Pregnancy detection kit and method for rapidly and quantitatively detecting relaxin in dogs and cats

Abstract

The invention relates to the technical field of dog and cat breeding health detection, in particular to a pregnancy detection kit and method for rapidly and quantitatively detecting dog and cat relaxin, and a sample processing unit is used for completing collection and pretreatment of dog and cat samples; a sample detection area, a quality control area, a water absorption area and a sample adding area are sequentially arranged on the detection card body; a standard curve reference module is arranged in the quantitative reference unit, and the standard curve reference module is a chip or a paper reference card used for storing standard curve data; the signal processing and output unit comprises a signal reading module, a result storage module and a transmission module, and the result storage and transmission module is used for recording detection data; the environment control unit comprises a temperature control module, the specific reaction efficiency and the signal stability are ensured, and the technical problems that in an existing dog and cat pregnancy detection technology, detection is slow, quantification is difficult, samples are limited, the environment is sensitive, and data are difficult to manage are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of canine and feline reproductive health detection, in particular to a pregnancy detection kit and method for rapid quantitative detection of canine and feline relaxin. BACKGROUND

[0002] As common companion animals and economically bred animals, early and accurate determination of the pregnancy state of dogs and cats is of great significance for feeding management, breeding plan development and pregnancy health monitoring. At present, the existing detection methods for canine and feline pregnancy mainly have the following shortcomings:

[0003] Poor timeliness: traditional palpation method can only preliminarily determine the pregnancy of dogs and cats after 4 weeks of pregnancy, and is easily affected by the experience of the operator and the body size of the dog and cat, with an accuracy rate of less than 60%; although ultrasonic detection can be performed about 3 weeks before pregnancy, it requires professional equipment (such as a veterinary B-ultrasound instrument) and technical personnel for operation, has high cost for single detection, and cannot realize quantitative analysis.

[0004] Obvious limitations of immune detection: the existing immune detection products based on relaxin are mostly qualitative test strips, which can only determine "pregnant / non-pregnant", and cannot quantify the pregnancy stage (such as early and middle stages) through the concentration of relaxin; and some products use a single blood sample collection method, which requires professional personnel to collect blood from a vein, is inconvenient to operate, lacks sample pretreatment optimization, is easily affected by red blood cell fragments and protein impurities in blood, and has a false positive rate of more than 8%.

[0005] Weak environmental adaptability: most detection test strips do not have an environmental control module, and the detection reaction is significantly affected by temperature - when the environmental temperature is lower than 15℃, the antigen-antibody binding efficiency decreases by more than 50%, and the detection time is prolonged to more than 30 minutes; when the temperature is higher than 35℃, the antibody is easily denatured and inactivated, the false negative rate suddenly increases, and the all-weather detection demand in different regions (such as northern winter and southern summer) cannot be met.

[0006] Lack of data management capability: the existing products have no detection data storage and transmission function, and the detection results need to be recorded manually, which is easy to cause data omission and misrecording; and the basic information such as breed, age and weight of the dog and cat cannot be associated, which is not conducive to long-term tracking and analysis of the pregnancy data of multiple dogs and cats in a breeding farm.

[0007] In view of the above technical problems, the present application realizes rapid quantitative detection of canine and feline relaxin by optimizing five core units of sample processing, detection reaction, quantitative reference, signal processing and environmental control, and fills the gap of "low cost, easy operation and high stability" in the quantitative detection products for canine and feline pregnancy. SUMMARY

[0008] In view of the deficiencies in the background art, the present application provides a rapid quantitative detection of canine and feline relaxin pregnancy test kit and method, which solves the technical problems of slow detection, difficult quantification, sample limitation, environmental sensitivity and difficult data management in the existing canine and feline pregnancy detection technology.

[0009] The technical solution of the present application is implemented as follows:

[0010] The rapid quantitative detection of canine and feline relaxin pregnancy test kit comprises:

[0011] The sample processing unit comprises a sample collection tool, a sample diluent container and a sample filtration assembly, the sample collection tool is a disposable blood collection tube or a urine collection tube or a saliva collection cotton swab with built-in anticoagulant or preservative, which is used for collecting and pretreating the canine and feline samples;

[0012] The detection reaction unit is a detection card body, the detection card body is provided with a sample detection area, a quality control area, a water absorption area and a sample addition area in sequence, the solid phase carrier of the detection area is fixed with detection antibodies for canine and feline relaxin, the solid phase carrier is a hydrophilic treated nitrocellulose membrane or nylon membrane or polyester membrane, the quality control area is fixed with capture antibodies which can bind with the detection antibodies, and the water absorption area is used to maintain the stable chromatography flow rate of the sample;

[0013] The quantitative reference unit is provided with a standard curve reference module, the standard curve reference module is a chip or a paper reference card for storing standard curve data, the standard curve data comprises signal intensity values corresponding to at least five concentration gradients of canine and feline relaxin standard products, and is used to provide a quantitative calculation reference;

[0014] The signal processing and output unit comprises a signal reading module, a result storage module and a transmission module, the signal reading module is a portable optical detector with built-in optical sensors, data processing units and display units, which is used to read signal intensity and calculate relaxin content, and the result storage and transmission module is used to record detection data and transmit the detection data to external equipment through Bluetooth or USB interface;

[0015] The environmental control unit comprises a temperature control module, the temperature control module is arranged outside the detection card body, and is used to stabilize the detection environment temperature at 20-30 DEG C, so as to ensure the specificity reaction efficiency and signal stability.

[0016] As a preferred scheme, the detection antibodies and the capture antibodies are fixed on the solid phase carrier by carbodiimide chemical coupling method, the loading capacity of the fixed antibodies is 0.8-1.5 mu g / cm, and the combination of the antibodies and the solid phase carrier is stable for more than 12 months under the condition of 4 DEG C sealed storage.

[0017] As a preferred solution, the optical sensor of the signal reading module detects the wavelength in the visible light range of 450-650 nm or the fluorescence range of 520-680 nm or the chemiluminescence range of 420-580 nm, the signal detection sensitivity is ≤0.05 ng / mL, and the relative error of the detection result is ≤5%.

[0018] As a preferred solution, the result storage and transmission module is built-in with a storage chip with a capacity of ≥1000 records, which is used for automatically associating the basic information input of dog and cat breeds, age, weight, etc., and the data transmission rate is ≥1 Mbps.

[0019] As a preferred solution, the detection card body is a sealed card shell structure, the card shell structure is provided with a sample addition hole corresponding to the sample addition area of the sample, the card shell structure is provided with an observation window corresponding to the detection area and the quality control area, and the card shell structure is made of ABS plastic resistant to temperature of-20-50℃.

[0020] The rapid quantitative detection method for detecting the pregnancy of dogs and cats based on the above device, comprising:

[0021] Sample collection and pretreatment: using a sample collection tool to collect blood, urine or saliva samples of dogs and cats, mixing the samples with a sample diluent, removing impurities through a sample filtration assembly to obtain a sample to be detected;

[0022] Specific reaction: adding the sample to be detected to the sample addition area of the detection card body, allowing the sample to be chromatographed along the solid phase carrier, allowing the relaxin in the sample to be detected to combine with the detection antibody labeled with a signal marker on the detection line to form a "relaxin-detection antibody" complex, and allowing the remaining detection antibody to combine with the capture antibody on the quality control line;

[0023] Signal reading and quantitative calculation: detecting the signal intensity generated by the markers on the detection line and the quality control line through the signal reading module, calling the standard curve data established by the known concentration of the relaxin standard sample in the standard curve reference module, comparing the detection signal intensity with the standard curve, and calculating the specific content of the relaxin in the sample to be detected;

[0024] Pregnancy determination: setting a relaxin content determination threshold, if the calculated relaxin content is greater than the threshold, it is determined that the dog or cat is pregnant, otherwise it is determined that the dog or cat is not pregnant, and the detection time, dog or cat information and determination result are recorded through the result storage module.

[0025] As a preferred solution, in the specific reaction, the signal marker is one of colloidal gold, fluorescein, horseradish peroxidase or acridinium ester chemiluminescent substance, and the specific reaction is carried out in an environment with a temperature of 20-30℃ and a relative humidity of 40%-60%, and the reaction time is 5-15 minutes.

[0026] As a preferred solution, in the signal reading and quantitative calculation, the standard curve reference module has a standard concentration gradient range of 0.1-100 ng / mL, and at least 3 parallel detections are performed for each concentration gradient, and the average signal intensity is used to establish the standard curve.

[0027] As a preferred solution, in the pregnancy determination, the relaxin content determination threshold is 5 ng / mL, when the relaxin content in the sample is greater than or equal to 5 ng / mL, it is determined to be pregnant, and when the relaxin content is less than 5 ng / mL, it is determined to be not pregnant, and the determination result can be displayed in real time by the display unit of the signal reading module.

[0028] As a preferred solution, in the sample collection and pretreatment, the dog and cat are subjected to 4-6 hours of fasting and water deprivation pretreatment before sample collection, the sample diluent is a phosphate buffer with a pH value of 7.2-7.4, and the filter membrane pore size of the sample filtration assembly is 0.22-0.45 μm.

[0029] The beneficial effects of the present application are:

[0030] I. Rapid and efficient detection: the total detection time is less than or equal to 15 minutes, which is more than 90% shorter than the traditional ELISA method (3-4 hours), meeting the "real-time detection" requirement; and without the need for professional laboratory equipment, the operator can start after simple training, suitable for families and basic pet clinics.

[0031] II. Accurate and reliable quantification: through the standard curve reference of 5 concentration gradients, combined with high-sensitivity optical sensors (detection limit ≤0.05 ng / mL), the relative error of the detection result is less than or equal to 5%, which can not only determine "whether pregnant", but also quantify the pregnancy stage (such as early, middle) through the relaxin concentration, providing accurate basis for pregnancy care.

[0032] III. Flexible and convenient sample: supporting blood, urine, and saliva sample collection, among which saliva collection is a non-invasive operation, avoiding stress reaction of dogs and cats; at the same time, through sample diluent optimization (pH 7.2-7.4 PBS) and filtration (0.22-0.45 μm filter membrane), impurity interference is reduced, and the sample processing qualified rate is greater than or equal to 98%.

[0033] IV. Strong environmental adaptability: the reaction environment is stabilized at 20-30℃ by the temperature control module, which can be used normally under storage environment of-20-50℃ and humidity environment of 40%-60%, suitable for different climate regions in north and south China, and the detection result stability (intra-batch CV ≤3%, inter-batch CV ≤5%) is significantly better than that of the traditional test strip without environmental control.

[0034] Five, data management is perfect: support ≥1000 detection record storage and dog cat basic information associated, data transmission rate ≥1Mbps, easy to breeding field on many dog cat pregnancy data system management, at the same time for pet doctor provides historical data traceability, auxiliary development of individualized breeding plan.

[0035] Six, long-term stability: detection antibody through carbodiimide chemical coupling method fixed, 4℃ sealed storage condition stability ≥12 months, reagent kit shelf life, reduce the user storage and procurement cost. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present application, the following will be described in the embodiments of the drawings needed to use a brief introduction, obviously, the following description in the drawings only some embodiments of the present application, for those skilled in the art, without creative labor, can also be obtained from these other drawings.

[0037] Figure 1 For the structure diagram of the rapid quantitative detection of canine and feline relaxin pregnancy test kit of the present application.

[0038] Figure 2 For the flowchart of the rapid quantitative detection of canine and feline relaxin pregnancy test kit of the present application.

[0039] In the figure: 1, sample processing unit; 2, detection reaction unit; 3, quantitative reference unit; 4, signal processing and output unit; 5, environmental control unit. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings of the embodiments of the present application Figure 1 、 2 , the technical solutions in the embodiments of the present application are described clearly and completely, obviously, the described embodiments only some embodiments of the present application, not all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of the present application.

[0041] Example 1, rapid quantitative detection of canine and feline relaxin pregnancy test kit, the reagent kit is designed by modularization, integrating sample processing, detection reaction, quantitative reference, signal processing and output, environmental control five units, the specific structure and parameters are as follows:

[0042] (1) sample processing unit 1, the unit is used for completing the collection, pretreatment and impurity removal of canine and feline samples, ensuring the purity and stability of the sample to be detected, including:

[0043] Sample collection tools: disposable consumables with built-in anticoagulants (such as EDTA-K2) or preservatives (such as 0.01% sodium azide), optional types include:

[0044] Disposable blood collection tubes: 2mL capacity, suitable for venous blood sample collection, anticoagulant prevents blood clotting, ensures relaxin activity;

[0045] Urine collection tube: 5mL capacity, with scale for accurate urine sample measurement, preservative inhibits bacterial growth and prevents sample deterioration;

[0046] Saliva collection cotton swab: head made of medical absorbent cotton, handle with built-in sample release channel, can be directly mixed with diluent after collection without additional squeezing operation, suitable for non-invasive collection in home environment.

[0047] Sample diluent container: 10mL capacity, contains pH 7.2-7.4 phosphate buffered saline (PBS), which is close to the physiological environment of dogs and cats, can maintain the natural conformation of relaxin and prevent its denaturation and inactivation; 0.1% bovine serum albumin (BSA) is added to the buffer to reduce non-specific binding of impurities and solid phase carriers in the sample and reduce background signal interference.

[0048] Sample filtration assembly: uses disposable filter with 0.22-0.45μm pore size, effectively removes cell debris, protein polymers and other impurities in the sample (such impurities usually ≥0.5μm in diameter), while allowing relaxin (molecular weight about 26kDa, diameter <0.01μm) to pass smoothly, ensuring the specificity of subsequent detection reactions.

[0049] (2) Detection reaction unit 2, which is the core reaction carrier of the kit, realizes antigen-antibody specific binding in a sealed detection card body, with the following specific structure:

[0050] Detection card body: made of ABS plastic resistant to -20-50℃, sealed card shell, with:

[0051] Sample addition hole: 3mm in diameter, aligned with the sample addition area, facilitating dropwise addition of the sample to be tested;

[0052] Observation window: 1.5cm x 0.5cm in area, corresponding to the detection area and the control area, allowing direct observation of the reaction results or identification by the signal reading module;

[0053] Sealed design prevents sample evaporation and external contamination, ABS plastic material ensures that the kit does not deform or crack during transportation and storage (such as low-temperature transportation and high-temperature storage).

[0054] Solid support: A hydrophilic nitrocellulose membrane (preferably with a pore size of 5-8 μm) is used. The hydrophilic treatment can stabilize the chromatography speed of the sample on the membrane (1-2 mm / min) and avoid uneven chromatography caused by the hydrophobicity of the membrane. Compared with nylon or polyester membranes, nitrocellulose membranes have a stronger adsorption capacity for proteins and a low non-specific binding rate, which can improve the detection sensitivity.

[0055] Detection zone (T line): Antibodies against canine and feline relaxin (such as mouse anti-canine and feline relaxin monoclonal antibody) are immobilized on a solid-phase support using carbodiimide chemical coupling. The antibody loading is controlled at 0.8-1.5 μg / cm. Carbodiimide can activate the carboxyl groups of the antibody and the hydroxyl groups of the nitrocellulose membrane to form stable amide bonds, ensuring that the antibody is firmly immobilized. Under sealed storage conditions at 4°C, the binding stability of the antibody to the solid-phase support is ≥12 months, avoiding the decline in detection performance caused by long-term storage of the kit.

[0056] Control zone (C line): Located downstream of the detection zone (away from the sample application area), it contains a capture antibody (such as goat anti-mouse IgG polyclonal antibody) that can bind to the detection antibody. Its loading is 1.2 times that of the detection antibody, ensuring that even if there is no relaxin in the sample to be tested, the remaining detection antibody can fully bind to the capture antibody to form a clear control line, which is used to verify the effectiveness of the detection process (if there is no signal on the control line, the detection result is invalid).

[0057] Water absorption zone: Located downstream of the quality control zone, it is made of superabsorbent resin (such as sodium polyacrylate) and absorbent paper. It has a water absorption capacity of ≥200μL and can quickly absorb the sample after chromatography through capillary action to maintain a stable chromatographic flow rate of the sample on the solid support (avoiding insufficient reaction due to excessive flow rate or prolonged detection time due to excessively slow flow rate).

[0058] (3) Quantitative reference unit 3, which provides a benchmark for quantitative detection, with the core being the standard curve reference module, specifically including:

[0059] Standard curve data: Includes at least five concentration gradients of canine and feline relaxin standards (concentration range 0.1-100 ng / mL, covering relaxin concentrations at different stages of pregnancy in dogs and cats: <1 ng / mL in non-pregnant dogs and cats, 3-5 ng / mL in early pregnancy, 10-30 ng / mL in mid-pregnancy, and 50-80 ng / mL in late pregnancy). Each concentration gradient is analyzed in at least three parallel runs, and the average signal intensity is used to construct a standard curve (the fitting equation is typically y = ax). 2 +bx+c, where y is the signal intensity and x is the relaxin concentration);

[0060] Storage medium: Optional built-in storage chip (integrated in the signal reading module, supporting real-time data retrieval) or paper reference card (printed with standard curve spectrum and concentration-signal correspondence table, suitable for scenarios without electronic reading equipment) to ensure that quantitative benchmarks can be quickly obtained during detection.

[0061] (4) Signal processing and output unit 4, which realizes the reading, quantitative calculation, data storage and transmission of detection signals, specifically including: signal reading module: a portable optical detector, built-in:

[0062] Optical sensor: The detection wavelength can be selected according to the type of signal marker—for colloidal gold / horseradish peroxidase (HRP) markers, the detection range is 450-650 nm in the visible light range; for fluorescein markers, the detection range is 520-680 nm in the fluorescence range; for acridine ester chemiluminescent substances, the detection range is 420-580 nm in the chemiluminescence range; the sensor's detection sensitivity is ≤0.05 ng / mL, ensuring that it can capture signals from low concentrations of relaxin (e.g., 3 ng / mL in early pregnancy).

[0063] Data processing unit: Built-in microcontroller (such as STM32 series), which can call standard curve data and calculate the specific content of relaxin in the sample to be tested through the correspondence between signal intensity and concentration. The relative error of the detection result is ≤5%.

[0064] Display unit: It adopts a 2.4-inch LCD touch screen, which can display relaxin concentration value, pregnancy determination result ("pregnant" / "not pregnant"), detection time and basic information of dogs and cats in real time.

[0065] Result storage and transmission module:

[0066] Storage chip: Built-in Flash chip with a capacity of ≥1000 records, supports automatic association of dog and cat breeds (such as Golden Retrievers and Ragdolls), age, weight and other basic information input (manual input via touch screen or import via QR code), and data retention period of ≥5 years;

[0067] Transmission interface: Supports Bluetooth 5.0 (transmission rate ≥1Mbps) and USB Type-C interface, which can transmit detection data to mobile APP, computer or cloud database in real time, facilitating data backup and batch analysis.

[0068] (5) Environmental control unit 5, which is used to maintain a stable environment for the detection reaction, and its core is the temperature control module:

[0069] Structural design: A flexible heating film (thickness < 0.5 mm) is attached to the outside of the test card body (corresponding to the test area and quality control area). The heating film has a power of 5W and works with a built-in temperature sensor (accuracy ± 0.5℃) to achieve closed-loop temperature control.

[0070] Temperature control range: The detection environment temperature is stabilized at 20-30℃. Within this temperature range, the binding rate of antigen (relaxin) and antibody is the fastest (the dissociation constant KD value is the lowest), and the specific binding rate is ≥95%. This can avoid reaction delay caused by low temperature (<15℃) or antibody denaturation caused by high temperature (>35℃).

[0071] When using a rapid quantitative pregnancy test kit for canine and feline relaxin:

[0072] ①. Sample processing unit preparation:

[0073] Disposable blood collection tubes: 2mL polyethylene tubes are used, with 0.5mg EDTA-K2 anticoagulant inside, and sealed after sterilization;

[0074] Saliva collection swab: The head is made of 0.1g medical absorbent cotton, the handle is made of PP material, and it has a built-in 0.5mL sample release channel;

[0075] Sample dilution solution: Prepare a phosphate buffer solution at pH 7.3 (containing 0.01 mol / L PBS, 0.1% BSA, and 0.01% sodium azide), and dispense it into 10 mL polyethylene bottles;

[0076] Sample filtration assembly: A 0.22μm mixed cellulose ester filter membrane is used and assembled in a disposable plastic filter.

[0077] ② Preparation of the detection reaction unit:

[0078] Test casing: Made of ABS plastic injection molding, measuring 6cm×3cm×0.5cm, with a 3mm sample dispensing hole and a 1.5cm×0.5cm observation window;

[0079] Solid support: Hydrophilic nitrocellulose membrane (6μm pore size) was selected and cut into strips of 3mm×4cm;

[0080] Antibody fixation: Mouse anti-canine and feline relaxin monoclonal antibody (detection antibody) was fixed in the detection area (T line) at a loading of 1.2 μg / cm using the carbodiimide chemical coupling method, and goat anti-mouse IgG polyclonal antibody (capture antibody) was fixed in the control area (C line) at a loading of 1.4 μg / cm. The samples were then sealed and dried at 4°C for 24 hours.

[0081] Absorbent Zone: Composite superabsorbent resin with absorbent paper, cut into strips of 3mm×1cm, and attach them downstream of the quality control zone.

[0082] ③ Preparation of quantitative reference unit:

[0083] Preparation of standards: Prepare canine and feline relaxin standards at concentrations of 0.1, 1, 10, 50, and 100 ng / mL using sample diluent (purchased from Sigma-Aldrich).

[0084] Standard curve establishment: Three parallel detections were performed for each concentration standard, and the signal intensity was measured using a signal reading module (with colloidal gold as the label, detection wavelength 520 nm). The average signal intensity was obtained as follows:

[0085]

[0086] Data storage: The above standard curve data (fitting equation y = 0.0002x) will be stored. 2 +0.017x+0.038, R 2 =0.998) is written to the storage chip and integrated into the signal reading module.

[0087] ④ Signal processing and output unit fabrication:

[0088] Signal reading module: The STM32F103 microcontroller is used as the data processing unit, paired with a 520nm wavelength optical sensor (sensitivity 0.01 absorbance unit), equipped with a 2.4-inch LCD touch screen, and the whole package is a portable detector of 10cm×5cm×2cm.

[0089] Storage and transmission: Built-in 16MB Flash chip (supports 1000+ records), integrated Bluetooth 5.0 module (transmission rate 1.2Mbps) and USB Type-C interface.

[0090] ⑤ Preparation of the environmental control unit:

[0091] Temperature control module: A 0.3mm thick flexible heating film (5W power) is used and attached to the corresponding detection area on the outside of the detection card shell. It is equipped with a DS18B20 temperature sensor (accuracy ±0.5℃) and achieves closed-loop temperature control of 20-30℃ through a microcontroller.

[0092] This invention also proposes a rapid quantitative method for detecting canine and feline relaxin for pregnancy, implemented using the aforementioned device, comprising:

[0093] (1) Sample collection and preprocessing

[0094] Sample collection preparation: Dogs and cats should be fasted and deprived of water for 4-6 hours to reduce the interference of food residue, salivary enzymes or urinary metabolites on the test results;

[0095] Sample collection: Select the sample type according to the scenario - in the pet hospital scenario, venous blood is preferred (collect 1mL using a disposable blood collection tube), and in the home scenario, saliva (swab the buccal mucosa inside the dog or cat's mouth with a saliva collection swab for 30 seconds) or urine (collect 2mL of midstream urine using a urine collection tube) is preferred.

[0096] Sample dilution and filtration: Add the collected sample (0.1 mL of blood / urine, or directly soak a saliva collection swab) to the sample dilution container (containing 1 mL of phosphate buffer) and shake to mix for 30 seconds; then filter the mixture through the sample filtration assembly (filter membrane pore size 0.22-0.45 μm) to remove impurities and obtain the sample to be tested.

[0097] (2) Specific reaction

[0098] Sample addition: Use a pipette to draw 0.1 mL of the sample to be tested and add it to the sample well of the test card body. The sample will be chromatographically deposited along the solid support (nitrocellulose membrane) towards the water absorption area through capillary action.

[0099] Antigen-antibody binding: The canine and feline relaxin in the sample to be tested first binds to the detection antibody labeled with a signal marker (such as colloidal gold or fluorescein) on the detection zone (T line) to form a "relaxin-detection antibody-signal marker" complex; the detection antibody that does not bind relaxin continues to chromatographically and binds to the capture antibody on the quality control zone (C line) to form a "detection antibody-capture antibody-signal marker" complex.

[0100] Reaction condition control: The temperature of the detection card is stabilized at 20-30℃ by the temperature control module of the environmental control unit, while ensuring that the relative humidity of the detection environment is 40%-60% (which can be monitored with the aid of a hygrometer). The reaction time is adjusted according to the signal markers - 5-10 minutes for colloidal gold / fluorescein markers and 10-15 minutes for horseradish peroxidase / acriminyl ester markers to ensure sufficient reaction and stable signal.

[0101] (3) Signal reading and quantitative calculation

[0102] Signal detection: Insert the detection card body into the card slot of the signal reading module, and the optical sensor automatically aligns with the detection area and the quality control area to detect the signal intensity (such as absorbance value, fluorescence intensity, and luminescence intensity) generated by the marker.

[0103] Quantitative calculation: The data processing unit calls the standard curve data built into the quantitative reference unit (such as the signal intensity values ​​corresponding to concentrations of 0.1, 1, 10, 50, and 100 ng / mL), and converts the detection signal intensity into the specific concentration value of relaxin in the sample to be tested by fitting the equation; if there is no signal in the quality control area, it will prompt "invalid detection" and the sample needs to be collected and tested again.

[0104] (4) Pregnancy determination and data management

[0105] Pregnancy determination: The threshold for determining relaxin level is set at 5 ng / mL. When the calculated relaxin concentration is ≥5 ng / mL, the result is determined to be "pregnant"; when the concentration is <5 ng / mL, the result is determined to be "not pregnant". The determination result is displayed in real time through the display unit of the signal reading module (e.g., green "pregnant" and red "not pregnant").

[0106] Data Management: The results storage module automatically records the test time (accurate to the minute), basic information of dogs and cats (breed, age, weight), and relaxin concentration value; users can transfer data to external devices (such as mobile phones and computers) via Bluetooth or USB interface to achieve data backup and long-term tracking.

[0107] Taking the testing of a 2-year-old female Golden Retriever (weighing 25kg) as an example, the steps are as follows:

[0108] Sample collection and pretreatment: The golden retriever was fasted and deprived of water for 6 hours before the test; 1 mL of venous blood was collected from the forelimb using a disposable blood collection tube and added to a container containing 1 mL of sample diluent, and shaken to mix for 30 seconds; the mixture was filtered through a 0.22 μm filter to obtain the sample to be tested.

[0109] Specific reaction: Pipette 0.1 mL of the sample to be tested into the sample well of the test card; activate the environmental control module to stabilize the temperature of the test card at 25℃ and react for 10 minutes in an environment with a relative humidity of 50%.

[0110] Signal reading and quantitative calculation: The detection card is inserted into the signal reading module. The sensor detects a signal intensity of 0.68 absorbance in the detection area and 0.85 absorbance in the quality control area (quality control valid); the standard curve fitting equation y = 0.0002x is called. 2 Substituting +0.017x + 0.038 into y = 0.68, the calculated relaxin concentration is 12.5 ng / mL.

[0111] Pregnancy determination and data management: Because the relaxin concentration is 12.5ng / mL ≥ 5ng / mL, the display unit prompts "pregnant"; the dog and cat information (breed: Golden Retriever, age: 2 years, weight: 25kg) is manually entered, and the storage module records the test time (May 10, 2024, 14:30) and concentration value (12.5ng / mL); the data is transmitted to the mobile APP via Bluetooth, and a test report is generated, indicating "mid-pregnancy (approximately 3-4 weeks), it is recommended to retest in 2 weeks".

[0112] In summary, this invention, through modular design and technical optimization, achieves rapid quantitative detection of canine and feline relaxin, solving many pain points of existing technologies and possessing significant practical value and promising prospects for widespread application.

[0113] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A pregnancy test kit for rapid quantitative detection of relaxin in dogs and cats, characterized by, The application relates to a canine and feline relaxin detection device, which comprises the following units: a sample processing unit, a detection reaction unit, a quantitative reference unit, a signal processing and output unit and an environment control unit. The sample processing unit comprises a sample collection tool, a sample diluent container and a sample filtering assembly, the sample collection tool is a disposable blood collection tube or a urine collection tube or a saliva collection cotton swab with built-in anticoagulant or preservative, and the sample collection tool is used for collecting and pretreating canine and feline samples. The detection reaction unit is a detection card body, the detection card body is sequentially provided with a sample detection area, a quality control area, a water absorption area and a sample adding area, a detection antibody for canine and feline relaxin is fixed on a solid carrier of the detection area, the solid carrier is a hydrophilic treated nitrocellulose membrane or a nylon membrane or a polyester membrane, the quality control area is fixed with a capture antibody which can be combined with the detection antibody, and the water absorption area is used for maintaining the stability of sample chromatography flow rate. The quantitative reference unit is internally provided with a standard curve reference module, the standard curve reference module is used for storing a chip or a paper reference card of standard curve data, the standard curve data comprises signal intensity values corresponding to canine and feline relaxin standard samples of at least five concentration gradients, and the standard curve data is used for providing a quantitative calculation reference. The signal processing and output unit comprises a signal reading module, a result storage module and a transmission module, the signal reading module is a portable optical detection instrument with built-in optical sensors, a data processing unit and a display unit, is used for reading signal intensity and calculating relaxin content, and the result storage and transmission module is used for recording detection data and transmitting the detection data to external equipment through a Bluetooth or USB interface. The environment control unit comprises a temperature control module which is arranged outside the detection card body and is used for stably maintaining the detection environment temperature at 20-30 DEG C, so as to ensure the specificity reaction efficiency and signal stability.

2. The rapid quantitative detection of relaxin in dogs and cats of pregnancy test kit according to claim 1, characterized in that, The detection antibody and the capture antibody are fixed on the solid carrier through a carbodiimide chemical coupling method, the antibody loading capacity after fixation is 0.8-1.5 mu g / cm, and the combination stability of the antibody and the solid carrier is greater than or equal to 12 months under the sealed storage condition at 4 DEG C.

3. The rapid quantitative detection of relaxin in dogs and cats of pregnancy test kit according to claim 1, characterized in that, The optical sensor of the signal reading module detects visible light in the range of 450-650 nm or fluorescence in the range of 520-680 nm or chemiluminescence in the range of 420-580 nm, the signal detection sensitivity is less than or equal to 0.05 ng / mL, and the relative error of the detection result is less than or equal to 5%.

4. The rapid quantitative detection of relaxin in dogs and cats of pregnancy test kit according to claim 1, characterized in that, The result storage and transmission module is internally provided with a storage chip with a capacity greater than or equal to 1000 records, is used for automatically associating the input of basic information such as canine and feline breeds, ages and weights, and has a data transmission rate greater than or equal to 1 Mbps.

5. The rapid quantitative detection of relaxin in dogs and cats of pregnancy test kit according to claim 1, characterized in that, The detection card body is a sealed card shell structure, the card shell structure is provided with a sample adding hole corresponding to the sample adding area, the card shell structure is provided with observation windows corresponding to the detection area and the quality control area, and the card shell structure is made of ABS plastic which is resistant to temperature of-20-50 DEG C.

6. A pregnancy test method for rapid quantitative detection of relaxin in dogs and cats, which is implemented based on a pregnancy test kit for rapid quantitative detection of relaxin in dogs and cats, characterized by, The application further relates to a canine and feline relaxin detection method. The sample collection tool is used to collect blood, urine or saliva samples of canines and felines, the sample is mixed with the sample diluent, impurities are removed through the sample filtering assembly, and a sample to be detected is obtained. In the specific reaction, the sample to be detected is added dropwise to the sample adding area of the detection card body, so that the sample is chromatographed along the solid carrier. The relaxin in the sample to be detected is combined with the detection antibody marked with a signal marker on the detection line to form a "relaxin-detection antibody" complex, and the remaining detection antibody is combined with the capture antibody on the quality control line. In the signal reading and quantitative calculation, the signal strength generated by the marker on the detection line and the quality control line is detected by the signal reading module, the standard curve data established by the known concentration of the relaxin standard sample is called from the standard curve reference module, the detection signal strength is compared with the standard curve, and the specific content of the relaxin in the sample to be detected is calculated. In the pregnancy determination, the relaxin content determination threshold is set. If the calculated content of the relaxin is greater than the threshold, it is determined that the dog or cat is pregnant, otherwise it is determined that the dog or cat is not pregnant. The detection time, dog or cat information and determination result are recorded by the result storage module.

7. The rapid quantitative detection of relaxin in pregnant detection method for dogs and cats according to claim 6, characterized in that, In the specific reaction, the signal marker is one of colloidal gold, fluorescein, horseradish peroxidase or acridinium ester chemiluminescent substance, and the specific reaction is carried out in an environment with a temperature of 20-30℃ and a relative humidity of 40%-60% for 5-15 minutes.

8. The rapid quantitative detection of relaxin of the pregnant detection method for dogs and cats according to claim 7, characterized in that, In the signal reading and quantitative calculation, the concentration gradient range of the standard sample of the standard curve reference module is 0.1-100 ng / mL, and at least 3 parallel detections are performed for each concentration gradient, and the average signal strength is used to establish the standard curve.

9. The rapid quantitative detection of relaxin of the pregnant detection method for dogs and cats according to claim 8, characterized in that, In the pregnancy determination, the relaxin content determination threshold is 5 ng / mL. When the content of the relaxin in the sample is ≥5 ng / mL, it is determined to be pregnant, and when the content of the relaxin is <5 ng / mL, it is determined to be not pregnant. The determination result can be displayed in real time by the display unit of the signal reading module.

10. The rapid quantitative detection of relaxin of the pregnant detection method for dogs and cats according to claim 9, characterized in that, In the sample collection and pretreatment, the dog or cat is pretreated with fasting and water deprivation for 4-6 hours before the sample is collected. The sample diluent is a phosphate buffer with a pH value of 7.2-7.

4. The pore size of the filter membrane of the sample filtration assembly is 0.22-0.45 μm.

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