and the total number of eggs per gram of feces are calculated.

[0125] Regarding the system in the above embodiments, the specific ways in which each module performs operations have been described in detail in the embodiments related to the method, and will not be elaborated here.

[0126] For the system embodiments, since they basically correspond to the method embodiments, the relevant parts can be referred to in the description of the method embodiments. The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this application according to actual needs. Those skilled in the art can understand and implement this without creative effort.

[0127] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein.

[0128] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A method for determining the number of parasite eggs in pet feces, characterized in that, Includes the following steps: Pet feces samples were mixed with a compound release agent and shaken to obtain a homogenized suspension. The homogenized suspension is subjected to multi-stage filtration to remove impurities and collect the insect egg enrichment solution. The insect egg enrichment solution was centrifuged at 1800 rpm for 10 minutes to enrich the insect eggs at the medium interface, forming an insect egg enrichment layer. Add insect egg activity staining solution to the egg enrichment layer for staining, and then collect the egg layer at a flow rate of 0.5 ml per minute to obtain the test egg suspension. The suspension of the insect eggs to be tested was injected into a quantitative counting plate, and a color fluorescence image was acquired under a fluorescence microscope. Then, color separation, target identification, deduplication of overlapping areas, and automatic counting were performed to obtain the number of live insect eggs and the number of dead insect eggs. The number of live eggs and the total number of eggs per gram of feces are calculated based on the number of live eggs and the number of dead eggs.

2. The method for determining the number of parasite eggs in pet feces as described in claim 1, characterized in that, The compound release agent consists of phosphate buffer, 0.5% by weight / volume of Tween-80 surfactant, and pepsin at a final concentration of 200 units per milliliter, and is preheated to 37 degrees Celsius before use. The oscillation process uses a vortex oscillator to oscillate at a speed of 2500 revolutions per minute for 5 minutes.

3. The method for determining the number of parasite eggs in pet feces as described in claim 1, characterized in that, The multi-stage filtration system, from top to bottom, includes a coarse filter with a pore size of 500 micrometers, a 300-micrometer impurity interception screen, and a 40-micrometer insect egg trapping screen. The specific method for collecting the insect egg enrichment solution is as follows: after gravity filtration is completed, a compound release agent is used to rinse the insect egg trap from the bottom to the top, and the rinsing solution is collected as the insect egg enrichment solution.

4. The method for determining the number of parasite eggs in pet feces as described in claim 1, characterized in that, During the centrifugation of the insect egg enrichment solution at a speed of 1800 rpm, the pre-filled double-layer density gradient medium in the centrifuge tube is as follows: the lower layer is 5 ml of sucrose-sodium iodide mixture with a density of 1.28 g / cm³, and the upper layer is 5 ml of sucrose solution with a density of 1.18 g / cm³. After centrifugation, the insect egg enrichment layer is located at the top of the upper medium and at the interface between the two mediums.

5. The method for determining the number of parasite eggs in pet feces as described in claim 1, characterized in that, The staining solution for the live eggs is a phosphate buffer containing fluorescein diacetate at a final concentration of 10 μg / mL and propidium iodide at a final concentration of 5 μg / mL. The staining process is as follows: 2 mL of staining solution is added to the egg enrichment layer, mixed well, and incubated at room temperature in the dark for 5 minutes. Live eggs fluoresce green, and dead eggs fluoresce red.

6. The method for determining the number of parasite eggs in pet feces as described in claim 1, characterized in that, The egg layer was collected at a constant flow rate of 0.5 ml per minute, with a collection volume of 3 ml, to obtain the egg suspension to be tested.

7. The method for determining the number of parasite eggs in pet feces as described in claim 1, characterized in that, The suspension of the test eggs was injected into a quantitative counting plate, and a color fluorescence image was acquired under a fluorescence microscope. Then, color separation, target identification, deduplication of overlapping areas, and automatic counting were performed to obtain the number of live and dead eggs, including: 20 μL of the test insect egg suspension was injected into a quantitative counting chamber with a depth of 0.2 mm and an area of ​​100 square millimeters. Panoramic color fluorescence images were captured using a fluorescence microscope equipped with a color cooled digital camera under 488 nm blue excitation light. The acquired panoramic color fluorescence image was decomposed into green and red channels, and adaptive threshold segmentation and connected component analysis were performed on them respectively. Based on area and roundness, candidate regions for live eggs and candidate regions for dead eggs were selected.

8. The method for determining the number of parasite eggs in pet feces as described in claim 7, characterized in that, The method further includes: When a panoramic color fluorescence image is stitched together from multiple adjacent fields of view, the overlapping areas of adjacent fields of view are identified, and candidate targets with a centroid distance less than a preset threshold are merged into the same insect egg, and duplicate records are deleted. Then, the number of targets retained in the green channel and the red channel are counted respectively as the number of live insect eggs and the number of dead insect eggs.

9. The method for determining the number of parasite eggs in pet feces as described in claim 1, characterized in that, The number of live worm eggs and the total number of worm eggs per gram of feces are calculated based on the number of live worm eggs and the number of dead worm eggs, including: The average number of live eggs measured by two independent counting chambers is taken as N_live, and the average number of dead eggs is taken as N_dead. The total volume of the egg layer is divided by the volume of the counting chamber to obtain the dilution factor, which is then divided by the weight of the fecal sample. The number of live eggs per gram of feces and the number of dead eggs per gram of feces are calculated separately. The two are added together to obtain the total number of eggs per gram of feces.

10. A system for determining the number of parasite eggs in pet feces, applied to the method for determining the number of parasite eggs in pet feces as described in claim 1, characterized in that, include: The sample pretreatment module is used to mix pet feces samples with a compound release agent and shake them to obtain a homogenized suspension. The homogenized suspension is subjected to multi-stage filtration to remove impurities and collect the insect egg enrichment solution. The insect egg enrichment solution was centrifuged at 1800 rpm for 10 minutes to enrich the insect eggs at the medium interface, forming an insect egg enrichment layer; insect egg activity staining solution was added to the insect egg enrichment layer for staining, and then the insect egg layer was collected at a flow rate of 0.5 ml per minute to obtain the insect egg suspension to be tested. The egg count module is used to acquire a color fluorescence image of the egg suspension to be tested on a quantitative counting plate, and then perform color separation, target recognition, deduplication of overlapping areas and automatic counting to obtain the number of live eggs and the number of dead eggs; based on the number of live eggs and the number of dead eggs, the number of live eggs and the total number of eggs per gram of feces are calculated.

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