Clostridium difficile triple detection kit based on colloidal gold immunochromatography and preparation method of clostridium difficile triple detection kit

Abstract

The invention relates to a clostridium difficile triple detection kit based on colloidal gold immunochromatography and a preparation method of the clostridium difficile triple detection kit, and belongs to the technical field of immunodetection. The clostridium difficile triple detection kit provided by the invention comprises a clostridium difficile triple detection card and a sample diluent, and the clostridium difficile triple detection card is formed by arranging and connecting three detection test strips in parallel; the detection test strip comprises a clostridium difficile toxin A detection test strip, a clostridium difficile toxin B detection test strip and a clostridium difficile glutamate dehydrogenase detection test strip. By using the clostridium difficile triple detection kit provided by the invention, three markers related to clostridium difficile can be detected at the same time, the detection limit of clostridium difficile toxin A is 0.25 ng / mL, the detection limit of clostridium difficile toxin B is 0.5 ng / mL, and the detection limit of clostridium difficile glutamate dehydrogenase is 0.5 ng / mL, so that the clostridium difficile triple detection kit has the advantages of good sensitivity, simplicity in operation, high sensitivity, high sensitivity, high sensitivity and the like. The method is suitable for large-scale popularization screening.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay detection, and in particular to a Clostridium difficile triple detection kit based on colloidal gold immunochromatography and a preparation method thereof. Background Art

[0002] Clostridium difficile, a Gram-positive anaerobic bacillus, is one of the main pathogens that cause antibiotic-associated diarrhea and pseudomembranous colitis. Its pathogenic mechanism is mainly through the production of toxin A (enterotoxin) and toxin B (cytotoxin), which can damage the intestinal mucosa, leading to severe inflammatory reactions, diarrhea, abdominal pain and even dehydration. What is more serious is that Clostridium difficile infection often occurs in patients who have received broad-spectrum antibiotics. While these antibiotics kill harmful bacteria, they also destroy the balance of normal flora in the intestines, providing a favorable environment for the reproduction of Clostridium difficile. Once the infection occurs, not only is the course of the disease long and the recurrence rate high, it may also cause life-threatening complications such as sepsis and toxic megacolon, posing a serious threat to the patient's health.

[0003] Given the high pathogenicity of C. difficile and its susceptibility to recurrence after antibiotic treatment, timely and accurate diagnosis is crucial to controlling the disease, reducing complications, and guiding treatment strategies. Early detection of C. difficile infection can quickly adjust the antibiotic regimen and avoid unnecessary use of broad-spectrum antibiotics, thereby alleviating patient suffering, shortening hospital stays, and reducing medical costs. In addition, timely detection and isolation of infected persons is also one of the key measures to prevent the spread of C. difficile in medical institutions. Therefore, the development of efficient and sensitive detection methods is of great significance to improving the clinical management of C. difficile infection and ensuring patient safety.

[0004] At present, the detection methods for Clostridium difficile are mainly divided into three categories: (1) Traditional bacterial culture method: Cultivate Clostridium difficile under anaerobic conditions in a specific culture medium, observe its growth and identify it. Although this method is classic, it is time-consuming and usually takes 48 hours or even longer to obtain results. It also requires high anaerobic conditions in the laboratory, which limits its ability to make rapid diagnoses; (2) PCR molecular detection technology: Use polymerase chain reaction (PCR) to amplify Clostridium difficile-specific gene fragments, such as toxin genes tcdA and tcdB, for rapid detection. The PCR method has high sensitivity and can detect extremely low concentrations of bacterial DNA in samples, but its disadvantages are that the equipment required is expensive, the operation technology is complex, and it requires professional personnel to operate. It is not suitable for use in primary medical institutions or large-scale screening; (3) Immunodetection technology: including enzyme-linked immunosorbent assay (ELISA) and immunochromatography, etc., which indirectly confirm infection by detecting Clostridium difficile toxin proteins in stool samples. This type of method is relatively fast, but may be affected by factors such as nonspecific antibody reactions or toxin degradation, resulting in false negative results.

[0005] Therefore, there is an urgent need for a detection method that is highly sensitive, rapid, simple, and low-cost to meet clinical needs. Summary of the invention

[0006] In order to solve the above technical problems, the present invention proposes a Clostridium difficile triple test kit based on colloidal gold immunochromatography, including a Clostridium difficile triple test card and a sample diluent. The Clostridium difficile triple test card is composed of three test strips arranged in parallel and connected, the test strips include Clostridium difficile toxin A test strip, Clostridium difficile toxin B test strip and Clostridium difficile glutamate dehydrogenase test strip, and three markers related to Clostridium difficile are detected at the same time.

[0007] The first object of the present invention is to provide a Clostridium difficile triple detection kit based on colloidal gold immunochromatography, wherein the Clostridium difficile triple detection kit comprises a Clostridium difficile triple detection card and a sample diluent, wherein the Clostridium difficile triple detection card is composed of three test strips arranged in parallel and connected, wherein the test strips comprise a Clostridium difficile toxin A test strip, a Clostridium difficile toxin B test strip, and a Clostridium difficile glutamate dehydrogenase test strip, wherein the test strips are provided with a sample pad, a binding pad, a coating film, and a water-absorbing pad on the bottom plate in sequence from the sample application end, and the coating film is provided with a Clostridium difficile toxin A detection line, a Clostridium difficile toxin B detection line, and a Clostridium difficile glutamate dehydrogenase detection line, respectively, wherein:

[0008] The conjugate pad of the Clostridium difficile toxin A test strip is sprayed with a colloidal gold-labeled Clostridium difficile toxin A detection antibody complex solution, the final concentration of the Clostridium difficile toxin A detection antibody is 15-22 μg / mL, and the test line is sprayed with a coating solution containing Clostridium difficile toxin A capture antibody, and the final concentration of the Clostridium difficile toxin A capture antibody in the coating solution is 1-1.5 mg / mL;

[0009] The conjugate pad of the Clostridium difficile toxin B test strip is sprayed with a colloidal gold-labeled Clostridium difficile toxin B detection antibody complex solution, the final concentration of the Clostridium difficile toxin B detection antibody is 12-17 μg / mL, and the test line is sprayed with a coating solution containing Clostridium difficile toxin B capture antibody, and the final concentration of the Clostridium difficile toxin B capture antibody in the coating solution is 1.4-2 mg / mL;

[0010] The conjugate pad of the Clostridium difficile glutamate dehydrogenase test strip is sprayed with a colloidal gold-labeled Clostridium difficile glutamate dehydrogenase detection antibody complex solution, the final concentration of the Clostridium difficile glutamate dehydrogenase detection antibody is 14-18 μg / mL, and the detection line is sprayed with a coating solution containing Clostridium difficile glutamate dehydrogenase capture antibody, and the final concentration of the Clostridium difficile glutamate dehydrogenase capture antibody in the coating solution is 1.1-1.5 mg / mL;

[0011] The sample diluent consists of sodium chloride, Tris, disodium hydrogen phosphate, dipotassium hydrogen phosphate, bovine serum albumin, a surfactant and a preservative.

[0012] Furthermore, a quality control line is arranged on the coating film of the test strip, and a coating solution containing a quality control detection antibody is sprayed on the quality control line, and the final concentration of the quality control detection antibody in the coating solution is 0.8-1.2 mg / mL.

[0013] Furthermore, the conjugate pad is sprayed with a quality control detection antibody complex solution labeled with colloidal gold, and the final concentration of the quality control detection antibody is 8-12 μg / mL.

[0014] Furthermore, the spraying amount of the colloidal gold-labeled Clostridium difficile toxin A detection antibody resolution, the colloidal gold-labeled Clostridium difficile toxin B detection antibody resolution and the colloidal gold-labeled Clostridium difficile glutamate dehydrogenase detection antibody resolution on the conjugate pad is 3-5 μL / cm 2 .

[0015] Furthermore, the spraying amount of the solution containing Clostridium difficile toxin A capture antibody, the solution containing Clostridium difficile toxin B capture antibody and the solution containing Clostridium difficile glutamate dehydrogenase capture antibody on the detection line is 1-3 μL / cm.

[0016] Furthermore, the sample pad and the conjugate pad are pretreated, and the pretreatment is to soak the sample pad and the conjugate pad in a sample pad treatment solution and a conjugate pad treatment solution respectively;

[0017] The sample pad treatment solution includes Tris-HCl buffer, polyvinyl pyrrolidone K30, surfactant, Tween 20, preservative and heterophilic antibody blocking agent HBR, and bovine serum albumin or casein;

[0018] The binding pad treatment solution includes Tris-HCl buffer, polyvinyl pyrrolidone K30, Tween 20 and a preservative, and bovine serum albumin or casein.

[0019] Furthermore, the amount of the sample pad treatment solution is 0.05-0.1 mL / cm 2 The amount of the conjugate pad treatment solution is 0.04-0.8 mL / cm 2 .

[0020] In one embodiment of the present invention, the quality control detection antibody is chicken IgY antibody.

[0021] In one embodiment of the present invention, the quality control capture antibody is a sheep anti-chicken IgY antibody. The second object of the present invention is to provide a method for preparing the above-mentioned Clostridium difficile triple detection kit, characterized in that it comprises the following steps:

[0022] Step S1, pre-treating the sample pad and the conjugate pad;

[0023] Step S2, preparing a colloidal gold solution and a colloidal gold labeled re-solution, and labeling and re-dissolving Clostridium difficile toxin A detection antibody, Clostridium difficile toxin B detection antibody, Clostridium difficile glutamate dehydrogenase detection antibody and quality control detection antibody with colloidal gold;

[0024] Step S3, preparing a coating diluent, and coating Clostridium difficile toxin A capture antibody, Clostridium difficile toxin B capture antibody, Clostridium difficile glutamate dehydrogenase capture antibody and quality control capture antibody;

[0025] Step S4, respectively mixing the colloidal gold-labeled Clostridium difficile toxin A capture antibody resolution, the Clostridium difficile toxin B detection antibody resolution and the Clostridium difficile glutamate dehydrogenase detection antibody resolution with the colloidal gold-labeled quality control detection antibody resolution, and spraying them on the conjugate pad after mixing;

[0026] Step S5, spraying the Clostridium difficile toxin A capture antibody coating solution, the Clostridium difficile toxin B capture antibody coating solution, and the Clostridium difficile glutamate dehydrogenase capture antibody coating solution on the test line, and spraying the quality control capture antibody on the quality control line;

[0027] Step S6, assembling the corresponding sample pad, binding pad, coating membrane and absorbent pad respectively to obtain Clostridium difficile toxin A test strip, Clostridium difficile toxin B test strip and Clostridium difficile glutamate dehydrogenase test strip;

[0028] Step S7, arranging the three test strips in parallel to assemble them to obtain the Clostridium difficile triple detection kit.

[0029] Further, in step S4, the OD value of the colloidal gold labeled Clostridium difficile toxin A detection antibody reconstitution solution is 45-60, the OD value of the colloidal gold labeled Clostridium difficile toxin B detection antibody reconstitution solution is 70-100, and the OD value of the colloidal gold labeled Clostridium difficile toxin glutamate dehydrogenase detection antibody reconstitution solution is 45-70.

[0030] Furthermore, in step S4, the OD value of the quality control detection antibody labeled with colloidal gold in the mixed solution is 35-50.

[0031] Beneficial effects of the present invention:

[0032] (1) The triple detection kit provided by the present invention can simultaneously detect three key indicators: Clostridium difficile toxin A, Clostridium difficile toxin B, and Clostridium difficile glutamate dehydrogenase, providing comprehensive information support for the rapid diagnosis of Clostridium difficile infection and avoiding the problem of false positives caused by simultaneous detection of three Clostridium difficile markers on the same test strip;

[0033] (2) The present invention optimizes the concentration of the detection antibody complex solution labeled with colloidal gold and the capture antibody on the detection line, so that the detection kit can achieve high-sensitivity detection of Clostridium difficile-related antigens, with a detection limit of 0.25 ng / mL for Clostridium difficile toxin A, a detection limit of 0.5 ng / mL for Clostridium difficile toxin B, and a detection limit of 0.5 ng / mL for Clostridium difficile glutamate dehydrogenase;

[0034] (3) The triple detection kit provided by the present invention is assembled from colloidal gold immunochromatographic test strips. It is only necessary to drip the treated sample onto the sample adding end of the test strip, and the result can be judged by observing the color change of the test line. No complicated instrument operation or professional training is required, which greatly simplifies the detection process.

[0035] (4) Compared with existing methods such as PCR molecular detection or bacterial culture, the triple detection kit provided by the present invention has low production cost and is easy to operate. It is suitable for use in primary medical institutions or large-scale screening and has good cost-effectiveness.

[0036] (5) By using the sample diluent provided in the triple detection kit provided by the present invention, different types of samples can be effectively processed to ensure the accuracy and reliability of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0038] Figure 1 It is a side view of the test strip of the present invention, wherein 1 is a sample pad, 2 is a conjugate pad, 3 is a cellulose acetate membrane, 31 is a test line, 32 is a quality control line, 4 is a water absorbent pad, and 5 is a bottom plate;

[0039] Figure 2 A top view of the triple test card of the present invention after assembly, wherein A represents the Clostridium difficile toxin A test strip, B represents the Clostridium difficile toxin B test strip, GDH represents the Clostridium difficile glutamate dehydrogenase test strip, T represents the test line, C represents the quality control line, and 61, 62, and 63 correspond to the sample addition holes of the Clostridium difficile toxin A test strip, the Clostridium difficile toxin B test strip, and the Clostridium difficile glutamate dehydrogenase test strip on the triple card, respectively. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0041] The partial reagent proportions used in the following examples are as follows:

[0042] Sample pad treatment solution: 40-80 mM Tris-HCl, 0.4-0.8 wt% casein or BSA, 0.8-1.5 wt% polyvinylpyrrolidone K30, 0.8-1.5 wt% surfactant S9, 0.04-0.1 wt% Tween 20, 0.02-0.05 wt% sodium azide or ProClin300 and 0.002-0.006 wt% heterophilic antibody blocking agent HBR;

[0043] Conjugate pad treatment solution: 40-80 mM Tris-HCl, 0.4-0.8 wt% casein or BSA, 0.8-1.5 wt% polyvinylpyrrolidone K30, 0.04-0.08 wt% Tween 20, 0.02-0.06 wt% sodium azide or ProClin300;

[0044] Colloidal gold solution: comprising chloroauric acid and sodium citrate, 0.01-0.04wt% chloroauric acid, and a mass ratio of chloroauric acid to sodium citrate of 1:(1-1.5);

[0045] Colloidal gold labeling solution: 50-100 mM Tris-HCl, 0.4-0.8 wt% casein or BSA, 15-25 wt% sucrose, 4-10 wt% trehalose, 0.02-0.06 wt% sodium azide or ProClin300;

[0046] Coating diluent: 8-15mM PBS buffer, 1.5-3wt% sucrose;

[0047] Sample diluent: 35-50g / L sodium chloride, 5-7g / L Tris, 0.35-0.5g / L anhydrous disodium hydrogen phosphate, 9-12g / L BSA, 0.4-0.6g / L anhydrous dipotassium hydrogen phosphate, 0.9-1.2mL / L surfactant S22, 0.15-0.3mL / L sodium azide or ProClin300, solution pH 7.0-8.0.

[0048] Example 1: Test strip preparation and assembly

[0049] 1. Pre-treat the sample pad and conjugate pad, prepare 0.05-0.1mL / cm 2 Sample pad treatment solution and 0.04-0.08mL / cm 2 The conjugate pad treatment solution is used to soak the sample pad and conjugate pad for at least 5 minutes. After dehydration, the sample pad and conjugate pad are placed in a 33-50°C oven to dry for at least 7 hours, and then sealed and stored in an environment with a humidity of ≤30%;

[0050] 2. The mouse anti-human Clostridium difficile toxin A detection antibody is labeled with colloidal gold and redissolved. Take 1 mL of colloidal gold solution and adjust the pH value to 7.5-8.0 with a 0.1 M potassium carbonate solution, add the mouse anti-human Clostridium difficile toxin A detection antibody to a concentration of 15-22 μg / mL, and mix for at least 10 minutes; add BSA to a concentration of 0.6-1 wt%, and mix for at least 10 minutes; perform low-temperature high-speed centrifugation, with centrifugation parameters of temperature 0-8°C, speed 9000-14000 rpm, and centrifuge for at least 5 minutes; remove the supernatant, add the colloidal gold labeling solution to the precipitate, and then break up and resuspend the precipitate to make the solution OD value 45-60;

[0051] 3. The mouse anti-human Clostridium difficile toxin B detection antibody was labeled with colloidal gold and redissolved. 1 mL of colloidal gold solution was adjusted to a pH of 8.0-8.5 with a 0.1 M potassium carbonate solution, and mouse anti-human Clostridium difficile toxin B detection antibody was added to a concentration of 12-17 μg / mL, and mixed for at least 10 min; BSA was added to a concentration of 0.6-1 wt%, and mixed for at least 10 min, and low-temperature high-speed centrifugation was performed, and the centrifugation parameters were a temperature of 0-8 ° C, a speed of 9000-14000 rpm, and centrifugation for at least 5 min; the supernatant was removed, and the colloidal gold labeling solution was added to the precipitate, and then the precipitate was broken up and resuspended to make the solution OD value 70-100;

[0052] 4. Label and redissolve the mouse anti-human Clostridium difficile glutamate dehydrogenase detection antibody, take 1 mL of colloidal gold solution, adjust the pH value of the colloidal gold solution to 7.5-8.0 with a 0.1 M potassium carbonate solution, add mouse anti-human Clostridium difficile glutamate dehydrogenase detection antibody to a concentration of 14-18 μg / mL, and mix for at least 10 minutes; add BSA to a concentration of 0.6-1 wt%, mix for at least 10 minutes, and perform low-temperature high-speed centrifugation, the centrifugation parameters are temperature 0-8°C, speed 9000-14000 rpm, and centrifugation for at least 5 minutes; remove the supernatant, add the colloidal gold labeling solution to the precipitate, then break up and resuspend the precipitate to make the solution OD value 45-70;

[0053] 5. Label and redissolve the quality control antibody chicken IgY antibody, take the colloidal gold solution and adjust the pH value of the colloidal gold solution to 8.5-9.0 with a 0.1M potassium carbonate solution, add the chicken IgY antibody to a concentration of 8-12 μg / mL, then mix for at least 10 minutes, add BSA to a concentration of 0.6-1wt%, then mix for at least 10 minutes, and perform low-temperature high-speed centrifugation with the centrifugation parameters of temperature 0-8°C, rotation speed 9000-14000rpm, and centrifugation for at least 5 minutes. in; the supernatant is removed by aspiration, and a colloidal gold-labeled reconstitution solution is added to the precipitate, and the colloidal gold-labeled chicken IgY antibody reconstitution solution is added to the mouse anti-human Clostridium difficile toxin A detection antibody gold-labeled reconstitution solution, the mouse anti-human Clostridium difficile toxin B detection antibody gold-labeled reconstitution solution, and the mouse anti-human Clostridium difficile glutamate dehydrogenase detection antibody gold-labeled reconstitution solution, respectively, so that the OD value of the colloidal gold-labeled chicken IgY antibody reconstitution solution in the three gold-labeled reconstitution solutions is 35-50. The three solutions after mixing are respectively gold-labeled reconstitution solution A, gold-labeled reconstitution solution B, and gold-labeled reconstitution solution GDH;

[0054] 6. Dilute the mouse anti-human Clostridium difficile toxin A capture antibody to 1-1.5 mg / mL with coating diluent to obtain mouse anti-human Clostridium difficile toxin A capture antibody coating solution; dilute the mouse anti-human Clostridium difficile toxin B capture antibody to 1.4-2 mg / mL to obtain mouse anti-human Clostridium difficile toxin B capture antibody coating solution; dilute the mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody to 1.1-1.5 mg / mL to obtain mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody coating solution; dilute the goat anti-chicken IgY antibody to 0.8-1.2 mg / mL to obtain goat anti-chicken IgY antibody coating solution;

[0055] 7. Spray the gold-labeled complex solution A, gold-labeled complex solution B and gold-labeled complex solution GDH on the three corresponding binding pads after treatment at a spray rate of 3-5 μL / cm, match the mouse anti-human Clostridium difficile toxin A capture antibody coating solution, mouse anti-human Clostridium difficile toxin B capture antibody coating solution, and mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody coating solution with the goat anti-chicken IgY antibody coating solution, and spray them on the three corresponding nitrocellulose membranes at a speed of 1-3 μL / cm, respectively, and form a detection line and a quality control line on each nitrocellulose membrane, then bake the treated nitrocellulose membrane at a temperature of 33-50°C for at least 6 hours, and then seal and store the dried nitrocellulose membrane in an environment with a humidity of ≤30%;

[0056] 8. Assemble the test strip. The positional relationship between the sample pad, conjugation pad, nitrocellulose membrane, water-absorbing pad and bottom plate is as follows: the sample pad, conjugation pad, water-absorbing pad and nitrocellulose membrane are all bonded to the bottom plate, wherein the nitrocellulose membrane is located near the middle of the bottom plate, one end of the nitrocellulose membrane is bonded to the water-absorbing pad with an overlap of 1-3 mm, and the other end is bonded to the conjugation pad with an overlap of 1-3 mm, and the other end of the conjugation pad is bonded to the sample pad with an overlap of 1-3 mm. The bottom plate is located at the bottom to support the entire test strip. The structure of the test strip is as follows: Figure 1 As shown;

[0057] 9. Arrange the three test strips in parallel to form a triple test card. The structure of the triple test box is as follows: Figure 2 shown.

[0058] Example 2: Use of test strips

[0059] (1) Dilute the Clostridium difficile toxin A antigen standard solution with sample diluent to the following concentrations: 1 μg / mL, 1 ng / mL, 0.25 ng / mL, 0.1 ng / mL; dilute the Clostridium difficile toxin B antigen standard solution with sample diluent to the following concentrations: 2.33 μg / mL, 2.5 ng / mL, 0.5 ng / mL, 0.25 ng / mL; dilute the Clostridium difficile glutamate dehydrogenase antigen with sample diluent to the following concentrations: 1.78 μg / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL.

[0060] (2) Take 80 μL of each of the 12 concentrations of antigen solutions prepared in step (1) and drip them onto the triple test card prepared in Example 1. Due to capillary action, the sample will move along the test strip to the conjugate pad and nitrocellulose membrane. Observe and display the results after 10 minutes (Note: color development is invalid after 15 minutes).

[0061] (3) Observation results: The test result is positive when the concentration of Clostridium difficile toxin A antigen is greater than or equal to 0.25 ng / mL, and negative when it is less than 0.25 ng / mL; the test result is positive when the concentration of Clostridium difficile toxin B antigen is greater than or equal to 0.5 ng / mL, and negative when it is less than 0.5 ng / mL; the test result is positive when the concentration of Clostridium difficile glutamate dehydrogenase antigen is greater than or equal to 0.5 ng / mL, and negative when it is less than 0.5 ng / mL.

[0062] Comparative Example 1

[0063] (1) A test strip is prepared according to the method described in Example 1, except that: in step 4, the coating solution is sprayed on the nitrocellulose membrane, and the mouse anti-human Clostridium difficile toxin A capture antibody coating solution, the mouse anti-human Clostridium difficile toxin B capture antibody coating solution, and the goat anti-chicken IgY antibody coating solution are sprayed on the same nitrocellulose membrane respectively to form a Clostridium difficile toxin A detection line, a Clostridium difficile toxin B detection line and a quality control line, and then the treated nitrocellulose membrane is placed at a temperature of 33-50°C and baked for at least 6 hours, and then the dried nitrocellulose membrane is placed in an environment with a humidity of ≤30% and sealed for storage.

[0064] According to the method described in Example 2, the Clostridium difficile toxin A antigen standard solution, the Clostridium difficile toxin B antigen standard solution, and the sample dilution solution of Example 1 were respectively tested, and it was found that when only the Clostridium difficile toxin B antigen standard solution was added, the Clostridium difficile toxin A detection line was positive, and when only the Clostridium difficile toxin A antigen standard solution was added, the Clostridium difficile toxin B detection line was positive. Therefore, the same test strip cannot specifically detect two markers, resulting in a false positive phenomenon.

[0065] (2) A test strip is prepared according to the method described in Example 1, except that: in step 4, the coating solution is sprayed on the nitrocellulose membrane, and the mouse anti-human Clostridium difficile toxin B capture antibody coating solution, the mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody coating solution, and the goat anti-chicken IgY antibody coating solution are sprayed on the same nitrocellulose membrane respectively to form a Clostridium difficile toxin B detection line, a Clostridium difficile glutamate dehydrogenase detection line and a quality control line, and then the treated nitrocellulose membrane is placed at a temperature of 33-50°C and baked for at least 6 hours, and then the dried nitrocellulose membrane is placed in a sealed state for storage in an environment with a humidity of ≤30%.

[0066] According to the method described in Example 2, the Clostridium difficile toxin B antigen standard solution, the Clostridium difficile glutamate dehydrogenase antigen standard solution, and the sample dilution solution of Example 1 were respectively tested, and it was found that when only the Clostridium difficile toxin B antigen standard solution was added, the Clostridium difficile glutamate dehydrogenase detection line was positive, while when only the Clostridium difficile glutamate dehydrogenase antigen standard solution was added, the Clostridium difficile toxin B detection line was positive. Therefore, the same test strip cannot specifically detect two markers, resulting in a false positive phenomenon.

[0067] (3) A test strip is prepared according to the method described in Example 1, except that: in step 4, the coating solution is sprayed on the nitrocellulose membrane, and the mouse anti-human Clostridium difficile toxin A capture antibody coating solution, the mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody coating solution, and the goat anti-chicken IgY antibody coating solution are sprayed on the same nitrocellulose membrane respectively to form a Clostridium difficile toxin A detection line, a Clostridium difficile glutamate dehydrogenase detection line and a quality control line, and then the treated nitrocellulose membrane is placed at a temperature of 33-50°C and baked for at least 6 hours, and then the dried nitrocellulose membrane is placed in a sealed state for storage in an environment with a humidity of ≤30%.

[0068] According to the method described in Example 2, the Clostridium difficile toxin A antigen standard solution, the Clostridium difficile glutamate dehydrogenase antigen standard solution, and the sample dilution solution of Example 1 were respectively tested, and it was found that when only the Clostridium difficile toxin A antigen standard solution was added, the Clostridium difficile glutamate dehydrogenase detection line was positive, while when only the Clostridium difficile glutamate dehydrogenase antigen standard solution was added, the Clostridium difficile toxin A detection line was positive. Therefore, the same test strip cannot specifically detect two markers, resulting in a false positive phenomenon.

[0069] It can be seen from this that preparing two test lines on the same test strip is prone to false positives, affecting the accuracy of the reagent. Considering the above situation, the present invention chooses to use different test strips to detect the three markers Clostridium difficile toxin A, Clostridium difficile toxin B and Clostridium difficile glutamate dehydrogenase.

[0070] Comparative Example 2

[0071] (1) A test strip was prepared according to the method described in Example 1, except that: in step 2, the antibody was labeled and reconstituted, and the OD value of the gold-labeled reconstituted solution of mouse anti-human Clostridium difficile toxin A monoclonal antibody 1 was 30, the OD value of the gold-labeled reconstituted solution of mouse anti-human Clostridium difficile toxin B monoclonal antibody 1 was 60, and the OD value of the gold-labeled reconstituted solution of mouse anti-human Clostridium difficile glutamate dehydrogenase monoclonal antibody 1 was 35.

[0072] According to the method described in Example 2, the Clostridium difficile toxin A antigen standard solution, Clostridium difficile toxin B antigen standard solution, and Clostridium difficile glutamate dehydrogenase antigen standard solution were respectively detected, and the minimum detection limits were: Clostridium difficile toxin A antigen concentration value greater than or equal to 0.5 ng / mL was positive, and less than 0.5 ng / mL was negative; Clostridium difficile toxin B antigen concentration value greater than or equal to 1 ng / mL was positive, and less than 1 ng / mL was negative; Clostridium difficile glutamate dehydrogenase antigen concentration value greater than or equal to 0.8 ng / mL was positive, and less than 0.8 ng / mL was negative.

[0073] (2) A test strip was prepared according to the method described in Example 1, except that: in step 3, the antibody coating solution was prepared, the mouse anti-human Clostridium difficile toxin A capture antibody was diluted to 0.5 mg / mL to obtain a mouse anti-human Clostridium difficile toxin A capture antibody coating solution; the mouse anti-human Clostridium difficile toxin B capture antibody was diluted to 1.2 mg / mL to obtain a mouse anti-human Clostridium difficile toxin B capture antibody coating solution; the mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody was diluted to 0.8 mg / mL to obtain a mouse anti-human Clostridium difficile glutamate dehydrogenase capture antibody coating solution.

[0074] According to the method described in Example 2, the Clostridium difficile toxin A antigen standard solution, Clostridium difficile toxin B antigen standard solution, and Clostridium difficile glutamate dehydrogenase antigen standard solution were respectively detected, and the minimum detection limits were: Clostridium difficile toxin A antigen concentration value greater than or equal to 0.5 ng / mL was positive, and less than 0.5 ng / mL was negative; Clostridium difficile toxin B antigen concentration value greater than or equal to 1.5 ng / mL was positive, and less than 1 ng / mL was negative; Clostridium difficile glutamate dehydrogenase antigen concentration value greater than or equal to 1 ng / mL was positive, and less than 1 ng / mL was negative.

[0075] It can be seen that reducing the amount of detection antibody on the conjugate pad or the amount of capture antibody on the nitrocellulose membrane will affect the detection performance of the reagent strip.

[0076] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A triple detection kit for Clostridium difficile based on colloidal gold immunochromatography, characterized in that: The Clostridium difficile triple test kit comprises a Clostridium difficile triple test card and a sample diluent, wherein the Clostridium difficile triple test card is composed of three test strips arranged in parallel and connected, wherein the test strips comprise a Clostridium difficile toxin A test strip, a Clostridium difficile toxin B test strip, and a Clostridium difficile glutamate dehydrogenase test strip, wherein the test strips are provided with a sample pad, a binding pad, a coating film, and a water-absorbing pad on the bottom plate in sequence from the sample application end, and the coating film is provided with a Clostridium difficile toxin A detection line, a Clostridium difficile toxin B detection line, and a Clostridium difficile glutamate dehydrogenase detection line, respectively, wherein: The conjugate pad of the Clostridium difficile toxin A test strip is sprayed with a colloidal gold-labeled Clostridium difficile toxin A detection antibody complex solution, the final concentration of the Clostridium difficile toxin A detection antibody is 15-22 μg / mL, and the test line is sprayed with a coating solution containing Clostridium difficile toxin A capture antibody, and the final concentration of the Clostridium difficile toxin A capture antibody in the coating solution is 1-1.5 mg / mL; The conjugate pad of the Clostridium difficile toxin B test strip is sprayed with a colloidal gold-labeled Clostridium difficile toxin B detection antibody complex solution, the final concentration of the Clostridium difficile toxin B detection antibody is 12-17 μg / mL, and the test line is sprayed with a coating solution containing Clostridium difficile toxin B capture antibody, and the final concentration of the Clostridium difficile toxin B capture antibody in the coating solution is 1.4-2 mg / mL; The conjugate pad of the Clostridium difficile glutamate dehydrogenase test strip is sprayed with a colloidal gold-labeled Clostridium difficile glutamate dehydrogenase detection antibody complex solution, the final concentration of the Clostridium difficile glutamate dehydrogenase detection antibody is 14-18 μg / mL, and the detection line is sprayed with a coating solution containing Clostridium difficile glutamate dehydrogenase capture antibody, and the final concentration of the Clostridium difficile glutamate dehydrogenase capture antibody in the coating solution is 1.1-1.5 mg / mL; The sample diluent consists of sodium chloride, Tris, disodium hydrogen phosphate, dipotassium hydrogen phosphate, bovine serum albumin, a surfactant and a preservative.

2. The Clostridium difficile triple detection kit according to claim 1, characterized in that: A quality control line is arranged on the coating film of the test strip, and a coating solution containing a quality control capture antibody is sprayed on the quality control line. The final concentration of the quality control capture antibody in the coating solution is 0.8-1.2 mg / mL.

3. The Clostridium difficile triple detection kit according to claim 2, characterized in that: The conjugate pad is sprayed with a quality control detection antibody complex solution labeled with colloidal gold, and the final concentration of the quality control detection antibody is 8-12 μg / mL.

4. The Clostridium difficile triple detection kit according to claim 1, characterized in that: The spraying amount of the colloidal gold-labeled Clostridium difficile toxin A detection antibody resolution, the colloidal gold-labeled Clostridium difficile toxin B detection antibody resolution and the colloidal gold-labeled Clostridium difficile glutamate dehydrogenase detection antibody resolution on the conjugate pad is 3-5 μL / cm 2 .

5. The Clostridium difficile triple detection kit according to claim 1, characterized in that: The spraying amount of the solution containing Clostridium difficile toxin A capture antibody, the solution containing Clostridium difficile toxin B capture antibody and the solution containing Clostridium difficile glutamate dehydrogenase capture antibody on the detection line is 1-3 μL / cm.

6. The Clostridium difficile triple detection kit according to claim 1, characterized in that: The sample pad and the conjugate pad are pretreated, and the pretreatment is to soak the sample pad and the conjugate pad in a sample pad treatment solution and a conjugate pad treatment solution respectively; The sample pad treatment solution includes Tris-HCl buffer, polyvinyl pyrrolidone K30, surfactant, Tween 20, preservative and heterophilic antibody blocking agent HBR, and bovine serum albumin or casein; The conjugate pad treatment solution includes Tris-HCl buffer, polyvinyl pyrrolidone K30, Tween 20 and a preservative, and bovine serum albumin or casein.

7. The Clostridium difficile triple detection kit according to claim 6, characterized in that: The amount of the sample pad treatment solution is 0.05-0.1 mL / cm 2 The amount of the conjugate pad treatment solution is 0.04-0.8 mL / cm 2 .

8. The method for preparing the Clostridium difficile triple detection kit according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step S1, pre-treating the sample pad and the conjugate pad; Step S2, preparing a colloidal gold solution and a colloidal gold labeled re-solution, and labeling and re-dissolving Clostridium difficile toxin A detection antibody, Clostridium difficile toxin B detection antibody, Clostridium difficile glutamate dehydrogenase detection antibody and quality control capture antibody with colloidal gold; Step S3, preparing a coating diluent, and coating Clostridium difficile toxin A capture antibody, Clostridium difficile toxin B capture antibody, Clostridium difficile glutamate dehydrogenase capture antibody and quality control capture antibody; Step S4, respectively mixing the colloidal gold-labeled Clostridium difficile toxin A detection antibody resolution, the Clostridium difficile toxin B detection antibody resolution and the Clostridium difficile glutamate dehydrogenase detection antibody resolution with the colloidal gold-labeled quality control detection antibody resolution, and spraying them on the conjugate pad after mixing; Step S5, spraying the Clostridium difficile toxin A capture antibody coating solution, the Clostridium difficile toxin B capture antibody coating solution, and the Clostridium difficile glutamate dehydrogenase capture antibody coating solution on the test line, and spraying the quality control capture antibody on the quality control line; Step S6, assembling the corresponding sample pad, binding pad, coating membrane and absorbent pad respectively to obtain Clostridium difficile toxin A test strip, Clostridium difficile toxin B test strip and Clostridium difficile glutamate dehydrogenase test strip; Step S7, arranging the three test strips in parallel to assemble them to obtain the Clostridium difficile triple detection kit.

9. The preparation method according to claim 8, characterized in that: In step S4, the OD value of the colloidal gold labeled Clostridium difficile toxin A detection antibody reconstitution solution is 45-60, the OD value of the colloidal gold labeled Clostridium difficile toxin B detection antibody reconstitution solution is 70-100, and the OD value of the colloidal gold labeled Clostridium difficile toxin glutamate dehydrogenase detection antibody reconstitution solution is 45-70.

10. The preparation method according to claim 8, characterized in that: In step S4, the OD value of the quality control detection antibody labeled with colloidal gold in the mixed solution is 35-50.

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