Direct immunofluorescence reagent and kit for detecting Cryptosporidium parvum and their application

Abstract

The present invention discloses a direct immunofluorescence reagent, a kit and an application thereof for detecting Cryptosporidium parvum, and belongs to the field of biotechnology. The present invention uses the whole worm protein of Cryptosporidium parvum as an antigen and adopts the hybridoma cell monoclonal antibody technology to prepare a monoclonal antibody against Cryptosporidium parvum; the monoclonal antibody against Cryptosporidium parvum obtained in step S1 is labeled with a fluorescent dye to obtain a direct immunofluorescence detection reagent for detecting Cryptosporidium parvum. The direct immunofluorescence detection method established by using the reagent of the present invention can be used for the detection and identification of Cryptosporidium parvum in clinical stool samples and the environment, and has high sensitivity, strong specificity, good stability, fast diagnosis speed, and good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a direct immunofluorescence reagent and a kit for detecting Cryptosporidium parvum and applications thereof. Background Art

[0002] Cryptosporidium is an intracellular parasitic protozoan. After entering the host, it mainly parasitizes on the brush edge of gastrointestinal epithelial cells. Cryptosporidiosis is widely distributed worldwide and is the main cause of diarrhea in many animals and humans. In infected animals with normal immune function, diarrhea is short-term, generally lasting about 2-3 weeks. In immunocompromised people, such as AIDS patients, it is accompanied by long-term and persistent diarrhea, which can be life-threatening. Cryptosporidium is mainly transmitted through fecal-oral transmission, and humans and animals are mainly infected by accidentally ingesting contaminated food and water. In low-income and middle-income countries, cryptosporidiosis is one of the important causes of moderate to severe diarrhea and even death. In high-income countries, cryptosporidiosis is the main cause of foodborne and waterborne outbreaks and is one of the pathogens that must be tested for drinking water in many countries. As a zoonotic parasitic protozoan, Cryptosporidium seriously threatens human life and health and brings huge economic losses to the development of animal husbandry. Therefore, rapid detection of Cryptosporidium is conducive to early detection and prevention of the disease, reducing the incidence rate, which is crucial to human health and animal husbandry development.

[0003] Currently, the commonly used methods for detecting Cryptosporidium include pathogen detection, immunological detection, and molecular detection. Among them, the sensitivity of pathogen detection is limited and it relies on professional technicians. Molecular detection has good sensitivity and can distinguish between species and subtypes, but it also has disadvantages such as being time-consuming and requiring high technical requirements. Immunological detection has higher sensitivity than other methods and has been widely used in the detection of Cryptosporidium. Among them, immunofluorescence detection based on monoclonal antibodies is a better detection method that can detect samples with a small number of oocysts. At present, commercial kits based on immunological methods have been launched abroad, such as those from Waterborne Company in the United States. The kit can detect Cryptosporidium oocysts by direct immunofluorescence. The antibodies produced by it are targeted at Cryptosporidium oocyst wall antigens and are used to detect Cryptosporidium in feces, food and environmental samples. The current international gold standard for detecting Cryptosporidium in water is the EPA 1623 method, which also involves the antibodies in the kit. The antibody is expensive and has a low titer (1:20), so it cannot be widely used. Therefore, the development of a higher-titer direct immunofluorescence reagent and kit for detecting Cryptosporidium parvum can greatly reduce the cost of testing, which is of great significance to the detection, diagnosis and prevention and control of cryptosporidiosis in human health, clinical, scientific research institutions and aquaculture. Summary of the invention

[0004] To solve the related problems, the primary purpose of the present invention is to provide a direct immunofluorescence reagent for detecting Cryptosporidium parvum.

[0005] Another object of the present invention is to provide a direct immunofluorescence kit for detecting Cryptosporidium parvum.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] A method for preparing a direct immunofluorescence detection reagent for detecting Cryptosporidium parvum comprises the following steps:

[0008] S1. Using the whole protein of Cryptosporidium parvum as an antigen, a monoclonal antibody against Cryptosporidium parvum is prepared by hybridoma cell monoclonal antibody technology;

[0009] S2. Labeling the anti-Cryptosporidium parvum monoclonal antibody obtained in step S1 with a fluorescent dye to obtain a direct immunofluorescence detection reagent for detecting Cryptosporidium parvum.

[0010] Preferably, in the step S1, the whole worm protein of Cryptosporidium parvum is obtained by the following method: taking Cryptosporidium parvum oocysts, centrifuging at 13200r / min for 3min at 4°C, treating with sodium hypochlorite solution on ice for 10min, centrifuging and removing the sodium hypochlorite solution, adding protease inhibitors and PBS, performing ultrasonic fragmentation on ice, and then repeatedly freezing and thawing in liquid nitrogen to obtain a whole worm protein solution of Cryptosporidium parvum.

[0011] More preferably, the concentration of the sodium hypochlorite solution is 1.3 wt %.

[0012] More preferably, the volume ratio of the protease inhibitor to PBS is 1:200.

[0013] Preferably, in step S1, the subclass of the monoclonal antibody against Cryptosporidium parvum is IgG1, which reacts only with the oocyst wall of Cryptosporidium parvum.

[0014] Preferably, in step S1, the specific steps of preparing anti-Cryptosporidium parvum monoclonal antibodies using hybridoma cell monoclonal antibody technology are:

[0015] (1) Whole worm protein of Cryptosporidium parvum is mixed with an equal volume of adjuvant, emulsified and injected subcutaneously at multiple points on the back of mice for immunization. Second to fourth immunizations are performed on the 14th, 28th, and 35th days after the first immunization, respectively. On the 7th day after the fourth immunization, blood is collected by tail cutting to separate serum, and the antibody titer of the serum is detected by indirect ELISA. After the serum antibody titer reaches 100,000, the mice are intraperitoneally immunized with a whole worm protein solution without adjuvant, and fusion is performed 3 days later;

[0016] (2) Cell fusion: Mouse spleen cells and SP2 / 0 myeloma cells were placed in a fusion bottle at a ratio of 5:1. After centrifugation, the supernatant was discarded. After the cell pellet was resuspended, preheated 50% PEG fusion agent was added along the tube wall. Serum-free DMEM medium was slowly added to terminate the fusion. After standing, centrifugation was performed. The cell pellet was suspended with HAT medium, mixed, added to a cell culture plate containing feeder cells, and cultured in a constant temperature CO2 incubator.

[0017] (3) Screening and subcloning of hybridoma cell lines: Use indirect ELISA and indirect immunofluorescence to simultaneously screen the target positive fusion cells, and perform two rounds of subcloning to obtain hybridoma cell lines that can stably secrete proteins against the oocyst wall of Cryptosporidium parvum; when using the indirect ELISA method, the whole worm protein of Cryptosporidium parvum is used as the detection antigen, and when using the indirect immunofluorescence method, Cryptosporidium parvum is used as the detection antigen;

[0018] (4) Preparation of monoclonal antibodies: Multiparous female BALB / c mice were selected and injected intraperitoneally with pristane. Seven days later, hybridoma cells were injected intraperitoneally. When the abdominal cavity of the mice was obviously bulging, the ascites of the mice was collected. The collected ascites was centrifuged and the supernatant was preserved. After purification, the monoclonal antibodies against Cryptosporidium parvum were obtained.

[0019] More preferably, in step (1), Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the second to fourth immunizations.

[0020] More preferably, in the step (2), the centrifugal conditions are: rotation speed 1000 r / min, time 5 min.

[0021] More preferably, in the step (2), the preheating temperature is 42°C.

[0022] More preferably, in step (4), the injection volume of pristane is 0.5 mL / head.

[0023] More preferably, in step (4), the injection volume of hybridoma cells is 1×10 6 Piece / piece.

[0024] More preferably, in the step (4), the centrifugal conditions are: rotation speed 10000 r / min, time 10 min.

[0025] More preferably, in the step (4), the storage temperature is -20°C.

[0026] Preferably, in the step S1, the specific steps of labeling the anti-Cryptosporidium parvum monoclonal antibody obtained in step S1 with a fluorescent dye are: adding a boric acid buffer to the diluted monoclonal antibody solution, and then adding it to the fluorescent dye reagent, incubating at room temperature in the dark, taking it out and placing it in a centrifugal column filled with a purification resin, shaking it slightly to completely mix the fluorescent dye-labeled antibody with the resin, and obtaining the fluorescently labeled antibody after centrifugation.

[0027] More preferably, the concentration of the diluted monoclonal antibody solution is 2 mg / mL.

[0028] More preferably, the ratio of the diluted monoclonal antibody solution, borate buffer and fluorescent dye is 0.5 mL: 40 μL: 50 μg.

[0029] A direct immunofluorescence detection reagent for detecting Cryptosporidium parvum is obtained by the above preparation method.

[0030] A direct immunofluorescence kit for detecting Cryptosporidium parvum, wherein the immunofluorescence reagent is the above-mentioned direct immunofluorescence reagent for detecting Cryptosporidium parvum.

[0031] The application of the direct immunofluorescence detection reagent or kit for detecting Cryptosporidium parvum in the detection of Cryptosporidium in the environment.

[0032] The environment includes but is not limited to water, soil, and feces.

[0033] The application comprises the following steps: using the above-mentioned direct immunofluorescence detection reagent or kit to detect the sample to be detected; observing under a fluorescence microscope, and judging it as positive if specific apple green fluorescence appears.

[0034] The technical principle of the present invention is as follows: the present invention uses the whole protein of Cryptosporidium parvum as an antigen, adopts the hybridoma cell monoclonal antibody technology to prepare an immunofluorescence reagent for detecting the oocyst wall of Cryptosporidium parvum, and further establishes a direct immunofluorescence detection method and a kit, which can be used for rapid detection of Cryptosporidium parvum. The direct immunofluorescence detection time is short, and the results can be reported in about 2.5 hours, with high accuracy and strong specificity, and it only reacts with the oocyst wall of Cryptosporidium parvum.

[0035] Compared with the prior art, the present invention has the following advantages and effects:

[0036] (1) The direct immunofluorescence detection method established using the reagent of the present invention can be used for the detection and identification of Cryptosporidium parvum in clinical stool samples and the environment.

[0037] (2) High sensitivity: The prepared monoclonal antibodies have high titers, thus ensuring that the prepared immunofluorescent antibodies have good sensitivity.

[0038] (3) High specificity: The prepared immunofluorescence reagent has no cross-reaction with other pathogens (Escherichia coli, Toxoplasma gondii, Eimeria) and only detects Cryptosporidium parvum, which has a high specificity.

[0039] (4) Good stability: Immunofluorescence reagents can be stored at -20°C for more than 12 months and have good stability.

[0040] (5) Fast diagnosis: The results can be reported in about 2.5 hours, which can save 1-2 hours compared with the indirect immunofluorescence detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a microscope image of the positive result of the reagent in the embodiment of the present invention in detecting Cryptosporidium parvum;

[0042] Figure 2 This is a microscope image of the negative result of the reagent in the embodiment of the present invention in detecting Escherichia coli;

[0043] Figure 3 This is a microscope image of a negative result of Toxoplasma gondii detected by the reagent of an embodiment of the present invention;

[0044] Figure 4 This is a microscope image of the negative result of the reagent in the embodiment of the present invention in detecting Eimeria;

[0045] Figure 5 This is a microscopic image of a negative result of a control test for Cryptosporidium parvum in an embodiment of the present invention;

[0046] Figure 6 A microscope image of a sample of an environment tested by a reagent according to an embodiment of the present invention;

[0047] Figure 7 This is a microscope picture of a clinical stool sample tested by the reagent of an embodiment of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0049] Example 1: Preparation of Cryptosporidium parvum whole worm protein antigen

[0050] Take an appropriate amount of Cryptosporidium parvum oocysts, centrifuge at 13200r / min for 3min at 4℃, treat with sodium hypochlorite solution (concentration of 1.3wt%) on ice for 10min, centrifuge and remove the sodium hypochlorite solution, wash twice with PBS, then add protease inhibitors and PBS (volume ratio 1:200), ultrasonically break on ice for 30min, and then freeze and thaw repeatedly in liquid nitrogen for 3 times to obtain Cryptosporidium parvum whole worm protein solution. Use NanodropTM One instrument detects whole insect protein concentration.

[0051] Example 2: Preparation of anti-Cryptosporidium parvum monoclonal antibodies

[0052] 1. Animal immunization: Three 6-week-old female BALB / c mice were selected, and the whole worm protein solution of Cryptosporidium parvum was mixed with an equal volume of adjuvant. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second to fourth immunizations. The protein amount was 50 μg / mouse. After emulsification, each mouse was immunized by multiple subcutaneous injections on the back. The second to fourth immunizations were performed on the 14th, 28th, and 35th days after the first immunization, respectively; on the 7th day after the fourth immunization, the tail was cut off and blood was collected to separate the serum. The antibody titer of the serum was detected by the indirect ELISA method. After the serum antibody titer reached 100,000, the mice were immunized by intraperitoneal impact with the whole worm protein solution without adjuvant, and fusion was performed 3 days later.

[0053] 2. Cell fusion: Place mouse spleen cells and SP2 / 0 myeloma cells in a fusion bottle at a ratio of 5:1. Centrifuge at 1000r / min for 5 minutes, discard the supernatant, beat the cell pellet evenly, add 1mL of 50% PEG fusion agent preheated to 42°C along the tube wall, slowly add 30mL of serum-free DMEM medium to terminate the fusion, let stand for 10 minutes, and centrifuge at 1000r / min for 5 minutes. Use HAT medium to suspend the cell pellet, mix it, and add 100μL per well to the 96-cell culture plate containing feeder cells prepared the day before, and culture it in a constant temperature CO2 incubator.

[0054] 3. Screening and subcloning of hybridoma cell lines: Indirect ELISA and indirect immunofluorescence were used to simultaneously screen the target positive fusion cells. The whole worm protein of Cryptosporidium parvum was used as the detection antigen when the indirect ELISA method was used, and Cryptosporidium parvum was used as the detection antigen when the indirect immunofluorescence method was used. After screening and two rounds of subcloning, hybridoma cells that can stably secrete proteins targeting the oocyst wall of Cryptosporidium parvum were obtained.

[0055] 4. Preparation of monoclonal antibodies: Multiparous female BALB / c mice were selected and intraperitoneally injected with pristane (0.5 mL / mouse). Seven days later, 1×10 6 The mouse ascites was collected when the mouse abdomen was obviously bulging about 7 days later. The collected mouse ascites was centrifuged at 10000g / min for 10min and the supernatant was stored at -20℃.

[0056] 5. Monoclonal antibody purification: Take Protein A + G Agarose to load the column, balance the column with 20mL TBS buffer, add mouse ascites to the chromatography column after passing through a 0.45μm filter membrane, mix it with Protein A + G Agarose, and mix it in a 4℃ chromatography cabinet for 30 minutes to make it fully hang on the column. After the mouse ascites flows through the column, rinse it with 20mL TBS buffer to elute non-specific impurities, and then elute the antibody with 6mL 50mM glycine (PH: 1.9). The collected antibodies are concentrated in a 10k ultrafiltration tube at 5000g / min and centrifuged for 30min, and the purified antibodies are replaced with PBS, the concentration is measured, and the antibodies are stored in a -80℃ refrigerator.

[0057] 6. Monoclonal antibody characterization

[0058] (1) According to the instructions of the Proteintech monoclonal antibody subtype identification kit, the subclass of the 2E1-C3 monoclonal antibody was determined to be IgG1.

[0059] (2) Identification of monoclonal antibody specificity: The obtained monoclonal antibodies were subjected to direct immunofluorescence detection with Cryptosporidium parvum, Escherichia coli, Toxoplasma gondii, and Eimeria, respectively. The results showed that the obtained monoclonal antibodies only reacted with the oocyst wall of Cryptosporidium parvum and had no cross-reaction with other pathogens. The identification results are shown in Table 1.

[0060] Table 1. DFA identification of specific anti-Cryptosporidium parvum monoclonal antibodies

[0061] Sample name Cryptosporidium parvum Escherichia coli Toxoplasma gondii Eimeria PBS result + - - - -

[0062] Note: “-” means no specific fluorescence, “+” means specific fluorescence.

[0063] Example 3: Preparation of immunofluorescence reagents

[0064] The monoclonal antibody against the oocyst wall of Cryptosporidium parvum was fluorescently labeled according to the Thermo Fisher labeling kit. 40 μL of boric acid buffer was added to 0.5 mL of antibody solution diluted to 2 mg / mL, and then the above solution was transferred to a centrifuge tube containing 50 μg of DyLight fluorescent dye. The tube was gently vortexed and centrifuged. After incubation at room temperature in the dark for 60 min, the tube was taken out and placed in a centrifuge column containing purification resin. The tube was slightly shaken to completely mix the labeled antibody and the resin. The fluorescently labeled antibody was obtained after centrifugation at 1000 g / min for 1 min at room temperature. The tube was aliquoted and stored at -20°C in the dark for later use.

[0065] Example 4: Establishment of direct immunofluorescence detection method

[0066] Take an appropriate amount of Cryptosporidium parvum oocysts, treat with sodium hypochlorite solution on ice for 10 minutes, centrifuge at 13200r / min for 3 minutes at 4°C, resuspend the oocysts in PBS, air-dry for 10 minutes, and then fix with methanol at room temperature for 15 minutes, wash with PBS three times, wash away the methanol, and then incubate with 5% TritonX-100 at 37°C for 40 minutes, and then dilute the labeled fluorescent antibody according to the appropriate concentration and take 30μL to each well, incubate at 37°C for 40 minutes, and then wash with PBS. Take the diluted DAPI dye solution in each well, incubate at 37°C for 5 minutes, and then wash with PBS. After the slide is dry, take 7μL of anti-fluorescence quencher in the center of each well, seal the slide and observe under a fluorescence microscope. The appearance of specific apple green fluorescence is judged as positive, and the control well is judged as negative if no specific apple green fluorescence appears.

[0067] Example 5: Identification of immunofluorescence reagent specificity

[0068] 1. Determination of the working concentration of the fluorescent reagent: Take an appropriate amount of Cryptosporidium parvum oocysts for spot drying, permeabilization, and then dilute the immunofluorescence reagent in multiples for direct immunofluorescence test. The dilution multiple that produces clear, bright, and specific apple green fluorescence is the maximum dilution multiple of the reagent. The results showed that the highest dilution multiple of the fluorescent reagent was 1:1200. To ensure the sensitivity and effectiveness of the fluorescent reagent, the working concentration was determined to be 1:400.

[0069] 2. Specificity detection of fluorescent reagent: After diluting the fluorescent antibody, Escherichia coli, Toxoplasma gondii, and Eimeria were taken as controls for direct immunofluorescence test. The results showed that only the oocyst wall of Cryptosporidium parvum showed specific apple green fluorescence under the fluorescence microscope, which was a positive reaction; other pathogens did not show specific apple green fluorescence, which was a negative reaction; the negative control wells also did not show specific green fluorescence, indicating that the prepared immunofluorescence reagent had good specificity and could be used for the detection and identification of Cryptosporidium parvum. The results are shown in Table 2.

[0070] Table 2. Specificity detection results of fluorescent reagents

[0071] Sample name Cryptosporidium parvum Escherichia coli Toxoplasma gondii Eimeria PBS result + - - - -

[0072] 3. Repeatability and stability test of immunofluorescence reagent: After the immunofluorescence reagent was packaged and stored at -20℃ for 3, 6, 9, and 12 months, a direct immunofluorescence test was performed to determine the shelf life and stability of the reagent. The results showed that the titer of the fluorescent antibody remained unchanged after the immunofluorescence reagent was stored at -20℃ for 12 months. Therefore, the storage period was temporarily set at -20℃ for 12 months.

[0073] Example 6: Application of direct immunofluorescence detection kit

[0074] 1. Detection of Cryptosporidium parvum in environmental samples

[0075] 5,000 Cryptosporidium parvum oocysts were artificially implanted into lake water from the environment, and tested using a direct immunofluorescence detection kit. The results showed that the Cryptosporidium parvum in the lake water emitted a clear, bright, and specific apple green fluorescence, indicating that the kit has good specificity and can be used for the detection of environmental samples.

[0076] 2. Detection of Cryptosporidium parvum in clinical stool samples

[0077] The direct immunofluorescence detection kit for Cryptosporidium parvum was used to detect 84 clinical stool samples from Ningxia, and the qPCR test results were used as a control. The results showed that the positive clinical stool samples containing 10,000 oocysts per gram of stool had a detection rate of 33% (4 / 12), and the positive clinical stool samples containing 100,000 oocysts or more per gram of stool had a detection rate of 100% (32 / 32), and bright, specific apple green fluorescence appeared. The positive samples containing 1,000 oocysts per gram of stool and the negative stool samples were not detected. The test results show that the kit has good specificity and can be used for the detection of clinical stool samples.

[0078] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a direct immunofluorescence detection reagent for detecting Cryptosporidium parvum, characterized in that: The steps include: S1. Using the whole protein of Cryptosporidium parvum as an antigen, a monoclonal antibody against Cryptosporidium parvum is prepared by hybridoma cell monoclonal antibody technology; S2, labeling the anti-Cryptosporidium parvum monoclonal antibody obtained in step S1 with a fluorescent dye, thereby obtaining a direct immunofluorescence detection reagent for detecting Cryptosporidium parvum; In the step S1, the whole worm protein of Cryptosporidium parvum is obtained by the following method: taking Cryptosporidium parvum oocysts, centrifuging at 13200r / min for 3 min under 4°C, treating with sodium hypochlorite solution on ice for 10 min, centrifuging and removing the sodium hypochlorite solution, adding protease inhibitors and PBS, performing ultrasonic fragmentation on ice, and then repeatedly freezing and thawing in liquid nitrogen to obtain a whole worm protein solution of Cryptosporidium parvum; the concentration of the sodium hypochlorite solution is 1.3wt%; the volume ratio of the protease inhibitor to PBS is 1:200; In the step S1, the subclass of the monoclonal antibody against Cryptosporidium parvum is IgG1, which reacts only with the oocyst wall of Cryptosporidium parvum; the specific steps of preparing the monoclonal antibody against Cryptosporidium parvum using hybridoma cell monoclonal antibody technology are as follows: (1) The whole worm protein of Cryptosporidium parvum was mixed with an equal volume of adjuvant, emulsified and injected subcutaneously at multiple points on the back of mice for immunization. The second to fourth immunizations were performed on the 14th, 28th and 35th days after the first immunization, respectively. On the 7th day after the fourth immunization, the tail was cut off and blood was collected to separate the serum. The antibody titer of the serum was detected by the indirect ELISA method. After the serum antibody titer reached 100,000, the mice were intraperitoneally immunized with the whole worm protein solution without adjuvant, and fusion was performed 3 days later. (2) Cell fusion: Place mouse spleen cells and SP2 / 0 myeloma cells in a fusion bottle at a ratio of 5:1, discard the supernatant after centrifugation, resuspend the cell pellet, add preheated 50% PEG fusion agent along the tube wall, slowly add serum-free DMEM medium to terminate fusion, let stand and then centrifuge; suspend the cell pellet with HAT medium, add to the cell culture plate containing feeder cells after mixing, and place in a constant temperature CO2 incubator for culture; (3) Screening and subcloning of hybridoma cell lines: Use indirect ELISA and indirect immunofluorescence to simultaneously screen the target positive fusion cells, and perform two rounds of subcloning to obtain hybridoma cell lines that can stably secrete proteins targeting the oocyst wall of Cryptosporidium parvum; The whole protein of Cryptosporidium parvum was used as the detection antigen when the indirect ELISA method was used, and the Cryptosporidium parvum was used as the detection antigen when the indirect immunofluorescence method was used; (4) Preparation of monoclonal antibodies: Select multiparous female BALB / c mice and inject them intraperitoneally with pristane. Seven days later, inject hybridoma cells intraperitoneally. When the abdominal cavity of the mice is obviously bulging, the mouse ascites is collected. The collected mouse ascites is centrifuged and the supernatant is preserved. After purification, the monoclonal antibody against Cryptosporidium parvum is obtained.

2. The method for preparing the direct immunofluorescence detection reagent for detecting Cryptosporidium parvum according to claim 1, characterized in that: In the step (1), Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the second to fourth immunizations; In the step (2), the centrifugal conditions are: speed 1000r / min, time 5 min; In the step (2), the preheating temperature is 42°C; In the step (4), the injection volume of pristane is 0.5 mL / mouse; In step (4), the injection volume of hybridoma cells is 1×10 6 Piece / piece; In the step (4), the centrifugal conditions are: speed 10000 r / min, time 10 min; In the step (4), the storage temperature is -20°C.

3. The method for preparing a direct immunofluorescence detection reagent for detecting Cryptosporidium parvum according to any one of claims 1 to 2, characterized in that: In the step S1, the specific steps of labeling the monoclonal antibody against Cryptosporidium parvum obtained in step S1 with a fluorescent dye are as follows: adding a boric acid buffer to the diluted monoclonal antibody solution, and then adding it to the fluorescent dye reagent, incubating at room temperature in the dark, taking it out and placing it in a centrifugal column filled with a purification resin, shaking it slightly to completely mix the fluorescent dye-labeled antibody with the resin, and obtaining the fluorescently labeled antibody after centrifugation.

4. The method for preparing the direct immunofluorescence detection reagent for detecting Cryptosporidium parvum according to claim 3, characterized in that: The concentration of the diluted monoclonal antibody solution is 2 mg / mL; The ratio of the diluted monoclonal antibody solution, borate buffer and fluorescent dye is 0.5 mL: 40 μL: 50 μg.

5. A direct immunofluorescence detection reagent for detecting Cryptosporidium parvum, characterized in that: The method is obtained by the preparation method described in any one of claims 1 to 4.

6. A direct immunofluorescence kit for detecting Cryptosporidium parvum, characterized in that: The immunofluorescence reagent is the direct immunofluorescence reagent for detecting Cryptosporidium parvum as described in claim 5.

7. Use of the direct immunofluorescence detection reagent for detecting Cryptosporidium parvum according to claim 5 or the direct immunofluorescence detection kit for detecting Cryptosporidium parvum according to claim 6 in detecting Cryptosporidium in an environment, characterized in that: The environment includes water, soil, and feces.

8. The use according to claim 7, characterized in that: The application comprises the following steps: using the direct immunofluorescence detection reagent or kit to detect the sample to be detected; observing under a fluorescence microscope, and judging it as positive if specific apple green fluorescence appears.

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