Preparation method of direct immunofluorescence detection kit, kit and joint detection method

Abstract

The present invention discloses a preparation method of a direct immunofluorescence detection kit for multi-pathogen joint detection, a kit for multi-pathogen joint detection, and a multi-pathogen joint detection method, and belongs to the field of immunoassay technology. The preparation method comprises the following steps: providing pathogen control slides and specimen slides for multi-pathogen joint detection; providing a sample diluent; providing a concentrated washing solution for cleaning the pathogen control slide carrying the sample; providing a fluorescein conjugate; and providing a fluorescent antibody diluent. The components of the fluorescent antibody diluent in the preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the multi-pathogen joint detection method provided by the present invention have a broad spectrum and good compatibility, and do not need to be adjusted due to different detection antigens, and the same fluorescent antibody diluent can meet the detection of all antigen substrates.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay detection, and in particular to a method for preparing a direct immunofluorescence detection kit for multi-pathogen joint detection, a kit for multi-pathogen joint detection, and a multi-pathogen joint detection method. Background Art

[0002] Respiratory tract infections include infectious inflammation of the nose, pharynx, larynx, trachea, bronchi and lungs, and are divided into upper respiratory tract infections and lower respiratory tract infections based on the larynx. Most respiratory tract infections are upper respiratory tract infections. According to statistics, more than 90% are caused by viruses, and a few are caused by bacteria. Lower respiratory tract infections are caused by microorganisms such as viruses, bacteria, mycoplasmas, and chlamydia. The main clinical manifestations are acute tracheobronchitis, chronic pneumonia, bronchiectasis, etc. During treatment, the pathogen causing the infection must be identified in order to select an effective treatment plan. One pathogen can cause multiple clinical symptoms, and the same clinical manifestation can be caused by multiple pathogens. Not all clinical symptoms can be accurately judged. According to statistics, among people with respiratory tract infections, the proportion of partial complex infections is about 20%. The detection of a single virus or bacteria and other pathogens can no longer assist in more accurate clinical judgments.

[0003] At present, the commonly used laboratory pathogen detection methods mainly include electron microscopy, separation and culture, antigen determination, specific antibody detection and molecular biology. Among them, electron microscopy detection of virus particles can identify pathogens to a certain extent, but this inspection method takes a long time, the positive rate is not high, and the time is long, which is not suitable for rapid clinical diagnosis. The separation and culture method takes a long time to detect, and false negatives will occur when the number of viruses contained in the specimen is small. It also has high requirements, such as material collection, environment, and operation technology, and is not popular in clinical practice. Antigen determination can clarify the evidence of infection and detect quickly, but its sensitivity is low; and although the detection of specific antibodies is specific and sensitive, it has a window period for early detection of infection and cannot provide timely auxiliary diagnosis; although the molecular biology method is highly sensitive and has rapid detection, it has high requirements for the experimental environment and is not easy to promote. Summary of the invention

[0004] In order to solve at least one aspect of the above problems and defects in the prior art, the present invention provides a method for preparing a direct immunofluorescence detection kit for multi-pathogen joint detection, a kit for multi-pathogen joint detection, and a multi-pathogen joint detection method. The technical solution is as follows:

[0005] One object of the present invention is to provide a method for preparing a direct immunofluorescence detection kit for multi-pathogen joint detection.

[0006] Another object of the present invention is to provide a kit for multi-pathogen joint detection.

[0007] Another object of the present invention is to provide a method for joint detection of multiple pathogens.

[0008] According to one aspect of the present invention, a method for preparing a direct immunofluorescence detection kit for multi-pathogen joint detection is provided, and the preparation method comprises the following steps:

[0009] Provide pathogen control slides and blank slides for multi-pathogen joint testing;

[0010] Provide sample diluent;

[0011] Provides concentrated wash solution for washing pathogen control slides loaded with specimens;

[0012] providing a fluorescein conjugate; and

[0013] Provide fluorescent antibody diluent;

[0014] Among them, the components of the fluorescent antibody diluent are: 0.1%~0.5% disodium hydrogen phosphate, 0.01%~0.05% sodium dihydrogen phosphate, 0.5%~1.5% sodium chloride, 0.01%~0.05% potassium chloride, 2%~8% sucrose, 10%~30% glycerol, 0.05%~0.3% casein sodium salt, 5%~20% bovine serum, 0.01%~0.1% Tween 20, 0.01%~0.1% TritonX-100, 0.005%~0.03% Evans blue, 0.01%~0.05% gentamicin, 0.05%~0.5% 4-aminoantipyrine, 0.005%~0.03% PC-300 and distilled water.

[0015] Specifically, 0.1% to 0.5% of disodium hydrogen phosphate is 0.2% to 0.4% of disodium hydrogen phosphate dodecahydrate, 0.01% to 0.05% of sodium dihydrogen phosphate is 0.02% to 0.04% of sodium dihydrogen phosphate dihydrate, and 2% to 8% of sucrose is 4% to 7% of sucrose.

[0016] Preferably, the fluorescein conjugate is a monoclonal antibody labeled with fluorescein.

[0017] During the test, the fluorescein conjugate is diluted with the fluorescent antibody diluent so that the concentration of the fluorescein conjugate in the fluorescent antibody diluent is 10-200µg / ml.

[0018] Specifically, the pathogen control slide is prepared by the following method, which includes the following steps:

[0019] Step S1: performing cell subculture on various pathogens respectively;

[0020] Step S2: preparing cell suspensions of at least two passaged cells of the plurality of pathogens respectively;

[0021] Step S3: applying the prepared cell suspension to different wells on the same glass slide, and after the cells in the cell suspensions in the wells of different glass slides are adsorbed, fixing the cells in the wells of different glass slides under the same conditions;

[0022] The multiple pathogens include any one of respiratory system infection pathogens and reproductive system infection pathogens and their combinations,

[0023] In step S3, the same conditions are to use anhydrous ethanol to fix for the same time at the same temperature, and then dry for the same time in the same humidity environment with a humidity of less than 50% to form a pathogen control slide.

[0024] Preferably, the same temperature condition is the same temperature selected from the range of 23°C to 27°C, the same fixed time is the same time selected from the range of 30min to 3h, and the same drying time is the same drying time selected from the range of 30min to 3h.

[0025] More preferably, the same temperature condition is 25°C, the same fixing time is 1 hour, the same drying time is 2 hours, and the anhydrous ethanol is -20°C anhydrous ethanol.

[0026] The humidity is less than 50% and is selected from the range of 10% to 20%.

[0027] In step S2, the preparation of the cell suspension includes the preparation of the bacterial suspension and the preparation of the non-bacterial infected cell suspension.

[0028] Specifically, the preparation of the bacterial suspension comprises the following steps:

[0029] The subcultured bacterial cells were placed in sterile saline to prepare a bacterial suspension;

[0030] The preparation of a cell suspension for infection with non-bacteria includes the following steps:

[0031] Step S21: digesting and separating at least two passage cells of the plurality of pathogens respectively with trypsin in a concentration range of 0.1% to 0.5%, and stopping the digestion and separation and aspirating the trypsin when at least two passage cells are detached from the bottom of the culture vessel;

[0032] Step S22: placing the at least two types of passaged cells after digestion and separation in a CO2 incubator with a humidity of ≥90%, a temperature of 37°C, and a CO2 concentration of 5% for adsorption for 1 h to 3 h;

[0033] Step S23: supplementing the at least two passaged cells after adsorption with a fetal bovine serum medium in a concentration range of 5% to 20%, and then continuing to infect the cells in a CO2 incubator with a humidity of ≥90%, a temperature of 37°C, and a CO2 concentration of 5% for 24 hours to 96 hours, until all pathogenic cells in at least two passaged cells become pathological;

[0034] Step S24: digest and separate the at least two passaged cells with pathological changes with trypsin in a concentration range of 0.1% to 0.5%, respectively; when the at least two passaged cells with pathological changes are detached from the bottom of the culture vessel, add fetal bovine serum culture medium in a concentration range of 1% to 5% to stop digestion and form a cell suspension.

[0035] Specifically, the preparation of the non-bacterial infected cell suspension comprises the following steps:

[0036] The primary cells of the plurality of pathogens are added to the corresponding passaged cells of the pathogens of the plurality of pathogens for infection for a predetermined time;

[0037] Digesting and separating at least two infected pathogen cells among the multiple pathogens to ensure cell viability, continuing to culture the at least two infected pathogen cells after digestion and separation until the cells become diseased, and then preparing the at least two infected pathogen cells that have become diseased into a cell suspension;

[0038] Among them, primary cells of various pathogens do not include bacterial cells.

[0039] The ratio of primary cells to passage cells ranges from 1:2 to 1:1, and the scheduled infection time ranges from 24h to 72h.

[0040] According to another aspect of the present invention, a kit for multi-pathogen joint detection is provided, wherein:

[0041] The kit is prepared according to any of the above methods for preparing a direct immunofluorescence kit for multi-pathogen joint detection.

[0042] According to another aspect of the present invention, a multi-pathogen joint detection method is provided, the joint detection method comprising the following steps:

[0043] Providing a kit, wherein the kit is the above kit;

[0044] Prepare the concentrated wash buffer in the kit and pretreat the samples;

[0045] The pretreated samples were added to different slide wells on the pathogen control slide, and then placed in a humidified box and incubated in an environment with a temperature range of 4°C to 37°C for 20min to 12h;

[0046] Dilute the labeled monoclonal antibody with the fluorescent antibody diluent in the kit;

[0047] The incubated pathogen control slide is washed with the prepared concentrated washing solution, and then the fluorescent antibody dilution containing monoclonal antibodies is added to different slide wells of the washed pathogen control slide. The pathogen control slide is then placed in a wet box and incubated in an environment with a temperature range of 4°C to 37°C for 20 minutes to 12 hours to obtain a pathogen control slide that can be used to interpret the results.

[0048] In the pathogen control slide capable of interpreting the results, a positive result is in the first color, and a negative result is in the second color, and the first color and the second color are two different colors.

[0049] The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection and the multi-pathogen joint detection method according to the present invention have at least one of the following advantages:

[0050] (1) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the fluorescent antibody diluent components in the multi-pathogen joint detection method provided by the present invention have a broad spectrum and good compatibility, and do not need to be adjusted due to different detection antigens, and the same fluorescent antibody diluent can meet the detection of all antigen substrates;

[0051] (2) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the multi-pathogen joint detection method provided by the present invention use the direct immunofluorescence method, which can reduce the non-specific reaction of the secondary antibody in the indirect method and improve its specificity;

[0052] (3) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the kit in the multi-pathogen joint detection method provided by the present invention have good specificity, and no blocking agent is required for result determination, thereby shortening the operation time;

[0053] (4) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the multi-pathogen joint detection method provided by the present invention can fix two or more pathogens such as viruses, bacteria, mycoplasmas and other different types of pathogenic microorganisms on the same pathogen control slide under the same reaction conditions, so that the differential diagnosis of multiple suspected pathogens can be completed simultaneously during the joint detection process, and multiple results can be obtained in a one-time operation;

[0054] (5) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the pathogen control slide preparation process in the multi-pathogen joint detection method provided by the present invention do not require a biochemical purification step, and the results can be interpreted by fluorescent two-color development, thus avoiding interference from non-specific fluorescence;

[0055] (6) In the preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the preparation of the pathogen control slide in the multi-pathogen joint detection method provided by the present invention, the cells do not need to be coated with a blocking agent to allow the antigen to be adsorbed onto the slide, while ensuring that no cross reaction occurs between different types of pathogens, and greatly shortening the time for preparing the pathogen control slide. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of the preferred embodiments in conjunction with the accompanying drawings, in which:

[0057] Figure 1 It is a flow chart of a method for preparing a direct immunofluorescence detection kit for multi-pathogen joint detection according to one embodiment of the present invention;

[0058] Figure 2 yes Figure 1 The flowchart of the preparation method of pathogen control slides for multi-pathogen joint detection is shown;

[0059] Figure 3 is used Figure 1 The flowchart of the method for joint detection of multiple pathogens using the test kit prepared by the preparation method shown. DETAILED DESCRIPTION

[0060] The technical solution of the present invention is further specifically described below by examples and in conjunction with the accompanying drawings. In the specification, the same or similar reference numerals indicate the same or similar components. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention and should not be construed as a limitation of the present invention.

[0061] See also Figure 1 , which shows a method for preparing a direct immunofluorescence detection kit for multi-pathogen joint detection according to an embodiment of the present invention, the preparation method comprising the following steps:

[0062] Provide pathogen control slides for multi-pathogen testing;

[0063] Provide specimen slides for multi-pathogen testing;

[0064] Provide sample diluent;

[0065] Provides concentrated wash solution for washing pathogen control slides loaded with specimens;

[0066] Provide fluorescein conjugates;

[0067] providing an effective amount of a monoclonal antibody; and

[0068] Provide fluorescent antibody diluent;

[0069] Among them, the components of the fluorescent antibody diluent are: 0.1%~0.5% disodium hydrogen phosphate, 0.01%~0.05% sodium dihydrogen phosphate, 0.5%~1.5% sodium chloride, 0.01%~0.05% potassium chloride, 2%~8% sucrose, 10%~30% glycerol, 0.05%~0.3% casein sodium salt, 5%~20% bovine serum, 0.01%~0.1% Tween 20, 0.01%~0.1% TritonX-100, 0.005%~0.03% Evans blue, 0.01%~0.05% gentamicin, 0.005%~0.03% PC-300 and distilled water.

[0070] In one example, the fluorescein conjugate is a monoclonal antibody labeled with fluorescein. When detecting an antigen, the fluorescein conjugate is diluted with a fluorescent antibody diluent so that the fluorescent antibody diluent contains an effective amount of the fluorescein conjugate. The concentration of the effective amount of the fluorescein conjugate is 10-200µg / ml, for example, 20µg / ml, 40µg / ml, 80µg / ml, 100µg / ml, 130µg / ml, 150µg / ml, 170µg / ml, and the like.

[0071] In one example, 0.1% to 0.5% disodium hydrogen phosphate is 0.2% to 0.4% disodium hydrogen phosphate dodecahydrate, 0.01% to 0.05% sodium dihydrogen phosphate is 0.02% to 0.04% sodium dihydrogen phosphate dihydrate, 2% to 8% sucrose is 4% to 7% sucrose used to protect the activity of antigens and antibodies, and casein sodium salt is Casein-Na used to protect antibody activity and block nonspecificity.

[0072] In one example, the antigen detection fluorescent antibody diluent is composed of 0.3% to 0.38% disodium hydrogen phosphate dodecahydrate, 0.022% to 0.03% sodium dihydrogen phosphate dihydrate, 0.7% to 0.98% sodium chloride, 0.015% to 0.025% potassium chloride, 4.5% to 6% sucrose, 15% to 25% glycerol, 0.08% to 0.25% Casein-Na, 8% to It is composed of 15% bovine serum, 0.03%~0.08% Tween 20 (Tween-20), 0.02%~0.07% TritonX-100, 0.007%~0.02% Evans blue, 0.07%~0.3% 4-aminoantipyrine, 0.01%~0.035% 80,000 gentamicin, 0.007%~0.02% PC300 and distilled water.

[0073] In one example, the fluorescein conjugate is a monoclonal antibody labeled with fluorescein, and the adsorbent is an IgG monoclonal antibody. Fluorescein includes fluorescein isothiocyanate (FITC) and phycoerythrin (PE), and the concentrated washing liquid is a phosphate buffer. Preferably, the fluorescein conjugate is an anti-human IgM monoclonal antibody labeled with FITC, and the adsorbent is a mouse human IgG monoclonal antibody. This example is an illustrative example, and those skilled in the art can use other fluoresceins, fluorescein conjugates, adsorbents, and concentrated washing liquids with the same function to replace the corresponding reagents. This example should not be understood as a limitation to the present invention.

[0074] In one example, when detecting an antigen, the fluorescent antibody diluent is used to dilute the fluorescein conjugate so that the concentration of the fluorescein conjugate in the fluorescent antibody diluent is 10-200µg / ml. A person skilled in the art will understand that the concentration of the labeled monoclonal antibody (i.e., the fluorescein conjugate) will vary depending on the titer of the labeled FITC antibody, but it only needs to be controlled within the range of 10-200µg / ml.

[0075] In one example, the sample diluent is phosphate buffered saline (PBS) including 10 mM sodium dihydrogen phosphate-sodium hydrogen phosphate system, 0.9% NaCl and pH 7.2, cells are washed, and 0.5% Triton is added to increase cell permeability.

[0076] In one example, the method for preparing a kit for multi-pathogen joint detection also includes providing a positive control and a negative control, where the negative control can be selected as 10% normal human serum, and the positive control is the pathogens in all slide wells on the same slide.

[0077] In one example, a pathogen control slide is formed by fixing different types of pathogen-positive cells under the same reaction conditions, and includes spotted wells (i.e., slide wells) containing positive control cells and spotted wells (i.e., slide wells) containing negative control cells.

[0078] In one example, the specimen slide is a pathogen blank slide, which is a blank, uncontaminated slide that has been pre-treated.

[0079] like Figure 2 As shown, the pathogen control slide used in the kit is prepared by the following method, which specifically includes the following steps:

[0080] Step S1: performing cell subculture on various pathogens respectively;

[0081] Step S2: preparing cell suspensions of at least two passaged cells of the plurality of pathogens respectively;

[0082] Step S3: Apply the prepared cell suspension to different wells on the same slide respectively, and after the cells in the cell suspensions in different wells are adsorbed, fix the cells in different wells under the same conditions (ie, different types of antigens are fixed in different wells).

[0083] In one example, the plurality of pathogens include at least two pathogenic microorganisms selected from any one of the pathogens of respiratory infection pathogens and reproductive system infection pathogens. Pathogenic microorganisms include viruses, bacteria, mycoplasmas, chlamydia, fungi, spirochetes, and rickettsia. For example, at least two pathogenic microorganisms in the respiratory infection pathogens include any combination of two or more of Mycoplasma pneumoniae, Chlamydia pneumoniae, influenza A virus, influenza B virus, respiratory syncytial herpes virus, Legionella pneumophila, etc. That is to say, in the same physiological function system (for example, in the same respiratory system, or in the same reproductive system), any combination of two or more of viruses, bacteria, chlamydia, mycoplasmas, etc.

[0084] In one example, the multiple pathogens may also include any combination of specific categories of a specific type of pathogenic microorganisms in any pathogen selected from respiratory system infection pathogens and reproductive system infection pathogens, such as any combination of two or more specific categories of respiratory system infection viruses, and any combination of two or more specific categories of reproductive system infection bacteria.

[0085] In one example, in step S3, the same condition is to use anhydrous ethanol to fix the same time under the same temperature condition, and then dry the same time under the same humidity environment with a humidity of less than 50% to form a pathogen control slide. Preferably, the same temperature condition is the same temperature selected from the temperature range of -30°C to -10°C, more preferably the temperature range is between -25°C and -15°C, such as -25°C, -22°C, -20°C, -17°C or -15°C, and particularly preferably the same temperature condition can be -20°C. In other words, when the ambient temperature is set between 23°C and 27°C, the humidity is maintained between 10% and 20% (preferably the humidity is 15%), by selecting -20°C anhydrous ethanol to fix the cells in different slide wells at the same time (for example, fix for 1 hour), the cells in different pathogen control slides do not cross-react with each other, so there is no need to use a blocking agent for cell coating. At the same time, the present invention adopts no biochemical purification step in the entire pathogen control slide preparation process, so that the antigen is close to the real three-dimensional conformation.

[0086] In one example, the fixed same time is the same time selected from the range of 30 minutes (min) to 3 hours (h), a more preferred time range is 50 min to 1.5 h, and a particularly preferred fixed same time is 1 h.

[0087] In one example, the cell suspension can also be fixed in a fume hood with cold acetone, maintaining the temperature in the fume hood at 20-30°C, such as 25°C, the humidity at 10%-30%, such as 15%, 20%, and the fixation time at 10-20 minutes. Of course, those skilled in the art will appreciate that the cell suspension can also be fixed with 1% sodium azide and stable water-soluble buffered glycerol. Since the above-mentioned cell fixative (i.e., cell blocking agent) has a specific effect on cells, the present invention preferably uses anhydrous ethanol precooled at -20°C for fixation.

[0088] In one example, preferably the same drying time is the same time selected from the range of 30min~3h. More preferably, the drying time is selected from the range of 1h~2.5h. Particularly preferably, the same drying time is 1.5h, 2h. The same humidity with a humidity less than 50% is the same humidity selected from the range of 20%~40%. For example, it can be cultured in an environment with any humidity of 20%, 25%, 30%, 35%, 40%. Preferably, all pathogens in the multiple pathogens are cultured in an environment with a humidity of 20%~30% (for example, 28% or 29%).

[0089] In one example, at least three slide wells are arranged on the slide, and the at least three slide wells include a blank control well and at least two slide wells with at least two antigens fixed thereon.

[0090] In one example, in step S1, the cell subculture includes subculture of bacterial cells and subculture of non-bacterial cells, the non-bacterial cells are other pathogen cells excluding bacterial cells, and the subculture of bacterial cells includes the following steps:

[0091] After the autoclaved culture medium solidifies, inoculate the bacterial strain, and then place it in a CO2 incubator with a humidity of less than 50%, a temperature of 37°C, and a concentration of 5% to grow into a biofilm and form bacterial passage cells for 36 to 48 hours;

[0092] Subculturing non-bacterial cells involves the following steps:

[0093] Step S11: primary cells of various pathogens are placed in different culture vessels, and then fetal bovine serum culture medium with a concentration ranging from 10% to 15% is added respectively, and then placed in a CO2 incubator with a humidity of ≥90% and a temperature of 37°C and a concentration of 5% for a first predetermined time for recovery;

[0094] Step S12: Digest and separate the revived pathogens with trypsin in a concentration range of 0.1% to 0.5%. When the cells of the pathogens are detached from the bottom of the culture vessel (e.g., the bottom of the culture bottle) and present a three-dimensional shape, the digestion is stopped and the trypsin is sucked out, and the cells are counted. Then, the cells are placed in a CO2 incubator with a humidity of ≥90% and a temperature of 37°C and a concentration of 5% for a second predetermined time. When the cell density in the culture vessel reaches 80% to 90%, the culture medium accumulated in the culture vessel is removed, and the cell subculture of the pathogens in the culture vessel is stopped.

[0095] In one example, the culture vessel includes a culture bottle and a culture dish, and those skilled in the art can make corresponding selections as needed.

[0096] In one example, the concentration of fetal bovine serum in step S11 can be selected to be 10%, 15% or 20%, and preferably, fetal bovine serum with a concentration of 10% is selected.

[0097] In one example, in step S11, the first predetermined time ranges from 24 hours to 48 hours. Preferably, the primary cells of the plurality of pathogens are revived by culturing for 48 hours.

[0098] In one example, in step S12, the concentration of trypsin is preferably 0.15%, 0.2%, 0.25%, or 0.3%, and more preferably, a concentration of 0.25% trypsin is selected to digest and separate multiple pathogen cells.

[0099] In one example, in steps S11 and S12, the humidity in the incubator is ≥ 90%. Preferably, the humidity in the incubator can be selected in the range of 95% to 98%, such as 95%, 97%, or 98%.

[0100] In one example, in step S12, the second predetermined time is less than the first predetermined time, and the range of the second predetermined time is 10h~48h. Preferably, 12h, 15h, 20h, 24h, 30h, 35h cultivation is selected, and 24h cultivation is more preferably selected.

[0101] In one example, bacterial cell subculture can also be achieved by the following method, which specifically includes the following steps: dissolving the special culture medium powder corresponding to different types of pathogens, sterilizing under high pressure at a temperature of 120℃~130℃ for 10min~20min, and then waiting for the culture medium to solidify, inoculating a certain amount of pathogens corresponding to the special culture medium, such as LP strains. Cultivating in a CO2 incubator with a humidity of less than 50%, a temperature of 37℃ and a concentration of 5% for a first predetermined time for recovery; culturing in a CO2 incubator with a humidity of less than 50%, a temperature of 37℃ and a concentration of 5% for 24h~48h, and stopping subculture when a biofilm, for example, grows.

[0102] In one example, the humidity in the incubator is ≥ 90%. Preferably, the humidity in the incubator can be selected in the range of 95% to 98%, such as 95%, 97%, or 98%.

[0103] In step S2, the preparation of the cell suspension includes the preparation of the bacterial suspension and the preparation of the non-bacterial cell suspension. The preparation of the bacterial cell suspension includes the following steps: placing the subcultured bacterial cells in sterile physiological saline to prepare a bacterial suspension.

[0104] The preparation of a cell suspension for infection with non-bacteria includes the following steps:

[0105] Step S21: digest and separate at least two passage cells of the plurality of pathogens (e.g., the passage cells obtained by culture in step S12) respectively with trypsin at a concentration ranging from 0.1% to 0.5%, preferably 0.25%, and stop digesting and separating when at least two passage cells are separated from the bottom of the culture vessel (e.g., the bottom of the culture bottle) and present a three-dimensional shape, and aspirate the trypsin;

[0106] Step S22: placing the at least two types of passaged cells after digestion and separation in a CO2 incubator with a humidity of ≥90%, preferably 96%, a temperature of 37°C and a concentration of 5%, for adsorption for 1 h to 3 h, preferably 2 h;

[0107] Step S23: Supplement the at least two passaged cells after adsorption with a fetal bovine serum medium in a concentration range of 5% to 20%, preferably 10%, and then continue to infect the cells in a CO2 incubator with a humidity ≥ 90%, preferably 96%, a temperature of 37°C and a concentration of 5% for 24h to 48h. Different infection times need to be set according to different pathogen cells (for example, the infection time of CoxB cells is 24h), until all pathogen cells in at least two passaged cells have pathological changes;

[0108] Step S24: Digest and separate the at least two passaged cells with pathological changes by using trypsin in a concentration range of 0.1% to 0.5%, preferably 0.25%. When the at least two passaged cells with pathological changes are separated from the bottom of the culture vessel (e.g., the bottom of the culture bottle) and present a three-dimensional shape, add a fetal bovine serum culture medium in a concentration range of 1% to 5% (preferably 2% fetal bovine serum culture medium) to stop digestion and form a cell suspension.

[0109] In one example, in steps S22 and S23, the humidity in the incubator is ≥ 90%, and preferably the humidity in the incubator can be selected in the range of 95% to 98%, such as 95%, 97%, 98%.

[0110] In one example, in step S2, the preparation of the non-bacterial infected cell suspension can also be prepared by the following method, which specifically includes the following steps:

[0111] The primary cells of the multiple pathogens are respectively added to the corresponding pathogen passage cells (i.e., the passage cells obtained by culturing in step S12) among the multiple pathogens for infection for a predetermined time;

[0112] At least two infected pathogen cells among the multiple pathogens are digested and separated to ensure cell viability, the at least two infected pathogen cells after digestion and separation are continuously cultured until the cells become diseased, and then the at least two infected pathogen cells with disease are prepared into a cell suspension.

[0113] Among them, the primary cells of various pathogens do not include bacterial cells, that is, they include, for example, viruses, mycoplasmas, chlamydia, etc.

[0114] The ratio of primary cells to transfer cells is in the range of 1:2 to 1:1, preferably 1:2, i.e., half the number of transfer cells of multiple pathogens are added to transfer cells of the same type as the primary cells for inoculation and infection. The infection time ranges from 24h to 72h, preferably 24h, 48h and 72h. Different infection times are selected according to different pathogen cell types.

[0115] See also Figure 3, which shows a method for joint detection of multiple pathogens using the above kit, the joint detection method comprising the following steps:

[0116] Step S100: providing a kit, which is the aforementioned kit;

[0117] Step S200: preparing the concentrated washing solution in the kit and pre-treating the sample;

[0118] Step S300: adding the pretreated samples to different slide wells on the pathogen control slide, and then placing them in a wet box and incubating them in an environment with a temperature range of 4°C to 37°C for 20 minutes to 12 hours;

[0119] Step S400: labeling the monoclonal antibody with the fluorescein conjugate in the kit, and then diluting the labeled monoclonal antibody with the fluorescent antibody diluent;

[0120] Step S500: Use the prepared concentrated washing solution to wash the incubated pathogen control slide, then add the fluorescent antibody dilution containing monoclonal antibodies to different slide wells of the washed pathogen control slide, and then put the pathogen control slide into a wet box and incubate it in an environment with a temperature range of 4°C to 37°C for 20min to 12h to obtain a pathogen control slide that can be used to interpret the results.

[0121] In the pathogen control slide capable of interpreting the results, a positive result is in the first color, and a negative result is in the second color, and the first color and the second color are two different colors.

[0122] In one example, in the pathogen control slide capable of interpreting the results, the positive result is a first color, the negative result is a second color, and the first color and the second color are two different colors. Preferably, the first color is red, and the second color is green.

[0123] In one example, in step S300, when the temperature is selected at 4°C, the incubation is performed for 8 to 12 hours; when the temperature is selected at 37°C, the incubation is performed for 1 hour; when room temperature is selected, the incubation can be performed for 2 hours.

[0124] In step S200, preprocessing the sample includes the following steps:

[0125] Step S201: Mix the sample and the adsorbent in the kit in an equal volume to the sample, and then centrifuge at a predetermined speed for a predetermined time;

[0126] Step S202: Add the adsorbed sample into the sample diluent in the reagent kit according to a predetermined ratio and mix well.

[0127] In one example, in step S201, the predetermined rotation speed is 200-700 rpm, and the predetermined centrifugal time is 3-7 min, preferably 5 min.

[0128] In one example, in step S202, the predetermined ratio is a volume ratio of the adsorbed sample to the sample diluent in a range of 1:5 to 1:3, preferably 1:4.

[0129] In one example, in step S200, the concentrated washing solution in the kit is configured by diluting the concentrated washing solution in the kit by 30 to 50 times, preferably 40 times.

[0130] Example 1

[0131] 1. The method for preparing pathogen control slides for respiratory infection joint testing comprises the following steps:

[0132] (1) Cell subculture:

[0133] Method 1: Pathogen-infected cell culture: Add DMEM culture medium containing 10% fetal bovine serum to the nasopharyngeal epithelial cells to be fixed, and then culture them in a CO2 incubator at 37°C, 90% humidity, and 5% CO2 concentration for 48 hours to recover; then digest and separate them with 0.25% trypsin, aspirate the trypsin and count 6 million / T75, and then culture them again in a 5% CO2 incubator at 37°C, 90% humidity for 24 hours for subculture.

[0134] Method 2: Legionella pneumophila LP cell culture: After the LP special culture medium powder is dissolved, it is sterilized under high pressure at 121℃ for 15 minutes. After cooling, it is poured into flat dishes, so that each dish contains about 15ml of culture medium. After the culture medium solidifies, 50uL of LP bacteria is inoculated and placed in a 5% CO2 incubator with a temperature of 37℃ and a humidity of 90% for 24 to 48 hours. After the bacterial film grows, the subculture is stopped.

[0135] (2) Pathogens infect cells:

[0136] Count the cells and discard the culture medium. Add various pathogens to the nasopharyngeal epithelial cells and place them in a 5% CO2 incubator at 37°C and 90% humidity for 2 hours for adsorption. Then add the culture medium to 16 ml and continue culturing until the cells become diseased.

[0137] (3) Preparation of cell suspension:

[0138] Method 1: After pathogen-infected cells become cytopathic, they are digested with 0.25% trypsin and 36 ml of DMEM medium containing 2% fetal bovine serum is added to prepare a cell suspension.

[0139] Method 2: Preparation of Legionella pneumophila LP bacterial suspension: scrape off the LP bacterial film, put it into sterile saline, blow and beat evenly to make a bacterial suspension.

[0140] (4) Pathogen fixation:

[0141] Method 1: Add the above pathogen-infected cell suspensions dropwise into the wells of the slide respectively. After the cells are adsorbed, remove the culture medium and fix them with -20°C anhydrous ethanol for 1 hour at an ambient temperature of 25°C and a humidity of 18%, and then air-dry.

[0142] Method 2: Drop the Legionella pneumophila LP cell suspension onto the wells of the glass slide and let it dry.

[0143] 2. The pretreatment of samples in the combined detection method for multiple pathogens in respiratory infections includes the following steps:

[0144] (1) Gently mix the specimen on a vortex mixer for 3 times, the time is controlled to be about 30 seconds. When it becomes turbid, transfer the specimen to a 1.5mL EP tube and centrifuge it at a speed of 400-600r / min for 5 minutes. Remove the supernatant, keep the precipitate, add an appropriate amount of sample diluent (such as phosphate buffer) about 300µl, and pipette to make it into a cell suspension. Then add 15µl of pre-treated cell suspension serum to each well of the specimen slide.

[0145] (2) Add 25 µl of pretreated cell suspension serum to each well of the pathogen control slide. Add 25 µl of undiluted positive control and 15 µl of undiluted negative control to two different cell wells. Dry and fix the cells in a drying oven for 1 hour, or air-dry at room temperature and fix with anhydrous ethanol in a -20°C refrigerator for 1 hour.

[0146] (3) Prepare fluorescent antibody diluent: add 50 g sucrose, 3.63 g disodium hydrogen phosphate, 0.24 g sodium dihydrogen phosphate, 9 g sodium chloride, 0.2 g potassium chloride, 0.5 g casein sodium salt, add distilled water to 600 ml, sterilize under high pressure, cool and add sterile fetal bovine serum 200 ml, 100 ml glycerol, 0.5 ml Tween-20, 0.5 ml TritonX-100, 0.01 g Evans blue, 0.2 ml gentamicin and 1 ml PC300, and then make up to 1000 ml to prepare fluorescent antibody diluent. The final concentration of gentamicin is 50 μg / ml with 80,000 μg of gentamicin.

[0147] 3. The interpretation method of the results of the combined test method for multiple pathogens in respiratory infections is as follows:

[0148] Method for determining the end point result: The pathogen control slide fixed with anhydrous ethanol in Example 2 is repeatedly washed three times in the washing solution and dried naturally. The fluorescent antibody conjugate is diluted 500 times with a fluorescent antibody diluent so that the concentration of the fluorescent antibody conjugate in the fluorescent antibody diluent is 10 μg / ml, and the diluted fluorescent monoclonal antibody is obtained.

[0149] In one example, the fluorescent antibody conjugate is 20 mg of monoclonal antibody labeled with FITC, which is resuspended in 4 ml of fluorescent antibody protective agent after dialysis. In one example, the fluorescent antibody protective agent is the protective agent after the antibody is coupled with FITC, that is, the protective agent of the mother solution, which is generally obtained by adding 50% glycerol and 10 mg / ml BSA to PBS.

[0150] After that, add, for example, 15µl, 20µl, or 25µl of diluted fluorescent monoclonal antibody to each well of the air-dried pathogen control slide, incubate at 37°C in a wet box for 30 minutes, rinse three times with PBS, air-dry, and observe the results under a fluorescence microscope (recommended filter wavelength is 470-495nm).

[0151] The results are interpreted as follows:

[0152] (1) Blank control: no green fluorescence, cells appear red.

[0153] (2) Positive control: The cell nucleus, cytoplasm or cell membrane of RSV, ADV, IFVA, IFVB and PIV1, 2, 3 showed apple green fluorescence.

[0154] (3) Negative control: RSV, ADV, IFVA, IFVB and PIV1, 2, 3 showed no fluorescence, and RSV, ADV, IFVA, IFVB and PIV1, 2, 3 cells appeared red.

[0155] Example 2

[0156] Common reproductive system infections are Chlamydia trachomatis and Ureaplasma urealyticum.

[0157] 1. Prepare pathogen control slides for reproductive system infection joint detection by the following preparation method:

[0158] (1) Cell subculture:

[0159] Pathogen-infected cell culture: The HaCaT cells to be fixed were cultured in a 5% CO2 incubator at 37°C and 95% humidity for 48 hours with DMEM medium containing 10% fetal bovine serum for recovery; after the cells in the culture dish grew into a dense monolayer of cells, they were digested with 0.25% trypsin, counted at 6 million / T75, and then cultured again in a 5% CO2 incubator at 37°C and 95% humidity for 24 hours for subculture.

[0160] (2) Pathogens infect cells:

[0161] The clinical specimens were repeatedly frozen and thawed three times at -80°C to break the cells and release Chlamydia trachomatis. The samples were centrifuged at 3000r / min for 5 minutes, and then the supernatant was taken to prepare the strain solution. The nutrient solution containing the dense monolayer of HaCaT cells was aspirated. Fresh growth culture medium was added to the cell wells, and then 0.1ml of clinical strain solution was added to the wells. After that, the samples were centrifuged at 32°C and 3000r / min for 1h, allowed to stand, and incubated in a 5% CO2 incubator at 37°C and 95% humidity for 2h. Chlamydia infection solution containing 1µg / ml actinomycin was added, and then incubated in a 5% CO2 incubator at 37°C and 95% humidity for 48-72h until the cells became lesions.

[0162] (3) Preparation of cell suspension:

[0163] Terminate the culture, aspirate the growth medium in the cell culture bottle, wash the cells 3 times with PBS solution, add 1 ml of 0.25% trypsin solution, the cell surface will appear frosted glass, discard the digestion solution, add 36 ml of DMEM medium containing 2% fetal bovine serum, and blow with a pipette to detach the cells and make a cell suspension.

[0164] (4) Pathogen fixation:

[0165] 30 µl of the above pathogen-infected cell suspension was dripped into the wells of the slide, and dried for 2 h (or naturally air-dried or blow-dried) at 20°C and humidity <20%, such as 17%. Fix with -20°C precooled anhydrous ethanol for 1 h.

[0166] 2. The method of pre-processing samples in the joint test for reproductive system infection is as follows:

[0167] (1) Gently mix the sample on a vortex mixer for 3 times for about 30 seconds. When it becomes turbid, transfer the sample to a 1.5 mL EP tube and centrifuge it at 400-600 r / min for 5 minutes. Remove the supernatant and keep the precipitate. Add an appropriate amount of sample diluent (e.g., phosphate buffer) (about 300 µl) and pipette to make it into a cell suspension.

[0168] (2) Prepare fluorescent antibody diluent: add 50 g sucrose, 3.63 g disodium hydrogen phosphate, 0.24 g sodium dihydrogen phosphate, 9 g sodium chloride, 0.2 g potassium chloride, 0.5 g casein sodium salt, add distilled water to 600 ml, sterilize under high pressure, cool and add sterile fetal bovine serum 200 ml, 100 ml glycerol, 0.5 ml Tween-20, 0.5 ml TritonX-100, 0.01 g Evans blue, 0.2 ml gentamicin and 1 ml PC300, and then make up to 1000 ml to prepare fluorescent antibody diluent. The final concentration of 80,000 gentamicin is 50 µg / ml.

[0169] (3) Add 30 µl of the above pathogen-infected cell suspension to each well of the slide, and dry for 2 h (or air dry or blow dry) at 20°C and humidity <20%, for example, 17%. Place in a wet box and fix with -20°C precooled anhydrous ethanol for 60 min.

[0170] 3. The interpretation method of the joint inspection results is as follows:

[0171] Method for determining the end point result: The pathogen control slide fixed with anhydrous ethanol in Example 3 is repeatedly washed three times in a washing solution and dried naturally. The fluorescent antibody conjugate is diluted 50 times with a fluorescent antibody diluent so that the concentration of the fluorescent antibody conjugate in the fluorescent antibody diluent is 100 μg / ml, and the diluted fluorescent monoclonal antibody is obtained.

[0172] In one example, the fluorescent antibody conjugate is 20 mg of monoclonal antibody labeled with FITC, which is resuspended in 4 ml of fluorescent antibody protective agent after dialysis. In one example, the fluorescent antibody protective agent is the protective agent after the antibody is coupled with FITC, that is, the protective agent of the mother solution, which is generally obtained by adding 50% glycerol and 10 mg / ml BSA to PBS.

[0173] Afterwards, add 15µl of diluted fluorescent monoclonal antibody to each well of the pathogen control slide, incubate at 37°C in a humidified chamber for 30 minutes, rinse three times with PBS, air-dry, and observe the results under a fluorescence microscope (recommended filter wavelength is 470-495nm).

[0174] Specific method for interpreting the results of the two-color system:

[0175] (1) Blank control: There is no green fluorescence in well 0, and the cells appear red.

[0176] (2) Positive control: apple green fluorescence appears in the nucleus, cytoplasm or cell membrane of Chlamydia trachomatis;

[0177] (3) Negative control: Chlamydia trachomatis cells appear red.

[0178] The method for preparing a direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the multi-pathogen joint detection method according to the present invention have at least one of the following advantages:

[0179] (1) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the fluorescent antibody diluent components in the multi-pathogen joint detection method provided by the present invention have a broad spectrum and good compatibility, and do not need to be adjusted due to different detection antigens, and the same fluorescent antibody diluent can meet the detection of all antigen substrates;

[0180] (2) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the multi-pathogen joint detection method provided by the present invention use the direct immunofluorescence method, which can reduce the non-specific reaction of the secondary antibody in the indirect method and improve its specificity;

[0181] (3) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the kit in the multi-pathogen joint detection method provided by the present invention have good specificity, and no blocking agent is required for result determination, thereby shortening the operation time;

[0182] (4) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the multi-pathogen joint detection method provided by the present invention can fix two or more pathogens such as viruses, bacteria, mycoplasmas and other different types of pathogenic microorganisms on the same pathogen control slide under the same reaction conditions, so that the differential diagnosis of multiple suspected pathogens can be completed simultaneously during the joint detection process, and multiple results can be obtained in a one-time operation;

[0183] (5) The preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the pathogen control slide preparation process in the multi-pathogen joint detection method provided by the present invention do not require a biochemical purification step, and the results can be interpreted by fluorescent two-color development, thus avoiding interference from non-specific fluorescence;

[0184] (6) In the preparation method of the direct immunofluorescence detection kit for multi-pathogen joint detection, the kit for multi-pathogen joint detection, and the preparation of the pathogen control slide in the multi-pathogen joint detection method provided by the present invention, the cells do not need to be coated with a blocking agent to allow the antigen to be adsorbed onto the slide, while ensuring that no cross reaction occurs between different types of pathogens, and greatly shortening the time for preparing the pathogen control slide.

[0185] Although some embodiments of the present general inventive concept have been shown and described, it will be appreciated by those skilled in the art that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing a direct immunofluorescence detection kit for multi-pathogen joint detection, the preparation method comprising the following steps: Provide pathogen control slides and specimen slides for multi-pathogen joint testing. Among them, cells in the cell suspensions in different slide wells on the pathogen control slide were fixed under the same conditions using anhydrous ethanol at −20°C; Provide sample diluent; Provides concentrated wash solution for washing pathogen control slides loaded with specimens; providing a fluorescein conjugate; and Provide fluorescent antibody diluent; The fluorescent antibody diluent comprises: 0.1% to 0.5% disodium hydrogen phosphate, 0.01% to 0.05% sodium dihydrogen phosphate, 0.5% to 1.5% sodium chloride, 0.01% to 0.05% potassium chloride, 2% to 8% sucrose, 10% to 30% glycerol, 0.05% to 0.3% casein sodium salt, 5% to 20% bovine serum, 0.01% to 0.1% Tween 20, 0.01% to 0.1% Triton X-100, 0.005% to 0.03% Evans blue, 0.01% to 0.05% gentamicin, 0.05% to 0.5% 4-aminoantipyrine, 0.005% to 0.03% PC-300 and distilled water. The pathogen control slides were prepared by the following steps: Step S1: performing cell subculture on various pathogens respectively; Step S2: preparing cell suspensions of at least two passaged cells of the plurality of pathogens respectively; Step S3: applying the prepared cell suspension to different wells on the same slide respectively, and after the cells in the cell suspensions in the different wells are adsorbed, fixing the cells in the different wells under the same conditions; The multiple pathogens include at least two pathogenic microorganisms selected from any one of respiratory system infection pathogens and reproductive system infection pathogens, In step S3, the same conditions are fixing with anhydrous ethanol for the same time under the same temperature conditions, and then drying for the same time under the same humidity environment with a humidity of less than 50% to form the pathogen control slide; The same temperature condition is that the ambient temperature is selected from the same temperature in the range of 23°C to 27°C, the same fixed time is that the fixed time is selected from the same time in the range of 30min to 3h, and the same drying time is that the drying time is selected from the same time in the range of 30min to 3h.

2. The preparation method according to claim 1, wherein The 0.1% to 0.5% disodium hydrogen phosphate is 0.2% to 0.4% disodium hydrogen phosphate dodecahydrate, the 0.01% to 0.05% sodium dihydrogen phosphate is 0.02% to 0.04% sodium dihydrogen phosphate dihydrate, and the 2% to 8% sucrose is 4% to 7% sucrose.

3. The preparation method according to claim 1, wherein The fluorescein conjugate is a monoclonal antibody labeled with fluorescein. During the detection, the fluorescent antibody diluent is used to dilute the fluorescent conjugate so that the concentration of the fluorescent conjugate in the fluorescent antibody diluent is 10-200 μg / ml.

4. The preparation method according to claim 1, wherein The same temperature condition is 25° C., the same fixing time is 1 h, and the same drying time is 2 h.

5. The preparation method according to claim 1, wherein The humidity less than 50% is selected from the range of 10% to 20%.

6. The preparation method according to claim 1, wherein In step S2, the preparation of the cell suspension includes the preparation of the bacterial suspension and the preparation of the non-bacterial infected cell suspension.

7. The preparation method according to claim 6, wherein: The preparation of the bacterial suspension comprises the following steps: The subcultured bacterial cells were placed in sterile saline to prepare a bacterial suspension; The preparation of a cell suspension for infection with non-bacteria includes the following steps: Step S21: digesting and separating at least two passage cells of the plurality of pathogens respectively with pancreatic enzymes in a concentration range of 0.1% to 0.5%, and stopping the digestion and separation when the at least two passage cells are detached from the bottom of the culture vessel and aspirating the pancreatic enzymes; Step S22: placing the at least two types of passaged cells after digestion and separation in a CO2 incubator with a humidity of ≥90%, a temperature of 37°C, and a CO2 concentration of 5% for adsorption for 1 h to 3 h; Step S23: supplementing the at least two passaged cells after adsorption with a fetal bovine serum medium in a concentration range of 5% to 20%, and then continuing to infect the cells in a CO2 incubator with a humidity of ≥90%, a temperature of 37°C, and a CO2 concentration of 5% for 24 hours to 96 hours, until all pathogenic cells in at least two passaged cells become pathological; Step S24: digest and separate the at least two passaged cells with pathological changes with trypsin in a concentration range of 0.1% to 0.5%, respectively; when the at least two passaged cells with pathological changes are detached from the bottom of the culture vessel, add fetal bovine serum culture medium in a concentration range of 1% to 5% to stop digestion and form a cell suspension.

8. The preparation method according to claim 6, wherein: The preparation of the non-bacterial infected cell suspension comprises the following steps: adding the primary cells of the plurality of pathogens to the corresponding pathogen passage cells of the plurality of pathogens respectively and infecting them for a predetermined time; Digesting and separating at least two infected pathogen cells among the multiple pathogens to ensure cell viability, continuing to culture the at least two infected pathogen cells after digestion and separation until the cells become diseased, and then preparing the at least two infected pathogen cells that have become diseased into a cell suspension; Among them, primary cells of various pathogens do not include bacterial cells. The ratio of primary cells to passage cells ranges from 1:2 to 1:1, and the scheduled infection time ranges from 24h to 72h.

9. A kit for multi-pathogen joint detection, characterized in that: The kit is prepared according to the preparation method of a direct immunofluorescence detection kit for multi-pathogen joint detection according to any one of claims 1-8.

10. A multi-pathogen joint detection method for non-diagnostic purposes, the joint detection method comprising the following steps: Providing a kit, wherein the kit is the kit according to claim 9; Prepare the concentrated washing solution in the kit and pre-treat the sample; The pretreated samples were added to different slide wells on the pathogen control slide, and then placed in a humidified box and incubated in an environment with a temperature range of 4°C to 37°C for 20min to 12h; diluting the fluorescein conjugate with the fluorescent antibody diluent; The incubated pathogen control slide is washed with the prepared concentrated washing solution, and then the fluorescent antibody dilution containing monoclonal antibodies is added to different slide wells of the washed pathogen control slide. The pathogen control slide is then placed in a wet box and incubated in an environment with a temperature range of 4°C to 37°C for 20 minutes to 12 hours to obtain a pathogen control slide that can be used to interpret the results. In the pathogen control slide capable of interpreting the results, a positive result is in a first color, and a negative result is in a second color, and the first color and the second color are two different colors.

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