Diluent for detecting clostridium difficile in excrement sample and application thereof

Abstract

The invention belongs to the technical field of pathogenic bacterium detection, and particularly discloses a diluent for detecting clostridium difficile in an excrement sample and application of the diluent. The diluent for detecting the clostridium difficile in the excrement sample is prepared from fetal calf serum, bovine serum albumin, a detergent and an anti-interference agent according to a specific proportion. Wherein efficient extraction and detection of the clostridium difficile GDH are realized through broad-spectrum protection of the FBS, directional stabilization of the BSA, formation of a multi-layer protection network through the FBS-BSA, targeted splitting decomposition of the detergent and specific removal of the anti-interference agent. According to the invention, the relation among the splitting efficiency, enzyme protection and interference elimination is balanced by precisely regulating and controlling the proportion of the related components, and a reliable technical scheme is provided for clinical rapid diagnosis of clostridium difficile. The kit provided based on the diluent has the advantages of being good in stability, high in accuracy, low in cost, convenient to use and the like, and the clostridium difficile in an excrement sample can be efficiently and rapidly detected by adopting the kit.

Description

Technical Field

[0001] The present invention belongs to the technical field of pathogenic bacteria detection, and relates to a diluent for detecting Clostridium difficile in fecal samples and an application thereof. Background Art

[0002] Clostridium difficile (CD) is an obligate anaerobic, Gram-positive bacillus that can be present in the normal intestinal flora. Excessive use of broad-spectrum antibiotics, immunosuppressants, or chemotherapy drugs can lead to intestinal dysbiosis, causing excessive proliferation of C. difficile and secretion of toxins, which can lead to Clostridium difficile infection (CDI). The main clinical symptoms of CDI include diarrhea, abdominal pain, and fever. Severe cases can cause pseudomembranous colitis, which can be accompanied by complications such as toxic megacolon, intestinal perforation, septic shock, and even death. Studies have reported that approximately 10-25% of cases of antibiotic-associated diarrhea, 50-75% of cases of antimicrobial-associated colitis, and 90-100% of cases of pseudomembranous colitis are caused by CDI.

[0003] Clostridium difficile glutamate dehydrogenase (CGDH) is a metabolic enzyme expressed at high levels in C. difficile and serves as a good antigenic marker for C. difficile in stool. A positive CGDH result confirms the presence of the bacterium in the stool specimen, while a negative result indicates its absence. Currently, methods for detecting C. difficile glutamate dehydrogenase (CGDH) include culture, enzyme-linked immunosorbent assay (ELISA), and molecular assays (such as PCR). Culture-based testing is time-consuming, requiring 2-4 days to produce results, making it unsuitable for rapid clinical testing. Conventional ELISA is cumbersome and time-consuming, typically requiring several hours to produce results, making it inefficient in emergency clinical testing scenarios. Molecular assays are expensive, complex, and time-consuming, limiting their application for large-scale, rapid testing. Summary of the Invention

[0004] In response to the shortcomings of the existing Clostridium difficile detection methods in the art, such as high detection cost, long detection cycle, cumbersome operation or inconvenience in clinical use, the present invention innovatively designs a diluent for detecting Clostridium difficile in fecal samples, and further provides a kit for rapid detection of Clostridium difficile in fecal samples.

[0005] To achieve the above-mentioned purpose, the embodiment of the present invention adopts the following technical solutions: In a first aspect, the present invention provides a diluent for detecting Clostridium difficile in a stool sample, comprising fetal bovine serum (FBS), bovine serum albumin (BSA), a detergent, and an anti-interference agent; Wherein, the detergent comprises at least one of Triton X100, Tween 20 or NP40; The mass ratio of bovine serum albumin (BSA) to detergent is 1:0.05 to 3:0.05; The mass ratio of the fetal bovine serum (FBS) to bovine serum albumin (BSA) is 5:1 to 1:1.

[0006] The diluent provided by the present invention for detecting Clostridium difficile in fecal samples is composed of a specific ratio of fetal bovine serum, bovine serum albumin, detergent, and anti-interference agent. Among them, through the broad-spectrum protection of FBS, the directional stabilization of BSA, the formation of a multi-layer protection network by FBS-BSA, the targeted cleavage of the detergent, and the specific removal of the anti-interference agent, efficient extraction and detection of Clostridium difficile GDH are achieved. The present invention balances the relationship between cleavage efficiency, enzyme protection, and interference elimination through precise regulation of the proportions of relevant components, providing a reliable technical solution for rapid clinical diagnosis.

[0007] FBS encapsulates and stabilizes C. difficile glutamate dehydrogenase, preventing its inactivation due to physical shear or chemical changes during dilution. Furthermore, the complex protein components in FBS compete with detergents for binding sites, reducing the detergent's potential damaging effects on CGDH while maintaining cell membrane lysis efficiency. BSA not only forms a molecular layer covering the CGDH surface, reducing its nonspecific binding to other components, but also inhibits CGDH self-aggregation or denaturation through steric hindrance. Detergents disrupt the C. difficile cell membrane through hydrophobic interactions, releasing intracellular CGDH. A specific mass ratio of BSA, FBS, and detergent balances lysis efficiency and enzyme protection, ensuring a detergent concentration sufficient to lyse cells without completely disrupting the CGDH structure. Anti-interference agents specifically block the binding sites of non-target substances in stool samples (such as bile acids, mucus, and other bacterial metabolites), reducing the risk of false positives.

[0008] The present invention controls the mass ratio of FBS:BSA to balance lysis efficiency and protection strength; and controls the ratio of BSA:detergent to improve the adaptability of different stool samples.

[0009] Furthermore, the mass ratio of the fetal bovine serum to the anti-interference agent is 1:0.004 to 1:0.05.

[0010] In the case where the mass ratio of BSA and detergent, and the mass ratio of FBS and BSA are determined as described above, the mass ratio of FBS and anti-interference agent is further defined, which helps to maintain the stability of the entire detection system. In the diluent system used to detect Clostridium difficile in fecal samples, FBS and BSA provide sufficient nutrition and protection for Clostridium difficile, maintain its activity and structure, thereby effectively extracting glutamate dehydrogenase, and enabling the anti-interference agent to play a good anti-interference role. If the ratio of anti-interference agent is too low, it may not be possible to completely eliminate the interference in the sample, resulting in inaccurate test results; and if the ratio of anti-interference agent is too high, it may affect the effect of fetal bovine serum on Clostridium difficile, and may even have a certain inhibition on the activity of glutamate dehydrogenase, thereby reducing the extraction efficiency.

[0011] The present invention can reasonably control the dosage of the anti-interference agent while ensuring the detection effect, thereby reducing the detection cost and improving the economy and practicality of the detection method.

[0012] Furthermore, the detergent includes at least one of Triton X100 or Tween 20.

[0013] Furthermore, the mass proportion of the fetal bovine serum in the diluent used to detect Clostridium difficile in fecal samples is 0.5% to 5%.

[0014] Furthermore, the mass proportion of the bovine serum albumin in the diluent used to detect Clostridium difficile in stool samples is 0.5% to 3%.

[0015] Furthermore, the mass proportion of the detergent in the diluent used for detecting Clostridium difficile in stool samples is 0.05% to 0.15%.

[0016] Furthermore, the diluent for detecting Clostridium difficile in stool samples also includes a buffer; the buffer includes Tris-HCl buffer, PBS ELISA buffer or PBS buffer.

[0017] Furthermore, the anti-interference agent includes IgG or a mixed antibody containing IgG.

[0018] Among them, IgG in the present invention includes mouse IgG.

[0019] In a second aspect, the present invention provides a kit for detecting Clostridium difficile in fecal samples, the kit comprising a diluent for detecting Clostridium difficile in fecal samples, a glutamate dehydrogenase capture antibody, an HRP-coupled glutamate dehydrogenase detection antibody, an HRP color development reagent, and a color development stop solution.

[0020] The kit provided by this invention not only offers advantages such as high sensitivity, excellent specificity, and strong anti-interference capabilities, but also comes pre-mixed with all components (diluent, antibodies, colorimetric reagent, etc.), eliminating the need for testers to dilute or mix the components themselves, thus avoiding test failures due to operational errors. The entire process, from sample processing to colorimetric termination, is standardized, shortening experimental time and increasing laboratory throughput. It can be stably stored for over one year at 2°C-8°C, meeting the needs of long-term clinical use and reducing the cost and management burden of frequent restocking.

[0021] The present invention provides a kit with good stability through coordinated optimization of components and pre-formulation standardized process, which significantly reduces the operation threshold and cost while improving detection accuracy.

[0022] Preferably, the glutamate dehydrogenase capture antibody comprises a mouse anti-Clostridium difficile glutamate dehydrogenase capture antibody and is coated in a 96-well plate.

[0023] In addition, the HRP-conjugated glutamate dehydrogenase detection antibody selected in the present invention can be used as the final antibody for direct color development reaction without the need for a secondary antibody. The results can be directly read using an enzyme-linked immunosorbent assay (ELISA) analyzer commonly used in clinical laboratories, achieving the goal of rapid analysis without the need for an expensive chemiluminescence analysis system.

[0024] The HRP-conjugated glutamate dehydrogenase detection antibody was prepared as follows: the enzyme-linked method was referred to ThermoScientific Traut's instructions; 0.1 mg of BM-CGDH-04 IgG1 antibody and 0.12 mg of HRP protein were reacted at room temperature for 2 hours and then transferred to 4°C for overnight reaction to obtain the HRP-conjugated glutamate dehydrogenase detection antibody.

[0025] In a third aspect, the present invention provides a method for detecting Clostridium difficile in a stool sample, using the above-mentioned kit for detecting Clostridium difficile in a stool sample to detect Clostridium difficile in the stool sample.

[0026] Furthermore, the detection is a quantitative detection.

[0027] Furthermore, the detection method comprises the following steps: Take one portion of the stool sample to be tested, add 4 to 6 portions of the stool sample diluent, and centrifuge to obtain the supernatant; The glutamate dehydrogenase capture antibody and the HRP-coupled glutamate dehydrogenase detection antibody were added to the supernatant in sequence, mixed and incubated in the dark for 15 min to 25 min, washed, and incubated in the dark for 20 min to 25 min after adding the HRP color development reagent. The reaction was terminated by adding the color development stop solution, and the results at a wavelength of 450 nm were read to calculate the content of Clostridium difficile glutamate dehydrogenase in the stool sample.

[0028] This kit is suitable for one-step detection of Clostridium difficile glutamate dehydrogenase in fecal samples. The total detection time is 35 minutes to 50 minutes. It can effectively solve the shortcomings of existing technologies such as high cost, long detection cycle, cumbersome operation or inconvenience in clinical use of Clostridium difficile detection.

[0029] The diluent provided by the present invention for detecting Clostridium difficile in stool samples balances the efficiency of Clostridium difficile lysis, protection of Clostridium difficile glutamate dehydrogenase, and elimination of matrix interference, ensuring detection accuracy. The kit provided by this diluent offers advantages such as good stability, high accuracy, low cost, and ease of use. This kit enables efficient and rapid detection of Clostridium difficile in stool samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 This is a standard curve diagram of CGDH in Example 5 of the present invention; Figure 2 This is a saturation curve of CDGH in the feces sample in Example 4 of the present invention; Figure 3 Figure 5 shows the stability test results of the kit in Example 5 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Horseradish peroxidase used in the present invention, HRP is purchased from Sigma, USA (P8375); FBS was purchased from Gibco, catalog number 10437-028; BSA was purchased from Sigma, bovine serum V; The anti-interference agent was mouse IgG purchased from Immuno Reagents Inc., with the product number MU-003-C; The detection antibody used was BM-CGDH-04 IgG1 antibody from ARXSciences; The capture antibody used was BM-CDHGH-6 IgG2 from ARX Sciences; Horseradish peroxidase color development solution was purchased from Thermo Scientific, catalog number P8250-25KU; The horseradish peroxidase color development stop solution was 2 M sulfuric acid; PBS ELISA buffer consists of: 10 mM PBS, 0.05% Tween 20, and the balance is deionized water.

[0034] Example 1 This embodiment provides a kit for detecting Clostridium difficile in stool samples, which consists of 100 mL of a diluent for detecting Clostridium difficile in stool samples, a 96-well plate coated with a glutamate dehydrogenase capture antibody, 6 mL of an HRP-conjugated glutamate dehydrogenase detection antibody solution, 12 mL of an HRP colorimetric reagent, 12 mL of a colorimetric stop solution, and 500 mL of PBS ELISA buffer.

[0035] The diluent used to detect Clostridium difficile in stool samples includes the following components: FBS 1%, BSA 1%, Tween200.05%, mIgG mixed antibody 0.05% and antibacterial agent Proclin 950 0.05%, and the balance is 20mM Tris-HCl buffer with a pH of 6.

[0036] The specific preparation method for a 96-well plate coated with a glutamate dehydrogenase capture antibody includes: selecting a standard high pH (9.5) 50mM carbonate buffer coating system, using BM-CDHGH-6 IgG2 antibody as the capture antibody, 3μg per well, and incubating at 4°C overnight. The plate is then blocked in 3% BSA as a blocking solution at room temperature for 30 minutes. After blocking, the 96-well plate is vacuum dried and sealed in a sealed bag containing 2 grams of silica gel desiccant.

[0037] Example 2 This embodiment provides a kit for detecting Clostridium difficile in stool samples, which consists of 100 mL of a diluent for detecting Clostridium difficile in stool samples, a 96-well plate coated with a glutamate dehydrogenase capture antibody, 6 mL of an HRP-conjugated glutamate dehydrogenase detection antibody, 12 mL of an HRP colorimetric reagent, 12 mL of a colorimetric stop solution, and 500 mL of PBS ELISA buffer.

[0038] The diluent used to detect Clostridium difficile in stool samples includes the following components: FBS 1%, BSA 1%, Tween200.05%, Triton X100 0.1%, mIgG mixed antibody 0.01% and antibacterial agent Proclin 950 0.05%, and the balance is 20mM Tris-HCl buffer with a pH of 6.

[0039] The preparation method of the 96-well plate coated with the glutamate dehydrogenase capture antibody is the same as that in Example 1.

[0040] Example 3 This embodiment provides a kit for detecting Clostridium difficile in stool samples, which consists of 100 mL of a diluent for detecting Clostridium difficile in stool samples, a 96-well plate coated with a glutamate dehydrogenase capture antibody, 6 mL of an HRP-conjugated glutamate dehydrogenase detection antibody, 12 mL of an HRP colorimetric reagent, 12 mL of a colorimetric stop solution, and 500 mL of PBS ELISA buffer.

[0041] The diluent used to detect Clostridium difficile in stool samples includes the following components: FBS 3%, BSA 1%, Tween 20 0.05%, mIgG mixed antibody 0.02% and antibacterial agent Proclin 950 0.05%, the balance is 20mM Tris-HCl buffer with a pH of 6 The preparation method of the 96-well plate coated with the glutamate dehydrogenase capture antibody is the same as that in Example 1.

[0042] Example 4 This embodiment provides a kit for detecting Clostridium difficile in stool samples, which consists of 100 mL of a diluent for detecting Clostridium difficile in stool samples, a 96-well plate coated with a glutamate dehydrogenase capture antibody, 6 mL of an HRP-conjugated glutamate dehydrogenase detection antibody, 12 mL of an HRP colorimetric reagent, 12 mL of a colorimetric stop solution, and 500 mL of PBS ELISA buffer.

[0043] The diluent used to detect Clostridium difficile in stool samples includes the following components: FBS 5%, BSA 1%, Tween200.05%, mIgG mixed antibody 0.02% and antibacterial agent Proclin 950 0.05%, and the balance is 20mM Tris-HCl buffer with a pH of 6.

[0044] The preparation method of the 96-well plate coated with the glutamate dehydrogenase capture antibody is the same as that in Example 1.

[0045] Example 5 This embodiment provides a method for detecting Clostridium difficile in a stool sample. This embodiment uses the kit provided in Example 1 as an example for illustration. The kits provided in Examples 2-4 can also achieve comparable results. The specific detection method is as follows: Step 1: Take out the reagents in the kit and the 96-well plate coated with the glutamate dehydrogenase capture antibody from the 2-8°C refrigerator and equilibrate them at room temperature.

[0046] Step 2: Take one aliquot (approximately 0.2g) of the stool sample to be tested and add 5 aliquots of a diluent for detecting Clostridium difficile in stool samples. Resuspend the sample and centrifuge it in a high-speed benchtop centrifuge at 13,000 rpm for 5 minutes. Collect the supernatant. This supernatant is stable at room temperature for three days or stored at -20°C for long-term storage. It can be frozen and thawed three or more times without clotting.

[0047] Step 3. Take 50 μL of the supernatant and add it to the wells of the above 96-well plate, then add 50 μL of HRP-coupled glutamate dehydrogenase detection antibody to each well and mix well. Cover with aluminum foil and keep it away from light at room temperature for 20 minutes. After removing the reaction solution, wash it 5 times with 350 mL of cleaning solution. After the last cleaning solution is removed, add 100 μL of HRP color reagent and keep it away from light for 20 minutes. Then add 100 μL of color stop solution to stop the reaction and read the results at a wavelength of 450 nm. According to Figure 1 The concentration of glutamate dehydrogenase in the stool sample to be tested was calculated using the standard curve of CGDH shown in Figure 1. The CGDH standard sample was diluted with the standard diluent prepared as follows: a normal human stool sample was diluted with the diluent for detecting Clostridium difficile in stool samples provided in Example 1 at a mass ratio of 1:5, centrifuged, and the supernatant was used as the standard diluent.

[0048] Each sample was measured three times in parallel and the mean value was taken.

[0049] The washing solution is PBS ELISA buffer.

[0050] Comparative Example 1 This comparative example provides a kit for detecting Clostridium difficile in fecal samples. The raw materials and their amounts in the kit are basically the same as those in Example 1, with the only difference being that "mIgG mixed antibody 0.05%" is replaced with "casein 0.5%". The remaining raw materials, their amounts, and preparation methods are the same as those in Example 1.

[0051] Comparative Example 2 This comparative example provides a kit for detecting Clostridium difficile in fecal samples. The raw materials and their amounts in the kit are basically the same as those in Example 2, except that "Triton X100 0.1%" is replaced by "SDS 0.1%". The remaining raw materials, their amounts, and the preparation method are the same as those in Example 1.

[0052] Comparative Example 3 This comparative example provides a kit for detecting Clostridium difficile in fecal samples. The raw materials and their amounts in the kit are basically the same as those in Example 3, except that "FBS 3%, mIgG mixed antibody 0.02%" is replaced with "FBS0%, mIgG mixed antibody 0.05%". The remaining raw materials, their amounts, and preparation methods are the same as those in Example 3.

[0053] Effect Example 1 Referring to the method of using the kit in Example 5, different diluents were used to dilute Clostridium difficile glutamate dehydrogenase (CDGH). This effect example investigated the application effect of the kit provided in Example 1-2 and Comparative Example 1-2 in detecting different concentrations of Clostridium difficile glutamate dehydrogenase (CDGH). The specific results are shown in Table 1.

[0054] Table 1

[0055] In Table 1, the CDGH standard was diluted in the Cal matrix using the diluent used for detecting Clostridium difficile in fecal samples to prepare dilutions of CDGH of corresponding concentrations; In Stool matrix, CDGH standard was diluted with healthy human stool samples to prepare CDGH dilution solutions of corresponding concentrations; The PBS ELISA buffer kit is based on the kit provided in Example 1, except that the diluent used for detecting Clostridium difficile in stool samples is replaced with PBS ELISA buffer, and the remaining raw materials and their amounts remain unchanged; The Thermo Scientific 37515 buffer-kit is the kit provided in Example 1, except that the diluent used for detecting Clostridium difficile in stool samples is replaced with Thermo Scientific 37515 buffer, and the remaining raw materials and their amounts remain unchanged.

[0056] It can be seen from the data in Table 1 that, compared with the comparative examples 1-2 in which the anti-interference agent or detergent was replaced, the kit protected by the examples 1-2 of the present invention can better detect CDGH in fecal samples.

[0057] Effect Example 2 In order to investigate the absence of FBS or the ratio of FBS to anti-interference agent, this effect example measured the application effect of the kits provided in Examples 3-4 and Comparative Example 3 in detecting CDGH in fecal samples. The specific results are shown in Table 2.

[0058] Table 2

[0059] Among them, the SD series refers to the dilution of the CDGH standard sample using the diluent for detecting Clostridium difficile in fecal samples provided in the corresponding comparative example or embodiment to prepare a dilution solution of CDGH with a corresponding series of concentrations; The PS series refers to diluting a normal human stool sample at a mass ratio of 1:5 using the diluent for detecting Clostridium difficile in fecal samples provided in the corresponding comparative example or embodiment, centrifuging, and taking the supernatant as the CDGH standard dilution to prepare a corresponding series of CDGH dilutions.

[0060] It can be seen from the data in Table 2 that the kit provided in Example 3 can better detect Clostridium difficile in stool samples.

[0061] Effect Example 3 This study verified the feasibility of CDGH detection in fecal samples. The details are as follows: 1 g of normal human stool sample was added with 5 mL of the diluent for detecting Clostridium difficile in stool sample provided in the kit provided in Example 2, mixed evenly and centrifuged for 5 minutes, and the supernatant was taken as the sample to be tested.

[0062] The CDGH standard was diluted using the normal human stool sample supernatant from Example 5 to prepare a CDGH dilution with a maximum concentration of 250 ng / mL. 50 μL of each sample concentration was added to a 96-well plate coated with the glutamate dehydrogenase capture antibody from the kit provided in Example 2. Similarly, another group was not treated with the CDGH standard to serve as a control group.

[0063] Then, add 50 μL of HRP-conjugated glutamate dehydrogenase detection antibody to each well and mix thoroughly. Cover with aluminum foil and incubate at room temperature for 20 minutes in the dark. Remove the reaction solution and wash five times with 350 mL of wash solution. After the final wash, add 100 μL of HRP colorimetric reagent and incubate in the dark for 20 minutes. Stop the reaction with 100 μL of colorimetric stop solution and read the results at 450 nm. The test results are shown in Table 3.

[0064] Table 3

[0065] As shown in Table 3, the concentrations of glutamate dehydrogenase in the fecal sample dilution group and the control group were positively correlated, and the negative sample could be used as a standard curve to eliminate the influence of the fecal sample.

[0066] Effect Example 4 This effect example measured the saturation curve of CDGH in fecal samples. Based on the standard curve prepared in Example 5, the highest concentration of CDGH was increased from 250 ng / mL to 500 μg / mL. The saturation curve of CDGH in fecal samples is shown in the figure below. Figure 2 and as shown in Table 4.

[0067] Table 4

[0068] Depend on Figure 2 As shown in Table 4, the quantitative analysis range of this method is 2000 ng / mL and below. Concentrations above 2000 ng / mL can be used for qualitative analysis.

[0069] Effect Example 5 This effect example investigates the stability of the kits provided in Examples 1-6. The stability test of the kits is conducted using an accelerated test at 37°C. The kits are placed at 37°C for 7 days to measure the activity of the reagents. Figure 3 shown.

[0070] The test kit was tested and found to maintain approximately 90% activity after being stored at 37°C for 7 days. It can be inferred that the kit can maintain approximately 90% activity after being stored at 4°C for 1 year.

[0071] Effect Example 6 This effect example compares the determination results of the kit provided in Example 2 with the clinical determination results. The experimental results show that the kit provided by the present invention has a high consistency with the clinical diagnosis results of the hospital. The specific results are as follows: The clinical samples in this invention were provided by the Second Affiliated Hospital of Hebei Medical College. After being assayed using the kit provided in Example 2 of this invention, they were sent to the hospital's laboratory research and development team for verification. Three batches of validation were conducted, with sample sizes of 6, 10, and 10, respectively.

[0072] The first batch of 6 clinical stool samples is used as an example for illustration. The kit provided in Example 2 of the present invention was used for measurement, and the measurement results and the hospital PCR result statistics are shown in Table 5.

[0073] Table 5

[0074] Among them, 4 samples were negative and 6 samples were positive in the second batch of 10 samples; all samples were negative in the third batch of 10 samples. The test results of the second and third batches of samples using the detection method of Clostridium difficile in fecal samples provided by the present invention were consistent with the test results of the hospital.

[0075] In summary, the detection method of Clostridium difficile in stool samples provided by the present invention and the hospital PCR results were used to measure 26 samples, and the results obtained were the same, with an accuracy rate of 100%.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A diluent for detecting Clostridium difficile in a stool sample, characterized in that: Including fetal bovine serum, bovine serum albumin, detergents and anti-interference agents; Wherein, the detergent comprises at least one of Triton X100, Tween 20 or NP40; The mass ratio of bovine serum albumin to detergent is 1:0.05 to 3:0.05; The mass ratio of the fetal bovine serum to bovine serum albumin is 5:1 to 1:

1.

2. The diluent for detecting Clostridium difficile in a stool sample according to claim 1, wherein: The mass ratio of the fetal bovine serum to the anti-interference agent is 1:0.004 to 1:0.

05.

3. The diluent for detecting Clostridium difficile in a stool sample according to claim 1, wherein: The mass proportion of the fetal bovine serum in the diluent for detecting Clostridium difficile in stool samples is 0.5% to 5%; and / or The mass proportion of the bovine serum albumin in the diluent for detecting Clostridium difficile in stool samples is 0.5% to 3%; and / or The mass proportion of the detergent in the diluent used for detecting Clostridium difficile in stool samples is 0.05% to 0.15%.

4. The diluent for detecting Clostridium difficile in a stool sample according to claim 1, wherein: Buffer is also included.

5. The diluent for detecting Clostridium difficile in a stool sample according to claim 1, wherein: The anti-interference agent includes IgG or mixed antibodies containing IgG.

6. A kit for detecting Clostridium difficile in stool samples, characterized in that: The invention comprises the diluent for detecting Clostridium difficile in stool samples according to any one of claims 1 to 5, as well as a glutamate dehydrogenase capture antibody, an HRP-coupled glutamate dehydrogenase detection antibody, an HRP color development reagent and a color development stop solution.

7. The kit for detecting Clostridium difficile in stool samples according to claim 6, wherein: The glutamate dehydrogenase capture antibody was coated in a 96-well plate.

8. A method for detecting Clostridium difficile in a stool sample, characterized in that: The kit for detecting Clostridium difficile in a stool sample according to claim 6 or 7 is used to detect Clostridium difficile in a stool sample.

9. The method for detecting Clostridium difficile in a stool sample according to claim 8, wherein: The detection is a quantitative detection.

10. The method for detecting Clostridium difficile in a stool sample according to claim 8, wherein: The detection method comprises the following steps: Take one portion of the stool sample to be tested, add 4 to 6 portions of the stool sample diluent, and centrifuge to obtain the supernatant; The glutamate dehydrogenase capture antibody and the HRP-coupled glutamate dehydrogenase detection antibody were added to the supernatant in sequence, mixed and incubated in the dark for 15 min to 25 min, washed, and incubated in the dark for 20 min to 25 min after adding the HRP color development reagent. The reaction was terminated by adding the color development stop solution, and the results at a wavelength of 450 nm were read to calculate the content of Clostridium difficile glutamate dehydrogenase in the stool sample.

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