Immunological animal origin detection kit based on lateral chromatography and detection method thereof
Through an immunologic animal origin detection kit based on lateral chromatography, combined with immunochromatography technology, the existing PCR detection technology is solved for a long time and complex operation, and a fast, simple and accurate animal origin detection is achieved.
Patent Information
- Application Number
- CN202510036423.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-05-13
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing animal-derived detection technology relies on PCR, and has problems such as strict detection environment, susceptible to pollution, cumbersome testing procedures and long-term time-consuming, making it difficult to meet the needs of fast and on-site testing.
Using an immunologic animal origin detection kit based on lateral chromatography, the unique preparation process of labeling pads, reaction membranes and sample pads, combined with immunochromatography technology, can achieve fast and simple animal origin detection.
It realizes that the inspection takes short time, easy operation, low equipment requirements, strong anti-pollution ability, and can quickly complete the inspection at room temperature of 15-30℃, and takes no more than 10 minutes, improving the detection efficiency and accuracy.
Smart Images
Figure CN119985965A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of animal-derived detection, and in particular to an immunological animal-derived detection kit based on lateral flow chromatography and a detection method thereof. Background Art
[0002] In the meat market, the prices of meat from different animal species vary greatly. Driven by profit, some unscrupulous merchants often sell low-priced meat (such as pork and duck) as high-priced meat (such as beef and mutton), which seriously damages the rights and interests of consumers and disrupts the market order. At present, animal-derived testing mostly relies on PCR technology, but PCR testing has prominent drawbacks. It not only has strict requirements on the testing environment and is easily contaminated, but also requires supporting professional equipment. The testing process is cumbersome and time-consuming, and it is difficult to meet the needs of rapid and on-site testing.
[0003] In view of this, this application is hereby filed. Summary of the invention
[0004] The object of the present invention is to provide an immunological animal-derived detection kit and a detection method thereof based on lateral flow chromatography, so as to solve the problems mentioned in the above background technology.
[0005] In order to solve the above technical problems, the present invention provides an immunological animal-derived detection kit based on lateral chromatography, comprising: a marker pad working solution: taking latex, mixing it with 19# and 20# diluents in sequence, centrifuging, ultrasonicating and ice bathing, adding a specific proportion of 1% EDAC and 1% NHS solution for activation, then mixing it with the labeling raw material, treating it with a blocking solution and a re-solution, fixing the volume, testing the OD value and storing it at 2°C-8°C; a marker pad working solution: diluting the marker pad working solution to 120OD value with 7# re-solution; a marker pad: spraying gold according to a specific spray volume, drying at 45±2°C for 12-24 hours; a reaction membrane: pasting and scratching the NC membrane ss12-15 according to the set parameters, diluting the B IOantibody and the porcine antibody with a specific diluent to make C and T lines, and packaging after oven treatment at 45±2°C; a sample pad: containing a specific formula solution, applying, drying, and sealing for storage.
[0006] Furthermore, when preparing the working mother solution of the marking pad, latex and 19# diluent are mixed in a volume ratio of 1:3, and centrifuged at 13500-14500*g for multiple times throughout the process, and the length of each centrifugation is 35 minutes, 35 minutes, 35 minutes, and 30 minutes according to the process; the precipitate is treated with an ultrasonic cleaner for 15-20 minutes, the ultrasonic cell disruptor has a power of 4%, an interval of 3s, and an ice bath for 10 minutes; the amount of 1% EDAC solution added is 20% of the latex volume, and the amount of 1% NHS solution is 10%, both of which are prepared with purified water and used immediately, and the mixing and activation are both 25-35 minutes; the marking raw material is diluted to 1 mg / ml with 20# diluent, and the amount added is twice the volume of the latex, and stirred for 1.5 hours.
[0007] Furthermore, the 4# blocking solution and the 7# re-solution are both boiled. When the 4# blocking solution is used, the precipitate is ultrasonically cleaned, crushed and treated with an ice bath before being fixed to 4 times the volume of the original latex and stirred for 25-35 minutes. The 7# re-solution is added and treated in the same way to ensure precise control of each link, maintain the stability and activity of the working mother solution, and facilitate subsequent dilution and labeling pad preparation.
[0008] Furthermore, in the dilution of the marking pad working solution, the 7# complex solution used is of qualified quality and free of impurities, the dilution operation is carried out in a clean environment, the OD value is monitored throughout the process, and the error is controlled within the range of ±5OD, ensuring that the concentration of the marking pad working solution is uniform and the performance is stable, so that the color development of the marking pad is accurate and reliable.
[0009] Furthermore, when preparing the marking pad, the spray volume is adjusted in the range of 0.8-2.0ul / cm, the parameters of the gold spray machine are calibrated in advance, and the spraying process is uniform and without breakpoints; during drying, the temperature and humidity of the oven are stable, the temperature fluctuation is controlled at ±2°C, and the humidity is <30%, which ensures the physical properties and marking effect of the marking pad and is conducive to subsequent chromatography reactions.
[0010] Furthermore, when preparing the reaction membrane, the pasting position accuracy of the NC membrane ss12-15 reaches ±0.2mm, and the feeding and cutting errors are controlled at ±0.5mm respectively; the membrane dilution liquid at each point is accurately prepared according to the formula, the antibody dilution concentration fluctuation does not exceed ±0.05mg / ml, the continuous film machine spray volume is stable, and the line spacing and C line position accuracy are within ±0.2mm, ensuring that the color development of the test line and the control line is standardized and the results are accurate.
[0011] Furthermore, the kit is packaged in an overall sealed package and contains a desiccant, which maintains low humidity to ensure stable and reliable quality during transportation and storage.
[0012] A detection method for an immunological animal-derived detection kit based on lateral flow chromatography, wherein the sample to be tested is diluted and then dripped onto a sample pad, reacts with antibodies according to the chromatography principle, and the result is determined based on the color development of the detection line and the control line.
[0013] Furthermore, the sample dilution is operated at a volume ratio of 1:5-1:10, and the drop amount is 50-100 μL to ensure sufficient chromatography and reaction of the sample and improve detection sensitivity and accuracy; the entire detection process is carried out at a room temperature of 15-30°C to reduce environmental interference.
[0014] Furthermore, it takes no more than 10 minutes from adding the sample to getting the result, which is efficient and fast. The test kit is stored at room temperature and away from light before testing, with a shelf life of no less than 12 months, which is convenient for storage and secondary verification.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] 1. Short detection time: Traditional PCR detection takes several hours, while this test kit only takes 5-10 minutes. In various links of meat circulation, such as market sampling and slaughterhouse rapid inspection, it can quickly give results, greatly reducing the detection time cost, making it impossible for problematic meat to hide, and effectively ensuring market circulation efficiency;
[0017] 2. Easy to operate: PCR testing requires operators to have professional knowledge and skills, but this test kit only requires simple training to use. Whether it is grassroots supervisors or ordinary merchants who conduct self-inspections, they can easily complete the operation, breaking down technical barriers and broadening the scope of people suitable for testing;
[0018] 3. Low equipment requirements: PCR testing cannot be separated from professional and expensive supporting equipment, which limits the testing scenarios. This test kit does not require additional equipment. With a simple drop sampling method, it can be tested in a simple environment or even outdoors, which is conducive to random inspections anytime and anywhere, and enhances regulatory flexibility;
[0019] 4. Strong anti-contamination ability: PCR is easily cross-contaminated by samples during testing, resulting in deviation of results and retesting. This kit does not have the problem of amplification contamination. Even if the surface of the meat sample is slightly contaminated, it can be sampled and tested after a simple rinse with clean water, ensuring accurate and reliable test results. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The schematic diagram of an immunological animal-derived detection kit based on lateral flow chromatography is shown in FIG. DETAILED DESCRIPTION
[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0022] See also Figure 1 The present invention provides a technical solution: an immunological animal-derived detection kit based on lateral flow chromatography and a detection method thereof, comprising:
[0023] 1. Preparation of components before assembling the kit:
[0024] 1. Preparation of labeling pad working stock solution:
[0025] 1.1. Preparation: Accurately measure a unit volume of latex and sufficient amount of 19# and 20# diluents. Prepare 1% EDAC solution and 1% NHS solution with purified water in advance. Make sure that 4# blocking solution and 7# re-solution have been boiled. Dilute the labeled material to 1mg / ml with 20# diluent for later use.
[0026] 1.2. Initial treatment: Pour the latex into a clean container, add 3 times the volume of 19# diluent, stir slowly with a glass rod or magnetic stirrer, then pour into a centrifuge tube, put into a centrifuge, set the centrifugal force to 13500-14500*g, centrifuge for 35 minutes, and carefully discard the supernatant after the end.
[0027] 1.3. Precipitate cleaning and volume adjustment: Add a small amount of 19# diluent to immerse the precipitate, place it in an ultrasonic cleaner for 15-20 minutes; then place the container in an ultrasonic cell disruptor, adjust the power to 4%, work at intervals of 3s, and simultaneously treat it in an ice bath for 10 minutes; after the treatment, add 19# diluent and adjust the volume to 4 times the original latex volume, centrifuge again for 35 minutes under the above centrifugation conditions, and discard the supernatant.
[0028] 1.4. Activation and addition of labeling materials: Replace with 20# diluent and repeat the precipitation, washing and volume adjustment operations; after completion, add 20% unit volume of 1% EDAC solution, mix with a vortex oscillator, and activate for 25-35 minutes; then add 10% unit volume of 1% NHS solution, mix and activate for 25-35 minutes, then centrifuge and discard the supernatant. Add appropriate amount of 20# diluent to 4 times the original latex volume, add 2 times the unit volume of latex labeling materials, stir for 1.5 hours, centrifuge again for 35 minutes and discard the supernatant.
[0029] 1.5. Blocking and re-dissolving: Add a small amount of boiled 4# blocking solution, and process according to the above-mentioned ultrasonic, crushing and ice bath process, dilute to 4 times the original latex volume, stir for 25-35 minutes, centrifuge for 30 minutes and discard the supernatant; finally, add a small amount of 7# re-dissolving solution, repeat the above process, test the OD value after diluting, record the data, and store at 2℃-8℃.
[0030] 2. Preparation of Marking Pad Working Solution:
[0031] Take out the marker pad working stock solution from the refrigerator, wait for it to return to room temperature, measure an appropriate amount of the marker pad working stock solution and the boiled 7# complex solution; use the 7# complex solution to gradually dilute the marker pad working stock solution, and use a professional photometer to detect the OD value while diluting until it reaches 120OD value, stir evenly, and set aside.
[0032] 3. Preparation of marking pad:
[0033] Estimate the required volume of latex marking pad working fluid according to the production scale, measure and pour into the special container of the gold spraying machine; adjust the parameters of the gold spraying machine, set the spray volume to 0.8-2.0ul / cm (the recommended spray volume is 1.5ul / cm for the first time), start the gold spraying machine, and spray the working fluid evenly on the marking pad; after spraying, immediately transfer the marking pad to an oven set at 45±2℃ and dry it for 12-24 hours; after drying, hand it over to the quality inspection personnel for inspection, determine the final spray volume, and feedback to the production personnel for mass production.
[0034] 4. Preparation of reaction membrane:
[0035] 4.1. Carefully install the NC film ss12-15 on the laminating machine, accurately adjust the laminating machine parameters, so that the NC film is adhered to 20±0.5mm from the bottom edge of the sheet, the running speed is set to 220r / min, the feeding setting is 42.0mm, and the cutting setting is 6.5mm.
[0036] 4.2. Dilute the Bioantibody to 0.5 mg / ml with 6# dot film diluent and 8# dot film diluent respectively as C line; dilute the porcine antibody to 0.5 mg / ml with 5# dot film diluent, 7# dot film diluent and 9# dot film diluent with a content of 4% of the total volume as T line; pour the diluted antibody solution into the corresponding container of the continuous film machine, adjust the spray volume of the film machine to 0.7-1.0 ul / cm, ensure that the adjacent lines are 5±0.5 mm apart, and the distance between the C line and the bottom edge is 34±0.5 mm, start the film machine to complete the coating; immediately after coating, put the reaction membrane into a 45±2℃ oven, bake for 12-24 hours, take it out after drying, and place it in a dry, dust-free environment for packaging and temporary storage.
[0037] 5.3204 Sample pad preparation:
[0038] Carefully place the glass fiber 2#K8964 sheet into the special mold, use a measuring cylinder to accurately measure 50 ml of 3204 sample pad solution and pour it into the mold; use a plastic card to slowly and evenly spread the solution to ensure that the glass fiber is fully soaked; transfer the coated sample pad to the drying room, set the temperature, and dry for 12-24 hours; after drying, quickly seal it and store it in a cool and dry place.
[0039] 2. Animal-derived testing operation steps:
[0040] 1. Sample preparation:
[0041] Take the meat sample to be tested. If there are obvious impurities, blood or pollution on the surface of the sample, rinse it with clean water and dry it with filter paper. Use a clean scalpel or scissors to cut an appropriate amount of sample (about 0.5-1g), put it into a clean centrifuge tube, add 5-10ml of sample diluent (select the appropriate diluent based on the characteristics of the sample), oscillate and mix, and make a sample suspension to be tested.
[0042] 2. Detection process:
[0043] 2.1. Take out the assembled lateral flow chromatography-based immunology animal-derived detection kit from the sealed package, place it horizontally on the clean laboratory bench, and check that the appearance of the kit is not damaged and the test strips are not damp; gently peel off the sample pad protective film on the kit.
[0044] 2.2. Use a pipette to absorb 50-100 μL of the sample suspension to be tested, and slowly and vertically drip it onto the sample pad of the reagent kit to avoid splashing of the sample or deviation of the dripping position; start timing immediately after the dripping is completed.
[0045] 3. Interpretation of results:
[0046] Observe the color development of the test line (T line) and the control line (C line) within 5-10 minutes: If a clear colored line appears at the T line position and a colored line also appears at the C line, it indicates that the sample contains the corresponding animal reactant, and it is judged as a positive result; if there is no colored line at the T line position, but a colored line appears at the C line, it indicates that the sample does not contain the corresponding animal reactant, and it is judged as a negative result; if no colored line appears at the C line, the test result is invalid and the test kit needs to be replaced and retested.
[0047] 3. Post-test processing:
[0048] 1. After testing, the test kits should be classified and collected according to the relevant regulations on medical waste or laboratory waste to avoid cross contamination. If samples of the test kit need to be retained for re-examination, they should be placed in a dry, dark, cool place, marked with key data such as the test date and sample information, and properly stored.
[0049] 2. Clean the laboratory table and wipe pipettes, scissors and other tools with 75% alcohol cotton balls to avoid contamination caused by residual samples; dispose of discarded sample suspensions and diluents harmlessly and follow the standard operating procedures for laboratory waste liquid treatment.
[0050] In summary: The present invention uses immunochromatography technology to specifically solve the technical difficulties faced by PCR detection when used for animal-derived detection. By fixing the required antibody pairs on the NC membrane and the colorimetric carrier respectively, and using a unique labeling pad, reaction membrane and sample pad preparation process, the contamination problem of PCR detection is successfully overcome; the reliance on complex equipment is abandoned to achieve on-site instant detection; the detection time is greatly shortened and the detection efficiency is improved; the operating threshold is lowered, making animal-derived detection more convenient, accurate and efficient, and meeting the needs of multiple detection scenarios.
[0051] It should be noted here that the animal-derived detection kit is an immunological detection method based on lateral flow chromatography, which can detect whether the corresponding animal reactant is present in the sample. In this test process, the corresponding animal reactant antibody 1 is fixed on the detection area of the reagent strip. When the sample is placed in the sample well, it reacts with the animal reactant antibody 2 color particles pre-embedded on the conjugate pad. This sample and antibody-
[0052] The color-developing particle mixture chromatographs along the reagent strip and interacts with the animal reactant antibody 1 immobilized on the test zone. If the sample contains the corresponding animal reactant, a colored line will appear in the test line area, indicating a positive result. If the sample does not contain the corresponding animal reactant, no colored line will appear in this area, indicating a negative result. As a validation control for the product, a colored line will always appear in the control line area, indicating that the appropriate volume of sample has been added and membrane chromatography has occurred.
[0053] The kit proposed by the present invention also includes a plastic card shell, absorbent paper, a dropper, a diluent, an outer aluminum foil bag, a paper box, a desiccant, and an instruction manual, which will not be repeated in the text.
Claims
1. An immunological animal-derived detection kit based on lateral flow chromatography, characterized in that: include: Marking pad working stock solution: Take latex, mix with 19# and 20# diluents in sequence, centrifuge, sonicate and ice bath, add 1% EDAC and 1% NHS solution in a specific ratio to activate, then mix with marking raw materials, treat with blocking solution and re-solution, make up to volume, test OD value and store at 2℃-8℃; Marking pad working solution: dilute the marking pad working stock solution to 120 OD value with 7# reconstitution solution; Marking pad: spray gold according to the specific spraying amount, dry at 45±2℃ for 12-24 hours; Reaction membrane: Paste and scratch the NC membrane ss12-15 according to the set parameters, dilute the BIO antibody and porcine antibody with specific diluent to make C and T lines, and package after drying in a 45±2℃ oven; Sample pad: Contains a specific formula solution, applied, dried, and sealed for storage.
2. The immunological animal-derived detection kit based on lateral flow chromatography according to claim 1, characterized in that: When preparing the working stock solution of the marking pad, latex and 19# diluent were mixed at a volume ratio of 1:3, and the whole process was repeated 13500- 14500*g centrifugation, each centrifugation time is 35 minutes, 35 minutes, 35 minutes, 30 minutes according to the process; ultrasonic cleaning instrument treatment precipitation is 15- 20min, ultrasonic cell disruptor power 4%, interval 3s, ice bath 10min; 1% EDAC solution added in an amount of 20% of the latex volume, 1% NHS solution 10%, both prepared with purified water before use, mixed and activated for 25-35min; labeled raw material with 20# diluent to 1mg / ml, added in an amount twice the volume of the latex, stirred for 1.5 hours.
3. The immunological animal-derived detection kit based on lateral flow chromatography according to claim 1, characterized in that: The 4# blocking solution and the 7# reconstitution solution are both boiled. When the 4# blocking solution is used, the precipitate is ultrasonically cleaned, crushed and treated in an ice bath, then the volume is fixed to 4 times the original latex volume and stirred for 25-35 minutes. The 7# reconstitution solution is added and treated in the same manner.
4. The immunological animal-derived detection kit based on lateral flow chromatography according to claim 1, characterized in that: In the dilution of the marking pad working solution, the 7# rehydration solution used is of qualified quality and free of impurities. The dilution operation is carried out in a clean environment, and the OD value is monitored throughout the process, with the error controlled within the range of ±5OD.
5. The immunological animal-derived detection kit based on lateral flow chromatography according to claim 1, characterized in that: When preparing the marking pad, the spray volume adjustment range is 0.8- 2.0ul / cm, the parameters of the gold spraying machine are calibrated in advance, the spraying process is uniform and without breakpoints; the temperature and humidity of the oven are stable during drying, the temperature fluctuation is controlled within ±2℃, and the humidity is <30%.
6. The immunological animal-derived detection kit based on lateral flow chromatography according to claim 1, characterized in that: When the reaction film is prepared, the NC film ss12- 15 The pasting position accuracy is ±0.2mm, and the feeding and cutting errors are controlled at ±0.5mm respectively; the membrane dilution liquid at each point is accurately formulated according to the formula, the antibody dilution concentration fluctuation does not exceed ±0.05mg / ml, the continuous film machine spray volume is stable, and the line spacing and C line position accuracy are ±0.2mm.
7. The immunological animal-derived detection kit based on lateral flow chromatography according to claim 1, characterized in that: The test kit is packaged in an overall sealed package, and contains a desiccant and a quality inspection card. The desiccant maintains low humidity, and the quality inspection card is used to randomly inspect the performance of the test kit to ensure stable and reliable quality during transportation and storage.
8. As claimed in claim 1- A detection method of an immunological animal-derived detection kit based on lateral flow chromatography as described in any one of the claims 7, characterized in that: The sample to be tested is diluted and dripped onto the sample pad, reacts with the antibody according to the chromatography principle, and the result is determined according to the color development of the detection line and the control line.
9. The detection method of the immunological animal-derived detection kit based on lateral flow chromatography according to claim 8, characterized in that: The sample dilution is operated at a volume ratio of 1:5-1:10, and the drop amount is 50-100 μL to ensure sufficient chromatography and reaction of the sample; the entire detection process is carried out at a room temperature of 15-30°C.
10. The detection method of the immunological animal-derived detection kit based on lateral flow chromatography according to claim 8, characterized in that: It takes no more than 10 minutes from adding the sample to getting the result. The test kit should be stored at room temperature and away from light before testing, and the shelf life is not less than 12 months.