Clostridium difficile marker joint detection card based on high-concentration colloidal gold

Abstract

The invention relates to a clostridium difficile marker combined detection card based on high-concentration colloidal gold, and belongs to the technical field of immunodetection. The high-concentration colloidal gold with the OD value of 100 is used for marking a detection antibody, the treatment process of the combination pad and the sample pad is adjusted, the marking process is optimized, the antibody concentration is accurately controlled, and the prepared detection card can detect three clostridium difficile markers including clostridium difficile toxin A, clostridium difficile toxin B and clostridium difficile glutamate dehydrogenase; the lowest detection limit for clostridium difficile toxin A is 0.1 ng / mL, the lowest detection limit for clostridium difficile toxin B is 0.25 ng / mL, and the lowest detection limit for clostridium difficile glutamate dehydrogenase is 0.1 ng / mL, so that the problems of limited sensitivity and insufficient signal amplification of a conventional colloidal gold test strip in the prior art are solved, the detection sensitivity and stability are remarkably improved, and the detection cost is reduced. The kit is suitable for clinical rapid diagnosis of clostridium difficile related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay technology, and particularly to a Clostridium difficile marker joint detection card based on high-concentration colloidal gold. BACKGROUND

[0002] Clostridium difficile is an anaerobic gram-positive spore-forming bacterium that is a normal flora in the human intestine, and the overgrowth of the bacterium can be caused by the use of antibiotics. With the widespread use of broad-spectrum antibiotics, the incidence of Clostridium difficile associated diarrhea has been increasing, and outbreaks have occurred in recent years. At present, Clostridium difficile has become the main pathogen causing hospital-acquired diarrhea and pseudomembranous colitis, and poses a particularly serious threat to the elderly, immunocompromised individuals, and patients who have long been hospitalized or used antibiotics. Therefore, rapid detection of Clostridium difficile is of great significance for timely diagnosis and prevention.

[0003] Current detection of Clostridium difficile infection mainly relies on enzyme-linked immunosorbent assay (ELISA), real-time fluorescent quantitative PCR, and cytotoxicity test. ELISA detects Clostridium difficile toxin A and Clostridium difficile toxin B through antibody-antigen reaction, has high specificity, but requires professional equipment and a long detection time. PCR technology realizes rapid detection by amplifying nucleic acid sequences, is suitable for nucleic acid level analysis, but the equipment is expensive and requires high sample purity. As the gold standard, cytotoxicity test can accurately detect toxin activity, but the operation is complex and time-consuming. In recent years, lateral flow immunochromatography technology has been favored due to its portability and rapidity, and colloidal gold-labeled test strips are widely used for disease detection. This technology labels colloidal gold with antibodies to form a colored line on the NC membrane, is simple to operate, and the results are intuitive. However, the existing colloidal gold test strip has limited sensitivity in detecting low concentrations of Clostridium difficile toxin A, Clostridium difficile toxin B, and glutamate dehydrogenase, and the signal amplification is insufficient. Moreover, the components of each processing solution lack targeted improvement and optimization, making it difficult to meet the detection needs of ultra-low concentration antigens. SUMMARY

[0004] To solve the above technical problems, the present application provides a Clostridium difficile marker joint detection card based on high-concentration colloidal gold, a preparation method and application. The preparation process includes labeling three specific detection antibodies with high-concentration colloidal gold with an OD value of 100, adjusting the processing of the conjugate pad and the sample pad, optimizing the colloidal gold labeling process, and controlling the concentration of the detection antibodies and the capture antibodies. The prepared Clostridium difficile marker joint detection card can realize rapid detection of Clostridium difficile within 10-15 minutes.

[0005] The first object of the present application is to provide a preparation method of a Clostridium difficile marker combined detection card, the Clostridium difficile marker comprising Clostridium difficile toxin A, Clostridium difficile toxin B and Clostridium difficile glutamate dehydrogenase, the preparation method comprising the following steps:

[0006] Step S1, pretreating the sample pad and the binding pad;

[0007] Step S2, preparing a reconstitution solution and a blocking solution, respectively labeling Clostridium difficile toxin A detection antibodies, Clostridium difficile toxin B detection antibodies and Clostridium difficile glutamate dehydrogenase detection antibodies with high-concentration colloidal gold, adding the blocking solution for reaction, taking the precipitate and adding the reconstitution solution to obtain Clostridium difficile toxin A colloidal gold labeling reconstitution solution, Clostridium difficile toxin B colloidal gold labeling reconstitution solution and Clostridium difficile glutamate dehydrogenase colloidal gold labeling reconstitution solution, and the OD value of the high-concentration colloidal gold is 100;

[0008] Step S3, preparing a coating diluent, coating Clostridium difficile toxin A capture antibodies, Clostridium difficile toxin B capture antibodies and Clostridium difficile glutamate dehydrogenase capture antibodies to obtain the coating diluent, and respectively spraying the obtained coating diluent on the coating film to set a detection line;

[0009] Step S4, spraying the Clostridium difficile toxin A colloidal gold labeling reconstitution solution, the Clostridium difficile toxin B colloidal gold labeling reconstitution solution and the Clostridium difficile glutamate dehydrogenase colloidal gold labeling reconstitution solution obtained in step S1 on the binding pad;

[0010] Step S5, assembling the corresponding sample pad, binding pad, coating film and water absorption pad to obtain Clostridium difficile toxin A, Clostridium difficile toxin B and Clostridium difficile glutamate dehydrogenase test strips, and parallelly arranging and assembling the obtained test strips to obtain the Clostridium difficile marker combined detection card.

[0011] Further, in step S1, the sample pad is soaked in a sample pad pretreatment solution, and the sample pad pretreatment solution comprises Tris-HCl, casein, polyvinylpyrrolidone K30, surfactant S9, Tween-20, preservative ProClin 300 and heterophilic antibody blocker HBR-5.

[0012] Further, in step S1, the binding pad is soaked in a binding pad pretreatment solution, and the binding pad pretreatment solution comprises Tris-HCl, casein, polyvinylpyrrolidone K30, Tween-20 and preservative ProClin 300.

[0013] Further, in step S2, when the high-concentration colloidal gold is labeled, the final concentration of the C. difficile toxin A detection antibody is 45-55 μg / mL, the final concentration of the C. difficile toxin B detection antibody is 55-65 μg / mL, and the final concentration of the C. difficile glutamate dehydrogenase detection antibody is 55-65 μg / mL.

[0014] Further, in step S2, the OD value of the C. difficile toxin A colloidal gold labeling reconstitution solution is 55-65, the OD value of the C. difficile toxin B colloidal gold labeling reconstitution solution is 85-100, and the OD value of the C. difficile glutamate dehydrogenase colloidal gold labeling reconstitution solution is 65-80.

[0015] Further, in step S2, the step of labeling and reconstituting the quality control detection antibody with colloidal gold is further included, and the OD value of the colloidal gold is 1-5.

[0016] Further, in step S3, the step of coating the quality control capture antibody and spraying the quality control line on the coated membrane is further included.

[0017] Further, in step S4, the step of mixing the quality control detection antibody colloidal gold reconstitution solution with the C. difficile toxin A colloidal gold labeling reconstitution solution, the C. difficile toxin B colloidal gold labeling reconstitution solution, and the C. difficile glutamate dehydrogenase colloidal gold labeling reconstitution solution, respectively, and spraying the obtained mixed solution on the conjugate pad is further included.

[0018] Further, the OD value of the obtained mixed solution is 25-35.

[0019] A second object of the present application is to provide a C. difficile marker joint detection card prepared by the above preparation method.

[0020] A third method of the present application is to provide the use of the above C. difficile marker joint detection card in C. difficile detection.

[0021] Further, the use includes the following steps:

[0022] (1) preparing a sample diluent, wherein the sample diluent comprises NaCl, Tris, anhydrous disodium hydrogen phosphate, BSA, anhydrous potassium hydrogen phosphate, a surfactant S22, and a preservative ProClin 300;

[0023] (2) diluting the sample to be tested with the sample diluent;

[0024] (3) adding the diluted sample to be tested into the sample addition hole, and due to the capillary effect, the sample solution will move along the test strip towards the absorbent pad, and the result is observed after 10 minutes.

[0025] Further, if the quality control line does not develop color, even if the detection line develops color, the detection result is also invalid.

[0026] Advantages of the present application:

[0027] The present application uses high-concentration colloidal gold with an OD value of 100, and combines meticulous adjustment and optimization of antibody labeling and detection card preparation processes, so that the prepared Clostridium difficile marker joint detection card has a minimum detection limit of 0.1 ng / mL for Clostridium difficile toxin A, a minimum detection limit of 0.25 ng / mL for Clostridium difficile toxin B, and a minimum detection limit of 0.1 ng / mL for Clostridium difficile glutamate dehydrogenase, thereby solving the problems of limited sensitivity and insufficient signal amplification of conventional colloidal gold test strips in the prior art, significantly improving the detection sensitivity and stability, and providing a more reliable tool for clinical rapid diagnosis of diseases caused by Clostridium difficile. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to make the content of the present application easier to be clearly understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which:

[0029] Figure 1 A side view of the Clostridium difficile detection card provided by the present application is shown, in which 1 is a sample pad, 2 is a conjugate pad, 3 is a coating film, 31 is a detection line, 32 is a quality control line, 4 is a water absorption pad, and 5 is a bottom plate.

[0030] Figure 2 A top view of the Clostridium difficile detection card provided by the present application is shown, in which A is a Clostridium difficile toxin A test strip, 61 is a corresponding sample addition hole, B is a Clostridium difficile toxin B test strip, 62 is a corresponding sample addition hole, GDH is a Clostridium difficile glutamate dehydrogenase test strip, 63 is a corresponding sample addition hole, T is a detection line, and C is a quality control line. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting to the present application.

[0032] The present application provides a preparation method of a Clostridium difficile detection card, comprising the following steps:

[0033] Step S1, pretreating the sample pad and the conjugate pad;

[0034] Step S2, prepare a reconstitution solution and a blocking solution, respectively label the C. difficile toxin A detection antibody, the C. difficile toxin B detection antibody and the C. difficile glutamate dehydrogenase detection antibody with high-concentration colloidal gold, add the blocking solution for reaction, take the precipitate and add the reconstitution solution to obtain the C. difficile toxin A colloidal gold-labeled reconstitution solution, the C. difficile toxin B colloidal gold-labeled reconstitution solution and the C. difficile glutamate dehydrogenase colloidal gold-labeled reconstitution solution, and the OD value of the high-concentration colloidal gold is 100;

[0035] Step S3, prepare a coating diluent, coat the C. difficile toxin A capture antibody, the C. difficile toxin B capture antibody and the C. difficile glutamate dehydrogenase capture antibody respectively, and spray the obtained coating diluents on the coating membranes respectively;

[0036] Step S4, spray the C. difficile toxin A colloidal gold-labeled reconstitution solution, the C. difficile toxin B colloidal gold-labeled reconstitution solution and the C. difficile glutamate dehydrogenase colloidal gold-labeled reconstitution solution obtained in step S1 on the binding pads respectively;

[0037] Step S5, assemble the sample pads, the binding pads, the coating membranes and the water absorption pads to obtain the C. difficile toxin A test strip, the C. difficile toxin B test strip and the C. difficile glutamate dehydrogenase test strip respectively, and assemble the obtained test strips in parallel to obtain the C. difficile marker joint detection card.

[0038] Example 1: Preparation of the C. difficile marker joint detection card

[0039] The embodiment provides a preparation method of a C. difficile marker joint detection card, and specifically includes the following steps:

[0040] I. Pretreatment of sample pads and binding pads

[0041] (1) Pretreatment of sample pads

[0042] Prepare a sample pad pretreatment solution, and the components of the sample pad pretreatment solution are as follows: 50 mM Tris-HCl, 0.5 wt% casein, 1 wt% polyvinylpyrrolidone K30, 1 wt% surfactant S9, 0.05 wt% Tween-20, 0.02 wt% preservative ProClin300 and 0.003 wt% heterophil antibody blocker HBR-5, and the pH value of the solution is adjusted to 8.0-9.0.

[0043] According to the proportion of 70 μL / cm 2 , add the sample pad pretreatment solution to the sample pad, soak the sample pad for 10 min, dry the sample pad after spinning, and then dry the sample pad in a 45℃ oven for 7 hours.

[0044] (2) Pretreatment of binding pads

[0045] Prepare the conjugate pad pretreatment solution with the following components: 50 mM Tris-HCl, 0.5 wt% casein, 1 wt% polyvinylpyrrolidone K30, 0.05 wt% Tween-20, and 0.02 wt% preservative ProClin 300, and adjust the pH to 8.0-9.0.

[0046] Add the conjugate pad pretreatment solution at a ratio of 65 μL / cm 2 to the conjugate pad, soak for 10 min, spin dry, and then dry the conjugate pad in an oven at 45°C for 7 hours.

[0047] II. Labeling and reconstitution of detection antibody and control antibody

[0048] (1) Solution preparation

[0049] Prepare a high-concentration colloidal gold solution with an initial OD value of 100 for labeling the detection antibody.

[0050] Prepare a regular colloidal gold solution with an initial OD value of 1-5 for labeling the control antibody.

[0051] Prepare a labeling reconstitution solution with the following components: 50 mM Tris-HCl, 0.5 wt% casein, 20 wt% sucrose, 5 wt% trehalose, and 0.02 wt% preservative ProClin 300, and adjust the solution pH to 8.0-9.0.

[0052] Prepare a blocking solution with the following components: 1 wt% casein and 100 mM boric acid, and adjust the pH to 8.0-8.5.

[0053] (2) Labeling and reconstitution of detection antibody

[0054] Labeling and reconstitution of C. difficile toxin A detection antibody: Dilute the high-concentration colloidal gold solution to an OD value of 50-60 using ultrapure water, then adjust the solution pH to 7.5-8.0 using a 0.1 M potassium carbonate solution, add the C. difficile toxin A detection antibody to a final concentration of 45-55 μg / mL. Add an equal volume of blocking solution, react for 15-20 min to block the remaining sites, then centrifuge to obtain the precipitate, add the labeling reconstitution solution to the precipitate to obtain a C. difficile toxin A detection antibody labeling reconstitution solution with an OD value of 55-65.

[0055] Labeling and reconstitution of C. difficile toxin B detection antibody: dilute OD value of high concentration colloidal gold solution to 50-60 using 0.01 M borate buffer solution with pH value of 8.4, then add C. difficile toxin B detection antibody to make the final concentration of the detection antibody 55-65 μg / mL. Add equal volume of blocking solution, react for 15-20 min to block the remaining sites, then centrifuge to obtain the precipitate, add labeling reconstitution solution to the precipitate to obtain C. difficile toxin B detection antibody labeling reconstitution solution with OD value of 85-100.

[0056] Labeling and reconstitution of C. difficile glutamate dehydrogenase detection antibody: dilute OD value of high concentration colloidal gold solution to 50-60 using 0.01 M phosphate buffer solution with pH value of 7.4, then add C. difficile glutamate dehydrogenase detection antibody to make the final concentration of the detection antibody 55-65 μg / mL. Add equal volume of blocking solution, react for 15-20 min to block the remaining sites, then centrifuge to obtain the precipitate, add labeling reconstitution solution to the precipitate to obtain C. difficile glutamate dehydrogenase detection antibody labeling reconstitution solution with OD value of 65-80.

[0057] (3) Labeling and reconstitution of quality control antibody

[0058] (a) take 1 mL of prepared 0.01 wt% colloidal gold solution with particle size of 30-40 nm, adjust the pH value of the colloidal gold solution to 8.5-9.0 using 0.1 M potassium carbonate solution;

[0059] (b) add quality control detection antibody to the solution prepared in step (a) to make the concentration of the quality control detection antibody 8-10 μg / mL, then mix for 10 min;

[0060] (c) add BSA to the solution prepared in step (b) to make the concentration of BSA 0.8-1 wt%, then mix for 10 min;

[0061] (d) perform low-temperature high-speed centrifugation on the solution prepared in step (c), with centrifugation parameters of temperature 0-8 °C, rotation speed 10000-13000 rpm, and centrifugation time 5 min;

[0062] (e) remove the supernatant of the solution obtained in step (d), then dilute with gold labeling reconstitution solution to make the OD value 35-45.

[0063] Three, preparation of coating film

[0064] Prepare coating diluent, which is composed of 1x phosphate buffer solution containing 2 wt% sucrose.

[0065] The C. difficile toxin A capture antibody, the C. difficile toxin B capture antibody, the C. difficile glutamate dehydrogenase capture antibody and the quality control capture antibody were respectively diluted to 0.8 mg / mL, 0.8 mg / mL, 0.8 mg / mL and 1 mg / mL using the coating diluent, and then the C. difficile toxin A capture antibody coating diluent, the C. difficile toxin B capture antibody coating diluent, the C. difficile glutamate dehydrogenase capture antibody coating diluent and the quality control capture antibody coating diluent were respectively sprayed on the coating membrane at a speed of 1.0 μL / cm to form the test line (T) and the quality control line (C), and the sprayed coating membrane was placed in a 37°C oven for baking for 7 hours.

[0066] Four, preparation of the conjugate pad

[0067] The C. difficile toxin A detection antibody reconstituted solution, the C. difficile toxin B detection antibody reconstituted solution and the C. difficile glutamate dehydrogenase detection antibody reconstituted solution were mixed with the quality control antibody labeled reconstituted solution to obtain three mixed solutions, and the OD value of the colloidal gold in the mixed solution was adjusted to 25-35. The mixed solution was sprayed on the corresponding conjugate pad at a speed of 3.0 μL / cm, and then the sprayed conjugate pad was placed in a 37°C oven for baking for 7 hours.

[0068] Five, assembly of the detection card

[0069] The corresponding sample pad, conjugate pad, coating membrane and water absorption pad were assembled to obtain the C. difficile toxin A test strip, the C. difficile toxin B test strip and the C. difficile glutamate dehydrogenase test strip. The three test strips were arranged in parallel to obtain the C. difficile marker joint detection card.

[0070] Example 2: detection method of the C. difficile marker joint detection card

[0071] 1. Preparation of sample diluent

[0072] The components of the sample diluent are: 43.83 g / L of NaCl, 6.055 g / L of Tris, 0.4 g / L of anhydrous disodium hydrogen phosphate, 10 g / L of BSA, 0.4 g / L of anhydrous potassium hydrogen phosphate, 1 ml / L of surfactant S22, and 0.2 ml / L of preservative ProClin 300, and the pH value of the solution is 7.4.

[0073] 2. Dilution of antigen

[0074] The C. difficile toxin A antigen was diluted to concentrations of 1 μg / mL, 1 ng / mL, 0.25 ng / mL and 0.1 ng / mL using the above sample diluent;

[0075] The C. difficile toxin B antigen was diluted to concentrations of 2.33 μg / mL, 2.5 ng / mL, 0.5 ng / mL and 0.25 ng / mL, respectively, using the sample diluent described above;

[0076] The C. difficile glutamate dehydrogenase antigen was diluted to concentrations of 1.78 μg / mL, 1 ng / mL, 0.5 ng / mL and 0.1 ng / mL, respectively, using the sample diluent described above.

[0077] 3. Detection of the antigen

[0078] 80 μL of each of the 12 concentrations of the antigen solution prepared above was taken, and 4 reagent cards (12 test strips) prepared in Example 1 were taken. The antigen solution was added to the sample application hole, and the sample solution moved along the test strip toward the absorbent pad by capillary action. The results were observed after 10 minutes (color development was ineffective after 15 minutes).

[0079] 4. Interpretation of results

[0080] When the concentration of the C. difficile toxin A antigen was 0.1 ng / mL or more, the test line (T) and the control line (C) were colored, and the test result was positive. When the concentration was less than 0.1 ng / mL, the control line (C) was colored, and the test line (T) was not colored, and the test result was negative.

[0081] When the concentration of the C. difficile toxin B antigen was 0.25 ng / mL or more, the test line (T) and the control line (C) were colored, and the test result was positive. When the concentration was less than 0.25 ng / mL, the control line (C) was colored, and the test line (T) was not colored, and the test result was negative.

[0082] When the concentration of the C. difficile glutamate dehydrogenase antigen was 0.1 ng / mL or more, the test line (T) and the control line (C) were colored, and the test result was positive. When the concentration was less than 0.1 ng / mL, the control line (C) was colored, and the test line (T) was not colored, and the test result was negative.

[0083] If the control line (C) was not colored, even if the test line (T) was colored, the test result was also invalid.

[0084] It is apparent that the above-described embodiments are merely illustrative for the purposes of clarity and are not to be regarded as limiting the mode of execution. Modifications and changes adaptable to the above-described explanation can be made by those of ordinary skill in the art. It is not necessary or possible to exhaust all the modes of execution. The changes or modifications thus inferred are still within the scope of the present invention.

Claims

1. A method for preparing a Clostridium difficile marker combined detection card, characterized in that: The Clostridium difficile marker includes Clostridium difficile toxin A, Clostridium difficile toxin B and Clostridium difficile glutamate dehydrogenase, and the preparation method includes the following steps: Step S1, pre-treating the sample pad and the conjugate pad; Step S2, preparing a reconstitution solution and a blocking solution, labeling the Clostridium difficile toxin A detection antibody, the Clostridium difficile toxin B detection antibody, and the Clostridium difficile glutamate dehydrogenase detection antibody with high concentration colloidal gold, adding the blocking solution to react, taking the precipitate and adding the reconstitution solution to obtain Clostridium difficile toxin A colloidal gold-labeled reconstitution solution, Clostridium difficile toxin B colloidal gold-labeled reconstitution solution, and Clostridium difficile glutamate dehydrogenase colloidal gold-labeled reconstitution solution, respectively, wherein the OD value of the high concentration colloidal gold is 100; Step S3, preparing coating diluents, coating Clostridium difficile toxin A capture antibody, Clostridium difficile toxin B capture antibody, and Clostridium difficile glutamate dehydrogenase capture antibody respectively to obtain coating diluents, spraying the obtained coating diluents on the coating membrane, and setting the detection line; Step S4, spraying the Clostridium difficile toxin A colloidal gold labeled reconstitution solution, the Clostridium difficile toxin B colloidal gold labeled reconstitution solution, and the Clostridium difficile glutamate dehydrogenase colloidal gold labeled reconstitution solution obtained in step S1 onto the conjugate pad respectively; Step S5: Assemble the corresponding sample pad, conjugate pad, coating membrane and absorbent pad to obtain Clostridium difficile toxin A, Clostridium difficile toxin B and Clostridium difficile glutamate dehydrogenase detection test strips respectively, and arrange the obtained test strips in parallel to obtain the Clostridium difficile marker combined detection card.

2. The preparation method according to claim 1, wherein: In step S1, the sample pad is immersed in a sample pad pretreatment solution, wherein the sample pad pretreatment solution includes Tris-HCl, casein, polyvinylpyrrolidone K30, surfactant S9, Tween-20, preservative ProClin 300 and heterophilic antibody blocker HBR-5.

3. The preparation method according to claim 1, wherein: In step S1 , the conjugate pad is immersed in a conjugate pad pretreatment solution, wherein the conjugate pad pretreatment solution comprises Tris-HCl, casein, polyvinylpyrrolidone K30, Tween-20 and preservative ProClin 300.

4. The preparation method according to claim 1, wherein: In step S2, when high concentration colloidal gold labeling is performed, the final concentration of the Clostridium difficile toxin A detection antibody is 45-55 μg / mL, the final concentration of the Clostridium difficile toxin B detection antibody is 55-65 μg / mL, and the final concentration of the Clostridium difficile glutamate dehydrogenase detection antibody is 55-65 μg / mL.

5. The preparation method according to claim 1, wherein: In step S2, the OD value of the Clostridium difficile toxin A colloidal gold labeled reconstitution solution is 55-65, the OD value of the Clostridium difficile toxin B colloidal gold labeled reconstitution solution is 85-100, and the OD value of the Clostridium difficile glutamate dehydrogenase colloidal gold labeled reconstitution solution is 65-80.

6. The preparation method according to claim 1, wherein: Step S2 also includes the steps of labeling and redissolving the quality control detection antibody with colloidal gold, wherein the OD value of the colloidal gold is 1-5.

7. The preparation method according to claim 6, characterized in that: Step S4 also includes mixing the quality control detection antibody colloidal gold complex solution with the Clostridium difficile toxin A colloidal gold labeled complex solution, the Clostridium difficile toxin B colloidal gold labeled complex solution and the Clostridium difficile glutamate dehydrogenase colloidal gold labeled complex solution, and spraying the resulting mixed solution on the conjugate pad.

8. The preparation method according to claim 7, characterized in that: The OD value of the obtained mixed solution was 25-35.

9. A Clostridium difficile marker combined detection card prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the Clostridium difficile marker combined detection card according to claim 9 in the detection of Clostridium difficile.

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