Excrement detection bag as well as preparation method and application thereof
By designing feces detection bags, combining feces filters and sample pipelines, using the combination of colloidal gold-labeled antibodies and nitrocellulose membranes, the problem that feces detection products can only detect a single or a small number of indicators in the prior art has been solved, and multiple items have been achieved. Simultaneous detection of biomarkers improves detection efficiency and sensitivity and reduces operational complexity.
Patent Information
- Application Number
- CN202411227816.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-03
- Publication Date
- 2025-01-03
AI Technical Summary
Existing fecal testing products can only detect a single or up to two indicators, which are complex in operation and low in sensitivity. They are prone to odor sensitivity and sample overflow problems during fecal sample processing, making it difficult to detect multiple biomarkers at the same time.
Design a fecal detection bag containing 6 kinds of test strip combinations and vacuum negative pressure chambers. The fecal filter and sample pipe are combined, and the combination of colloidal gold-labeled antibodies and nitrocellulose membranes can achieve a variety of biomarkers. Simultaneous detection reduces operation difficulty and improves sensitivity.
The results of multiple detection indicators can be obtained by achieving one-time detection, reducing operation difficulty, improving detection efficiency, and significantly improving the sensitivity of the test strips by extending the reaction time, avoiding sample overflow and odor sensitivity problems.
Smart Images
Figure CN119246848A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of feces detection, and specifically discloses a feces detection bag and a preparation method and application thereof. Background Art
[0002] Stool testing is one of the three routine in vitro diagnostic tests. By testing human feces, we can obtain information such as the digestive system function, pathological changes, and microbial and parasitic infections of the test subject.
[0003] Most of the stool testing products on the market provide test results for a single indicator, such as fecal occult blood test kits, transferrin test kits, calprotectin test kits, rotavirus test kits, adenovirus test kits, norovirus test kits, fecal occult blood / transferrin dual test kits, etc. A single test can only produce test results for one or at most two indicators. If the tester wants to test multiple biomarkers in stool, he or she not only needs to purchase different types of test kits from different manufacturers, but also needs to follow the operating instructions of the test kits from different manufacturers to sample, process and add stool, which will inevitably increase the test time and difficulty of operation.
[0004] The above-mentioned colloidal gold test kits all require the use of a stool collector to manually collect stool, and then the collected stool is placed in a special stool dilution tube for mixing, dilution and processing, and then the processed stool dilution in the stool dilution tube is manually added to the sample well of the test card to start the reaction of the test card. Most operators are very sensitive to the smell of stool during the process of stool collection, processing and sampling, and will experience nausea and vomiting; during the process of adding the stool sample to the stool dilution tube for mixing, and in the process of adding the processed stool to the sample well of the test card, the stool sample will overflow.
[0005] Most of the colloidal gold test strips on the market are made by drying and fixing colloidal gold-labeled antibodies on glass fibers to make gold-labeled pads (colloidal gold binding pads). After the target antigen in the feces binds to the gold-labeled antibody in the gold-labeled pad, it is quickly chromatographed onto the nitrocellulose membrane (NC membrane) to react with the coated antibody. Since the physical distance between the gold-labeled pad and the coated antibody is too short, a part of the target antigen binds to the coated antibody on the nitrocellulose membrane before it has time to fully react with the gold-labeled antibody in the gold-labeled pad, resulting in too little binding to the gold-labeled antibody in the gold-labeled pad, making the sensitivity of the test strip low.
[0006] In summary, it is of great significance to prepare a combined detection integrated stool detection bag. Summary of the invention
[0007] The object of the present invention is to provide a feces detection bag and a preparation method and application thereof, so as to solve the problems raised in the above background technology.
[0008] A method for preparing a stool detection bag comprises the following steps: Step 1: Preparation of test strip assembly: S1.1: Attach 6 different nitrocellulose membranes to the middle of the PVC base plate; attach the sample pad to the left side of the PVC base plate and cut into strips to obtain 6 different test strips; S1.2: 6 different test strips are evenly arranged on the annular absorbent pad; the right side of the PVC bottom plate of the test strip is attached to the annular absorbent pad, and the nitrocellulose membrane in the test strip overlaps the annular absorbent pad by 2 mm; and a test strip assembly is obtained; Step 2: Assembling the test strip assembly and the lower cover of the vacuum negative pressure chamber: S2.1: Take the vacuum negative pressure chamber lower cover equipped with the vacuum negative pressure chamber, slot, T-shaped buffer chamber, test strip fixing slot, and rubber ring groove and set aside; S2.2: The annular absorbent pad part of the test strip assembly is loaded into the vacuum negative pressure chamber on the lower cover B of the vacuum negative pressure chamber; wherein the 6 different test strips of the test strip assembly are correspondingly arranged in the test strip fixing grooves, and a pair of T-row buffer chambers are respectively arranged next to the test strip fixing grooves, and the T-row buffer chambers are arranged on both sides of the nitrocellulose membrane areas of the 6 different test strips; and a semi-finished product A is obtained; Step 3: Preparation of waterproof bag: The inner waterproof bag is connected to the feces filter net, and the outer waterproof bag is embedded outside the inner waterproof bag to form a space that can carry and contain the feces solution; 6 sample pipes containing different gold-labeled antibody probes are fixed between the outer waterproof bag and the inner waterproof bag; the end B of the sample pipe is extended into the lower part of the feces filter net; the end A of the sample pipe is heat-sealed with a sealing sheet to obtain a waterproof bag; Step 4: Assemble the waterproof bag and the lower cover of the vacuum negative pressure cabin in semi-finished product A: Insert the end A of the sample pipe in the waterproof bag into the rubber ring, fix it in the test strip fixing groove of the lower cover of the vacuum negative pressure chamber in the semi-finished product A, and connect it with the groove of the rubber ring on the test strip fixing groove; obtain the semi-finished product B; Step 5: Assembly of the upper cover of the vacuum negative pressure chamber and the lower cover of the vacuum negative pressure chamber in semi-finished product B: S5.1: Take the upper cover of the vacuum negative pressure chamber equipped with the observation window, spring button hole, spring button, vacuum hole, and plug-in protrusion and set aside for use; S5.2: Set 24 groups of plug-in protrusions on the B surface of the upper cover of the vacuum negative pressure chamber, and assemble them one by one with the 24 groups of slots on the lower cover of the vacuum negative pressure chamber in the semi-finished product B; vacuumize; and obtain a feces detection bag.
[0009] More optimally, in step 1, the six different nitrocellulose membranes corresponding to the six different test strips are respectively coated with six different detection lines, namely, T1 line, T2 line, T3 line, T4 line, T5 line, T6 line, and a quality control line C; The quality control line C is coated with goat anti-mouse IgG antibody; the 6 different detection lines are respectively coated with anti-human hemoglobin monoclonal antibody T1, transferrin monoclonal antibody T2, calprotectin monoclonal antibody T3, rotavirus monoclonal antibody T4, adenovirus monoclonal antibody T5, and norovirus monoclonal antibody T6; The six different test strips correspond to the detection items of the six sample pipes containing different gold-labeled antibody probes one by one; the corresponding different gold-labeled antibody probes are gold-labeled anti-human hemoglobin monoclonal antibody, gold-labeled transferrin monoclonal antibody, gold-labeled calprotectin monoclonal antibody, gold-labeled rotavirus monoclonal antibody, gold-labeled adenovirus monoclonal antibody, and gold-labeled norovirus monoclonal antibody; The corresponding test items for the 6 different test lines are fecal occult blood, transferrin, calprotectin, rotavirus, adenovirus, and norovirus.
[0010] More optimally, the observation windows are arranged on the A side of the upper cover of the vacuum negative pressure chamber, with a total of 6, for correspondingly observing 6 test items; each observation window is provided with a "T" and "C" mark; there are 6 spring buttons near the "T" mark, i.e. 6 start buttons; they are used to pierce the sealing sheet at the A end of the sample pipe, open the vacuum negative pressure chamber and the sample reaction pipe, and start the test strip reaction; when starting, the spring button is pressed into the spring button hole; The vacuum hole is used to evacuate the vacuum negative pressure cabin to form a negative pressure state.
[0011] More preferably, the preparation method of the gold-labeled antibody probe is: S6.1: Boil the HAuCl4 aqueous solution, add trisodium citrate aqueous solution, and continue boiling for 4 to 6 minutes after the solution changes to purple-red, cool, and add water to restore the initial volume to obtain colloidal gold; S6.2: Add K2CO3 to colloidal gold, adjust the pH to 7.4-7.6, add 6 different antibodies under stirring, react at room temperature for 30-35 minutes, add BSA solution after the reaction, react at room temperature for 20-25 minutes, centrifuge, collect the supernatant and add gold label working solution to obtain 6 different gold label solutions; S6.3: Add different gold-labeled solutions into the sample tube and dry them in a 37°C oven for ≥8 hours to obtain 6 different gold-labeled antibody probes.
[0012] More optimally, in the colloidal gold, the concentration of the HAuCl4 aqueous solution is 0.03%; the concentration of the trisodium citrate aqueous solution is 2%; the ratio of the HAuCl4 aqueous solution to the trisodium citrate aqueous solution is 600mL:14.2mL; and the particle size of the colloidal gold is 30~70nm.
[0013] More optimally, in the different gold label solutions, the 6 different antibodies are anti-human hemoglobin monoclonal antibody, transferrin monoclonal antibody, calprotectin monoclonal antibody, rotavirus monoclonal antibody, adenovirus monoclonal antibody, and norovirus monoclonal antibody; The ratio of colloidal gold to 6 different antibodies and gold-labeled working solution is 10 mL: 80-100 μg: 1 mL; the final concentration of BSA is 0.05%.
[0014] More preferably, the preparation method of the nitrocellulose membrane is: S7.1: Anti-human hemoglobin monoclonal antibody, transferrin monoclonal antibody, calprotectin monoclonal antibody, rotavirus monoclonal antibody, adenovirus monoclonal antibody, and norovirus monoclonal antibody were diluted with 0.01M PBS to obtain T1 solution, T2 solution, T3 solution, T4 solution, T5 solution, and T6 solution, respectively; goat anti-mouse IgG antibody was diluted with 0.01M PBS to obtain C solution; S7.2: T1 solution, T2 solution, T3 solution, T4 solution, T5 solution, and T6 solution were mixed with C solution respectively, and streaked on nitrocellulose membrane according to the streaking parameters of liquid output of 0.8-1μL / cm and speed of 80-100mm / s. The membrane was dried in an oven at 37°C for 8 hours to obtain 6 different nitrocellulose membranes coated with T1 line, T2 line, T3 line, T4 line, T5 line, T6 line and quality control line C.
[0015] More optimally, the T1 line, T2 line, T3 line, T4 line, T5 line, and T6 line on the six different nitrocellulose membranes are respectively close to one end of the sample pad, and the C line is close to one end of the annular absorbent pad; The concentration of the T1 solution is 1.0-1.2 mg / mL; the concentration of the T2 solution is 1.2-1.5 mg / mL; the concentration of the T3 solution is 1.0-1.2 mg / mL; the concentration of the T4 solution is 1.2-1.5 mg / mL; the concentration of the T5 solution is 1.2-1.5 mg / mL; the concentration of the T6 solution is 1.2-1.5 mg / mL; and the concentration of the C solution is 2.5-3.0 mg / mL.
[0016] A relatively optimized feces detection bag is prepared by the method for preparing a feces detection bag.
[0017] More optimally, the stool test bag is used for integrated detection of six items in stool, including occult blood, transferrin, calprotectin, rotavirus, adenovirus, and norovirus.
[0018] The present invention can assemble gold-labeled capture antibodies of multiple biomarkers as probes in the sample reaction pipeline to achieve a detection combination of multiple biomarkers, and can simultaneously detect fecal occult blood, transferrin, calprotectin, rotavirus, adenovirus, and norovirus. It can achieve one-time detection to simultaneously obtain the detection results of multiple detection indicators such as fecal occult blood, transferrin, calprotectin, rotavirus, adenovirus, and norovirus, thereby reducing the difficulty of operation for the detector and effectively improving the detection efficiency.
[0019] The present invention fixes the colloidal gold-labeled antibody in the sample channel, which is different from the traditional treatment method on the market of fixing the colloidal gold-labeled antibody on a glass fiber membrane to make a gold-labeled pad (colloidal gold binding pad). The physical reaction time between the target antigen to be tested in the feces and the gold-labeled antibody in the sample channel is prolonged, and the gold-labeled antibody fully reacts and combines with the target antigen to be tested, which can greatly improve the sensitivity of the entire test strip reaction system.
[0020] Reaction principle: the feces sample is carried by the feces filter 22, and a feces dissolving agent is added to soften the feces sample, and the target substance to be tested is dissolved in the feces dissolving agent; the spring button 8 is pressed to pierce the sealing sheet 16 at the end 17 of the sample pipe A, and the vacuum negative pressure chamber 5 and the sample reaction pipe are opened. The feces solution flows to the sample pipe due to the action of atmospheric pressure, and passes through the sample pad 1 for a second time to filter out fine feces residues, and is chromatographed onto the nitrocellulose membrane 2, thereby starting the test strip immunochromatography reaction.
[0021] If there are hemoglobin, transferrin, calprotectin, rotavirus, adenovirus, and norovirus antigens in the feces, they will be combined with the gold-labeled antibody probe 20 in the sample pipeline, namely, gold-labeled anti-human hemoglobin monoclonal antibody, gold-labeled transferrin monoclonal antibody, gold-labeled calprotectin monoclonal antibody, gold-labeled rotavirus monoclonal antibody, gold-labeled adenovirus monoclonal antibody, and gold-labeled norovirus monoclonal antibody, to form a target antigen-gold-labeled antibody complex; the target antigen-gold-labeled antibody complex continues to flow along the sample pipeline under the action of atmospheric pressure. When the liquid flows through the pipeline and is chromatographed on the nitrocellulose membrane area 2 of the six test strips, it reacts with the antibodies on T1-T6 respectively: the anti-human hemoglobin monoclonal antibody on the T1 line, the transferrin monoclonal antibody on the T2 line, the calprotectin monoclonal antibody on the T3 line, the rotavirus monoclonal antibody on the T4 line, the adenovirus monoclonal antibody on the T5 line, and the norovirus monoclonal antibody on the T6 line, forming a coated antibody-target antigen-gold-labeled antibody complex; and is fixed on the nitrocellulose membrane area 2 to form a color band visible to the naked eye.
[0022] Regardless of whether the marker to be tested exists in the stool sample, a colored band will appear in the quality control line C area. The colored band that appears in the quality control line C area is the standard for judging whether there is enough specimen and whether the chromatography process is normal. It also serves as the internal control standard of the reagent. If the quality control line C does not appear, the experiment is invalid. There are T-shaped buffer cabins 13 structures on both sides of the nitrocellulose membrane 2 area, which play a role in diversion and buffering to prevent the liquid from flowing too fast. The annular absorbent pad 3 on the vacuum negative pressure cabin 5 can absorb the fecal solution to prevent backflow. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 An exploded view of a feces detection bag provided in some embodiments of the present invention; Figure 2 A surface A of a vacuum negative pressure chamber upper cover of a feces detection bag provided in some embodiments of the present invention; Figure 3 A vacuum negative pressure chamber upper cover B of a feces detection bag provided in some embodiments of the present invention; Figure 4 A lower cover B of a vacuum negative pressure chamber of a feces detection bag provided in some embodiments of the present invention; Figure 5 A lower cover A of a vacuum negative pressure chamber of a feces detection bag provided in some embodiments of the present invention; Figure 6 A schematic diagram of a test strip assembly of a stool detection bag provided in some embodiments of the present invention; Figure 7 A schematic diagram of the assembly of a test strip assembly of a stool detection bag and a lower cover of a vacuum negative pressure chamber provided in some embodiments of the present invention; Figure 8 A schematic diagram of the connection between a test strip assembly of a stool test bag and a lower cover of a vacuum negative pressure chamber provided in some embodiments of the present invention and a sample pipe after the assembly; Fig. 9 A schematic diagram of the connection between a waterproof bag, a feces filter and a sample pipe of a feces detection bag provided in some embodiments of the present invention; Fig.10 From left to right are the analytical sensitivity results of fecal occult blood clinical positive samples, fecal occult blood 50-fold diluted samples, fecal occult blood 100-fold diluted samples, and normal human feces samples; Fig.11 From left to right, the analytical sensitivity results of transferrin 80-fold diluted samples, transferrin 40-fold diluted samples, transferrin clinically positive samples, and normal human feces samples; Fig.12 From left to right, the analytical sensitivity results of 60-fold diluted calprotectin samples, 30-fold diluted calprotectin samples, calprotectin clinically positive samples, and normal human stool samples; Fig.13 From left to right, the analytical sensitivity results of rotavirus 80-fold diluted samples, rotavirus 40-fold diluted samples, rotavirus clinically positive samples, and normal human stool samples; Fig.14 From left to right are the analytical sensitivity results of adenovirus clinical positive samples, adenovirus 40-fold diluted samples, adenovirus 80-fold diluted samples, and normal human feces samples; Fig.15 From left to right are the analytical sensitivity results of Norovirus 100-fold diluted samples, Norovirus 50-fold diluted samples, Norovirus clinically positive samples, and normal human stool samples; In the figure: 1-sample pad, 2-nitrocellulose membrane, 3-absorbent pad, 4-PVC bottom plate, 5-vacuum negative pressure chamber, 6-observation window, 7-spring button hole, 8-spring button, 9-rubber ring groove, 10-vacuum hole, 11-plug-in protrusion, 12-slot, 13-T-shaped buffer chamber, 14-test strip fixing groove, 15-rubber ring, 16-sealing sheet, 17-sample pipe A end, 18-outer waterproof bag, 19-inner waterproof bag, 20-gold-labeled antibody probe, 21-sample pipe B end, 22-feces filter. DETAILED DESCRIPTION
[0024] 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.
[0025] The specific embodiments of the present invention are as follows: Example 1: Preparation and application of feces detection bag: Part I: Preparation of sample tubes containing 6 different gold-labeled antibody probes 20: S1.1: Boil 600 mL of 0.03% HAuCl4 aqueous solution, add 14.2 mL of 2.0% trisodium citrate aqueous solution, the solution first changes from colorless to black, then to purple-red, continue boiling for 5 minutes, cool, add water to restore to the initial volume, and obtain colloidal gold; wherein the prepared colloidal gold particles have a size of 50 nm; scan the highest absorption peak of the colloidal gold particles by ultraviolet spectrophotometry, and the highest absorption peak is at 520-550 nm; S1.2: Prepare 6 different gold label solutions: S1.2-1: Add 0.2M K2CO3 to 10mL colloidal gold, adjust the pH to 7.4, add 100μg anti-human hemoglobin monoclonal antibody under stirring, and react at room temperature for 30 minutes; after the reaction, add 5% BSA to make the final concentration of BSA in the solution 0.05%, and react at room temperature for 20 minutes; centrifuge at 10000rpm to remove the supernatant and take the sediment, add 1mL gold label working solution 1 (the pH of gold label working solution 1 is 9.00; including 0.2M borate buffer, 2% glucose, 0.5% PEG2000, 0.01% TWEEN20, 0.25% BSA), and obtain a colloidal gold-labeled anti-human hemoglobin gold label solution; S1.2-2: Add 0.2M K2CO3 to 10mL colloidal gold, adjust the pH to 7.4, add 80μg transferrin monoclonal antibody under stirring, and react at room temperature for 30 minutes; after the reaction, add 5% BSA to make the final concentration of BSA in the solution 0.05%, and react at room temperature for 20 minutes; centrifuge at 10000rpm to remove the supernatant and take the sediment, add 1mL gold label working solution 2 (the pH of gold label working solution 2 is 8.00; including 0.2M borate buffer, 2% glucose, 0.5% PEG2000, 0.01% TX100, 0.25% BSA), and obtain a colloidal gold-labeled transferrin gold label solution; S1.2-3: Add 0.2M K2CO3 to 10mL colloidal gold, adjust the pH to 7.4, add 90μg of calprotectin monoclonal antibody under stirring, and react at room temperature for 30 minutes; after the reaction, add 5% BSA to make the final concentration of BSA in the solution 0.05%, and react at room temperature for 20 minutes; centrifuge at 10000rpm to remove the supernatant and take the sediment, add 1mL gold label working solution 3 (the pH of gold label working solution 3 is 9.00; including 0.2M borate buffer, 2% glucose, 0.5% PEG2000, 0.01% TX100, 0.25% BSA), and obtain colloidal gold labeled calprotectin gold label solution; S1.2-4: Add 0.2M K2CO3 to 10mL colloidal gold, adjust the pH to 7.4, add 100μg rotavirus monoclonal antibody under stirring, and react at room temperature for 30 minutes; after the reaction, add 5% BSA to make the final concentration of BSA in the solution 0.05%, and react at room temperature for 20 minutes; centrifuge at 10000rpm to remove the supernatant and take the sediment, add 1mL gold label working solution 4 (the pH of gold label working solution 4 is 9.00; including 0.2M borate buffer, 2% glucose, 0.5% PEG2000, 0.01% TWEEN20, 0.25% BSA), and obtain a colloidal gold-labeled rotavirus gold label solution; S1.2-5: Add 0.2M K2CO3 to 10mL colloidal gold, adjust the pH to 7.4, add 80μg adenovirus monoclonal antibody under stirring, and react at room temperature for 30 minutes; after the reaction, add 5% BSA to make the final concentration of BSA in the solution 0.05%, and react at room temperature for 20 minutes; centrifuge at 10000rpm to remove the supernatant and take the sediment, add 1mL gold label working solution 5 (the pH of gold label working solution 5 is 9.00; including 0.2M borate buffer, 2% glucose, 0.5% PEG2000, 0.01% TX100, 0.5% BSA), and obtain a colloidal gold-labeled adenovirus gold label solution; S1.2-6: Add 0.2M K2CO3 to 10mL of colloidal gold, adjust the pH to 7.4, add 90μg of norovirus monoclonal antibody under stirring, and react at room temperature for 30 minutes; after the reaction, add 5% BSA to make the final concentration of BSA in the solution 0.05%, and react at room temperature for 20 minutes; centrifuge at 10000rpm to remove the supernatant and take the sediment, add 1mL of gold label working solution 6 (the pH of gold label working solution 6 is 8.00; including 0.2M borate buffer, 2% glucose, 0.5% PEG2000, 0.01% TX100, 0.5% BSA), and obtain a colloidal gold-labeled norovirus gold label solution; S1.3: Preparation of sample tubes containing 6 different gold-labeled antibody probes 20: Take 6 sample pipes, and respectively push 100 μL of colloidal gold-labeled anti-human hemoglobin gold-labeled solution, 100 μL of colloidal gold-labeled transferrin gold-labeled solution, 100 μL of colloidal gold-labeled calprotectin gold-labeled solution, 100 μL of colloidal gold-labeled rotavirus gold-labeled solution, 100 μL of colloidal gold-labeled adenovirus gold-labeled solution, and 100 μL of colloidal gold-labeled norovirus gold-labeled solution to the inner pipe wall at the middle position of the sample pipe with a syringe, put it in a 37° C. oven, dry it for more than 8 hours, add a desiccant and seal it for storage; obtain a sample pipe containing 6 different gold-labeled antibody probes 20; Part II: Preparation of nitrocellulose membrane 2: S2.1: Anti-human hemoglobin monoclonal antibody, transferrin monoclonal antibody, calprotectin monoclonal antibody, rotavirus monoclonal antibody, adenovirus monoclonal antibody, and norovirus monoclonal antibody were diluted with 0.01M PBS to obtain T1 solution, T2 solution, T3 solution, T4 solution, T5 solution, and T6 solution, respectively; goat anti-mouse IgG antibody was diluted with 0.01M PBS to obtain C solution; S2.2: T1 solution, T2 solution, T3 solution, T4 solution, T5 solution, and T6 solution were respectively coated with nitrocellulose membrane with the C solution at the film-stripping parameters of 1 μL / cm and 80 mm / s, and dried in a 37°C oven for 8 hours to obtain 6 different nitrocellulose membranes 2; Among them, the T1 line, T2 line, T3 line, T4 line, T5 line, and T6 line on the six different nitrocellulose membranes 2 are respectively close to one end of the sample pad 1, and the C line is close to one end of the annular absorbent pad 3; T1 solution: dilute anti-human hemoglobin to 1.0 mg / mL with 0.01 M PBS at pH 7.4; T2 solution: dilute transferrin monoclonal antibody to 1.2 mg / mL with 0.01 M PBS at pH 7.4; T3 solution: dilute calprotectin monoclonal antibody to 1.0 mg / mL in 0.01 M PBS, pH 7.4; T4 solution: dilute the rotavirus monoclonal antibody to 1.5 mg / mL with 0.01 M PBS at pH 7.4; T5 solution: dilute the adenovirus monoclonal antibody to 1.2 mg / mL with 0.01 M PBS at pH 7.4; T6 solution: dilute the norovirus monoclonal antibody to 1.2 mg / mL with 0.01 M PBS at pH 7.4; Solution C: Dilute goat anti-mouse IgG antibody to 3.0 mg / mL in 0.01 M PBS, pH 7.4.
[0026] Part 3: Assembly of the stool test bag: Step 1: Preparation of test strip assembly: like Figure 1-9 The stool detection bag comprises a sample pad 1, a nitrocellulose membrane 2, a ring-shaped absorbent pad 3, a PVC bottom plate 4, a vacuum negative pressure chamber 5, an observation window 6, a spring button hole 7, a spring button 8, a rubber ring groove 9, a vacuum hole 10, a plug-in protrusion 11, a slot 12, a T-shaped buffer chamber 13, a test strip fixing groove 14, a rubber ring 15, a sealing sheet 16, a sample pipe A end 17, an outer waterproof bag 18, an inner waterproof bag 19, a gold-labeled antibody probe 20, a sample pipe B end 21, and a stool filter 22; S1.1: 6 different nitrocellulose membranes 2 coated with detection lines and quality control lines are respectively pasted in the middle of the PVC bottom plate 4; a sample pad 1 (glass fiber membrane model: Ahistrom8964) is pasted on the left side of the PVC bottom plate 4, and a strip cutter is used to cut 6 different test strips with a width of 10 mm. The schematic diagram of the structure of the test strip is shown in Figure 6 ; S1.2: 6 different test strips are evenly arranged on the annular absorbent pad 3; the annular absorbent pad 3 is attached to the right side of the PVC bottom plate 4 of the test strip, and the nitrocellulose membrane 2 in the test strip overlaps the annular absorbent pad 3 by 2 mm; a test strip assembly is obtained, see Figure 6 ; Among them, the model of the annular absorbent pad 3 is H-1; Step 2: Assembling the test strip assembly and the lower cover of the vacuum negative pressure chamber: S2.1: Take the vacuum negative pressure chamber lower cover provided with the vacuum negative pressure chamber 5, the slot 12, the T-shaped buffer chamber 13, the test strip fixing groove 14, and the rubber ring groove 9, and set it aside; S2.2: The annular absorbent pad 3 of the test strip assembly is loaded into the vacuum negative pressure chamber 5 on the lower cover B of the vacuum negative pressure chamber; wherein, the 6 different test strips of the test strip assembly are correspondingly arranged in the test strip fixing grooves 14, and a pair of T-row buffer chambers 13 are respectively arranged beside the test strip fixing grooves 14, a total of 6 pairs, and the 12 chambers play a role of diversion and buffering to prevent the liquid from flowing too fast; at the same time, the T-row buffer chambers 13 correspond to both sides of the nitrocellulose membrane 2 area of the 6 different test strips, and a semi-finished product A is obtained; Step 3: Preparation of waterproof bag: The inner waterproof bag 19 is connected to the feces filter 22, and the outer waterproof bag 18 is embedded outside the inner waterproof bag 19 to form a space that can carry and contain feces solution; 6 sample pipes containing different gold-labeled antibody probes 20 are fixed between the outer waterproof bag 18 and the inner waterproof bag 19; the end B 21 of the sample pipe extends into the lower part of the feces filter 22, and the sample pipe B ends 21 of ... Fig. 9 ; The sample pipe A end 17 is heat-sealed using a sealing sheet 16 to obtain a waterproof bag; Step 4: Assemble the waterproof bag and the lower cover of the vacuum negative pressure cabin in semi-finished product A: Insert the end 17 of the sample pipe A in the waterproof bag into the rubber ring 15, and fix it in the position of the upper rubber ring groove 9 of the test strip fixing groove 14 of the lower cover of the vacuum negative pressure chamber in the semi-finished product A to obtain the semi-finished product B; wherein, the 6 different sample pipes should correspond to the items of the 6 different test strips in the test strip fixing groove 14 according to the item types of the gold-labeled antibody probe 20 on the inner wall of the pipe; Step 5: Assembly of the upper cover of the vacuum negative pressure chamber and the lower cover of the vacuum negative pressure chamber in semi-finished product B: S5.1: Take the upper cover of the vacuum negative pressure chamber provided with the observation window 6, the spring button hole 7, the spring button 8, the vacuum hole 10, and the plug-in protrusion 11 and set it aside; S5.2: 24 groups of plug-in protrusions 11 are arranged on the surface B of the upper cover of the vacuum negative pressure chamber, which correspond one by one to the 24 groups of slots 12 on the surface B of the lower cover of the vacuum negative pressure chamber in the semi-finished product B, and the plug-in protrusions 11 are plugged and matched with the slots 12; S5.2: Vacuuming and sealing: Connect the vacuum hole 10 to the vacuum negative pressure chamber 5 near the position marked "C". The vacuum hole 10 can be used to evacuate the entire vacuum negative pressure chamber to form a negative pressure state; after all structures are assembled, use a vacuum packaging bag to evacuate and seal the vacuum negative pressure chamber; cover the lower cover A side of the vacuum negative pressure chamber, see Figure 5, affix double-sided adhesive strips to fix the stool test bag on the outer wall of the toilet during testing. Then put in the desiccant, put it into an aluminum foil bag and seal it; get the stool test bag; Among them, the upper cover A of the vacuum negative pressure cabin is shown in Figure 2 There are 6 observation windows, and the project names are printed on the upper part of the observation windows to distinguish 6 different projects: fecal occult blood FOB, transferrin TRF, calprotectin CAL, rotavirus (RV), adenovirus ADV, and norovirus NV, which correspond to the test strip detection lines T1, T2, T3, T4, T5, and T6 respectively. The corresponding gold-labeled antibody probes on the inner wall of the sample channel are gold-labeled anti-human hemoglobin antibody, gold-labeled transferrin antibody, gold-labeled calprotectin antibody, gold-labeled rotavirus antibody, gold-labeled adenovirus antibody, and gold-labeled norovirus antibody; Each observation window 6 is provided with "T" and "C" marks; there are 6 spring buttons 8, i.e. 6 start buttons, at the position near the "T" mark; pressing this button can pierce the sealing sheet 16 at the end 17 of the sample pipe A, open the vacuum negative pressure chamber 5 and the sample reaction pipe, and start the test strip reaction; during the start-up process, the spring button 8 is pressed into the spring button hole 7; near the "C" position there is a vacuum hole 10 connected to the vacuum negative pressure chamber 5, through which the entire vacuum negative pressure chamber can be evacuated to form a negative pressure state; after all the structures are assembled, the vacuum negative pressure chamber is evacuated and sealed with a vacuum packaging bag, and a double-sided adhesive tape is affixed to the A side of the lower cover of the vacuum negative pressure chamber, such as Figure 5 As shown, during the test, the feces detection bag is fixed on the outer wall of the toilet, and then a desiccant is put in and sealed in an aluminum foil bag.
[0027] Part 4: Application of fecal test bags: Step 1: Sample preparation: Prepare clinically positive fecal occult blood samples, 50-fold diluted fecal occult blood samples, 100-fold diluted fecal occult blood samples, and normal human stool; clinically positive transferrin samples, 40-fold diluted transferrin samples, 80-fold diluted transferrin samples, and normal human stool; clinically positive calprotectin samples, 30-fold diluted calprotectin samples, 60-fold diluted calprotectin samples, and normal human stool; clinically positive rotavirus samples, 40-fold diluted rotavirus samples, 80-fold diluted rotavirus samples, and normal human stool; clinically positive adenovirus samples, 40-fold diluted adenovirus samples, 80-fold diluted adenovirus samples, and normal human stool; clinically positive norovirus samples, 50-fold diluted norovirus samples, 100-fold diluted norovirus samples, and normal human stool; Step 2: Sample testing: The prepared stool test bag is used to test the sample in step 1; S2.1: Lift the toilet seat, open the aluminum foil bag, take out the stool test bag, put the test bag into the toilet, tear off the adhesive strip on the back of the vacuum negative pressure chamber 5, stick it on the outer wall of the toilet, press it with your hands to fit it, and cover the toilet seat; S2.2: Place the sample prepared in step 1 into a stool test bag, add a stool dissolving agent, and wait for 5 minutes to allow the stool sample to fully dissolve; S2.3: After 5 minutes, press the six spring buttons 8 respectively, and the spring buttons 8 will pierce the sealing sheet 16 at the end 17 of the sample pipe A to start the reaction; S2.4: Wait for 20 minutes to determine the result through the observation window 6; Step 3: Result judgment: Taking the occult blood project as an example, other projects are similar; positive: a red line appears at the detection line T1 line and the quality control line C position in the result observation window, indicating that human hemoglobin (fecal occult blood) exists in the sample; negative: only a red line appears at the quality control line C position, indicating that there is no human hemoglobin (fecal occult blood) in the sample; invalid: no red line appears at the quality control line position, indicating that the result is invalid and should be retried.
[0028] Table 1
[0029] (Note: + represents the presence of stripes, - represents the absence of stripes, and from + to ++++++ represents the gradual strengthening of the stripe color); Conclusion: From Table 1 above, we can see that when the dilution ratio of clinical positive samples of human hemoglobin (fecal occult blood) is 1:100, the T1 line band can still be observed, indicating that the analytical sensitivity of detecting human hemoglobin (fecal occult blood) can reach 1:100. Fig.10 .
[0030] When the dilution ratio of transferrin clinical positive samples was 1:80, the T2 line band could still be observed, indicating that the analytical sensitivity of transferrin detection could reach 1:80; the results are as follows Fig.11 .
[0031] When the dilution ratio of the clinical positive sample of calprotectin was 1:60, the T3 line band could still be observed, indicating that the analytical sensitivity of calprotectin detection could reach 1:60; the results are as follows Fig.12 .
[0032] When the dilution ratio of rotavirus clinical positive samples was 1:80, the T4 line band could still be observed, indicating that the analytical sensitivity of rotavirus detection can reach 1:80; the results are as follows Fig.13 .
[0033] When the dilution ratio of adenovirus clinical positive samples was 1:80, the T5 line band could still be observed, indicating that the analytical sensitivity of adenovirus detection could reach 1:80; the results are as follows Fig.14 .
[0034] When the dilution ratio of clinically positive samples of norovirus was 1:100, the band of T6 line could still be observed, indicating that the analytical sensitivity of detecting norovirus could reach 1:100; the results are as follows Fig.15 .
[0035] In summary, the stool detection bag of the present invention has a high analytical sensitivity for clinical positive samples of fecal occult blood, transferrin, calprotectin, rotavirus, adenovirus, and norovirus.
[0036] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.
[0037] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for preparing a feces detection bag, characterized in that: The steps include: Step 1: Preparation of test strip assembly: S1.1: Attach 6 different nitrocellulose membranes (2) to the middle of the PVC base plate (4); attach the sample pad (1) to the left side of the PVC base plate (4), and cut into strips to obtain 6 different test strips; S1.2: 6 different test strips are evenly arranged on the annular absorbent pad (3); the right side of the PVC base plate (4) in the test strip is attached to the annular absorbent pad (3), and the nitrocellulose membrane (2) in the test strip overlaps the annular absorbent pad (3) by 2 mm; and a test strip assembly is obtained; Step 2: Assembling the test strip assembly and the lower cover of the vacuum negative pressure chamber: S2.1: Take the lower cover of the vacuum negative pressure chamber provided with the vacuum negative pressure chamber (5), the slot (12), the T-shaped buffer chamber (13), the test strip fixing groove (14), and the rubber ring groove (9) and set aside; S2.2: The annular absorbent pad (3) of the test strip assembly is partially loaded into the vacuum negative pressure chamber (5) on the lower cover B of the vacuum negative pressure chamber; wherein the six different test strips of the test strip assembly are correspondingly arranged in the test strip fixing grooves (14), and a pair of T-row buffer chambers (13) are respectively arranged next to the test strip fixing grooves (14), and the T-row buffer chambers (13) are arranged on both sides of the nitrocellulose membrane (2) areas of the six different test strips; and a semi-finished product A is obtained; Step 3: Preparation of waterproof bag: The inner waterproof bag (19) is connected to the feces filter (22), and the outer waterproof bag (18) is embedded outside the inner waterproof bag (19) to form a space capable of carrying and containing feces solution; 6 sample pipes containing different gold-labeled antibody probes (20) are fixed between the outer waterproof bag (18) and the inner waterproof bag (19); the B end (21) of the sample pipe is extended into the lower part of the feces filter (22); the A end (17) of the sample pipe is heat-sealed using a sealing sheet (16) to obtain a waterproof bag; Step 4: Assemble the waterproof bag and the lower cover of the vacuum negative pressure cabin in semi-finished product A: Insert the sample pipe A end (17) in the waterproof bag into the rubber ring (15), fix it in the test strip fixing groove (14) of the lower cover of the vacuum negative pressure chamber in the semi-finished product A, and connect it with the rubber ring groove (9) on the test strip fixing groove (14); obtain the semi-finished product B; Step 5: Assembly of the upper cover of the vacuum negative pressure chamber and the lower cover of the vacuum negative pressure chamber in semi-finished product B: S5.1: Take the upper cover of the vacuum negative pressure chamber provided with the observation window (6), the spring button hole (7), the spring button (8), the vacuum hole (10), and the plug-in protrusion (11) and set aside; S5.2: 24 groups of plug-in protrusions (11) are arranged on the surface B of the upper cover of the vacuum negative pressure chamber, and plugged and assembled with the 24 groups of slots (12) of the lower cover of the vacuum negative pressure chamber in the semi-finished product B in a one-to-one correspondence; vacuuming is performed; and a feces detection bag is obtained.
2. The method for preparing a stool detection bag according to claim 1, characterized in that: In step 1, the six different nitrocellulose membranes (2) corresponding to the six different test strips are coated with six different detection lines, namely, T1 line, T2 line, T3 line, T4 line, T5 line, T6 line, and a quality control line C; The quality control line C is coated with goat anti-mouse IgG antibody; the 6 different detection lines are respectively coated with anti-human hemoglobin monoclonal antibody T1, transferrin monoclonal antibody T2, calprotectin monoclonal antibody T3, rotavirus monoclonal antibody T4, adenovirus monoclonal antibody T5, and norovirus monoclonal antibody T6; The six different test strips correspond to the detection items of the six sample pipes containing different gold-labeled antibody probes (20) one by one; the corresponding different gold-labeled antibody probes (20) are gold-labeled anti-human hemoglobin monoclonal antibody, gold-labeled transferrin monoclonal antibody, gold-labeled calprotectin monoclonal antibody, gold-labeled rotavirus monoclonal antibody, gold-labeled adenovirus monoclonal antibody, and gold-labeled norovirus monoclonal antibody; The corresponding test items for the 6 different test lines are fecal occult blood, transferrin, calprotectin, rotavirus, adenovirus, and norovirus.
3. The method for preparing a feces detection bag according to claim 1, characterized in that: The observation windows (6) are arranged on the A surface of the upper cover of the vacuum negative pressure chamber, with a total of 6, for correspondingly observing 6 test items; each observation window (6) is provided with a "T" and a "C" mark; there are 6 spring buttons (8) near the "T" mark, i.e. 6 start buttons; they are used to pierce the sealing sheet (16) at the A end (17) of the sample pipeline, open the vacuum negative pressure chamber (5) and the sample reaction pipeline, and start the test strip reaction; when starting, the spring button (8) is pressed into the spring button hole (7); The vacuum hole (10) is used to evacuate the vacuum negative pressure cabin (5) to form a negative pressure state.
4. The method for preparing a stool detection bag according to claim 1, characterized in that: The preparation method of the gold-labeled antibody probe (20) is as follows: S6.1: Boil the HAuCl4 aqueous solution, add trisodium citrate aqueous solution, and continue boiling for 4 to 6 minutes after the solution changes to purple-red, cool, and add water to restore the initial volume to obtain colloidal gold; S6.2: Add K2CO3 to colloidal gold, adjust the pH to 7.4-7.6, add 6 different antibodies under stirring, react at room temperature for 30-35 minutes, add BSA solution after the reaction, react at room temperature for 20-25 minutes, centrifuge, collect the supernatant and add gold label working solution to obtain 6 different gold label solutions; S6.3: Add different gold-labeled solutions into the sample tube and dry them in a 37°C oven for ≥8 hours to obtain 6 different gold-labeled antibody probes (20).
5. The method for preparing a stool detection bag according to claim 4, characterized in that: In the colloidal gold, the concentration of the HAuCl4 aqueous solution is 0.03%; the concentration of the trisodium citrate aqueous solution is 2%; the ratio of the HAuCl4 aqueous solution to the trisodium citrate aqueous solution is 600mL:14.2mL; and the particle size of the colloidal gold is 30-70nm.
6. The method for preparing a stool detection bag according to claim 4, characterized in that: In the different gold label solutions, the six different antibodies are anti-human hemoglobin monoclonal antibody, transferrin monoclonal antibody, calprotectin monoclonal antibody, rotavirus monoclonal antibody, adenovirus monoclonal antibody, and norovirus monoclonal antibody; The ratio of colloidal gold to 6 different antibodies and gold-labeled working solution is 10 mL: 80-100 μg: 1 mL; the final concentration of BSA is 0.05%.
7. The method for preparing a stool detection bag according to claim 1, characterized in that: The preparation method of the nitrocellulose membrane (2) is as follows: S7.1: Anti-human hemoglobin monoclonal antibody, transferrin monoclonal antibody, calprotectin monoclonal antibody, rotavirus monoclonal antibody, adenovirus monoclonal antibody, and norovirus monoclonal antibody were diluted with 0.01M PBS to obtain T1 solution, T2 solution, T3 solution, T4 solution, T5 solution, and T6 solution, respectively; Goat anti-mouse IgG antibody was diluted with 0.01 M PBS to obtain solution C; S7.2: T1 solution, T2 solution, T3 solution, T4 solution, T5 solution, and T6 solution are mixed with C solution respectively, and streaked on nitrocellulose membrane according to the parameters of liquid output of 0.8~1μL / cm and membrane speed of 80~100mm / s, and dried in an oven at 37℃ for 8 hours to obtain 6 different nitrocellulose membranes coated with T1 line, T2 line, T3 line, T4 line, T5 line, T6 line and quality control line C (2).
8. The method for preparing a stool detection bag according to claim 7, characterized in that: The T1 line, T2 line, T3 line, T4 line, T5 line, and T6 line on the six different nitrocellulose membranes (2) are respectively close to one end of the sample pad (1), and the C line is close to one end of the annular absorbent pad (3); The concentration of the T1 solution is 1.0-1.2 mg / mL; the concentration of the T2 solution is 1.2-1.5 mg / mL; the concentration of the T3 solution is 1.0-1.2 mg / mL; the concentration of the T4 solution is 1.2-1.5 mg / mL; the concentration of the T5 solution is 1.2-1.5 mg / mL; the concentration of the T6 solution is 1.2-1.5 mg / mL; and the concentration of the C solution is 2.5-3.0 mg / mL.
9. A feces detection bag prepared according to the method for preparing a feces detection bag according to any one of claims 1 to 8.
10. An application of a feces detection bag, characterized in that: The stool testing bag as described in claim 9 is used for integrated detection of six items in stool: fecal occult blood, transferrin, calprotectin, rotavirus, adenovirus, and norovirus.