A microbiological inspection method based on fluorescent immunomicrospheres

Abstract

The present invention relates to the field of rapid microbial detection. In order to solve the problem that immunomagnetic beads need to be separated from microorganisms and then stained in the immunomagnetic bead detection method of microorganisms under the prior art, and the operation is complicated, a microbial inspection method based on fluorescent immunomicrospheres is disclosed, which includes the following steps: dissolving fluorescent quantum dots in an organic solvent, adding swollen superparamagnetic beads, and removing the solvent in the mixed solution after a period of reaction to obtain fluorescent superparamagnetic beads; activating the fluorescent superparamagnetic beads and mixing them with antibodies of the microorganism to be tested for incubation, and then sealing and treating to obtain fluorescent immunomagnetic beads; mixing the sample to be tested with a bacterial fluorescent dye or a fungal fluorescent dye, and then adding fluorescent immunomagnetic beads for mixed incubation, and adding the sample to be tested as a test liquid to a well plate after magnetic separation and washing to observe the fluorescence of the fluorescent immunomagnetic beads that capture the microorganism. The method does not need to separate the immunomagnetic beads from the microorganisms during the detection process, can be directly detected, is easy to operate, and has a short detection time.

Description

Technical Field

[0001] The invention relates to the field of rapid microbial testing, and in particular to a microbial testing method based on fluorescent immunomicrospheres. Background Art

[0002] Microorganisms are a group of tiny organisms with small size and simple structure. Their size cannot be observed by naked eyes and can only be seen with the help of a microscope. Microorganisms are a large biological group, covering many species, involving many fields such as health, medicine, industry and agriculture, and environmental protection. Microbial contamination is the most important factor causing pathogenic diseases. Therefore, as an important means of health management and safety index evaluation, the establishment of a fast, accurate, and sensitive modern microbial detection method plays a more important role in safety monitoring. Traditional microbial inspection methods have problems such as complex operation, long test time, and large amount of consumables. For example, plate culture requires multiple operation steps such as medium preparation, sterilization, pouring plates, inoculation, culture, counting, separation, and identification, and the test operation is complicated. The conventional total colony count culture time is 48h, which is the shortest time among traditional methods, and some fastidious bacteria culture time even exceeds 14 days. Therefore, in order to meet the urgent needs of microbial safety analysis, it is necessary to develop a fast, accurate, economical, and easy-to-operate microbial inspection method.

[0003] Immunomagnetic beads are surface-modified magnetic beads with superparamagnetism, on which there are specifically bound antibodies. With the promotion and application of magnetic separation immunoassay technology, people are increasingly using immunomagnetic separation of pathogenic microorganisms, and the use of immunomagnetic beads also provides a method for microbial detection. Most of the existing methods of using immunomagnetic beads for microbial detection are to first capture microorganisms from samples with immunomagnetic beads, and then separate the microorganisms and immunomagnetic beads to detect the presence of microorganisms. For example, the "Universal Pathogenic Microorganism Rapid Magnetic Separation Identification Method and Kit Based on Fluorescence Staining" disclosed in the Chinese patent literature has a publication number of CN110596383A, and the identification method is as follows: (1) preparing universal bridged magnetic beads: a) preparing magnetic beads-streptavidin crosslinks; b) preparing biotin-Ab2 crosslinks; c) making universal bridged magnetic beads: mixing and incubating the magnetic beads-streptavidin crosslinks and the biotin-Ab2 crosslinks to obtain universal bridged magnetic beads; (2) preparing specific capture magnetic beads: mixing the universal bridged magnetic beads with the mouse-derived Ab1 of the pathogenic microorganism to be tested; (3) mixing and incubating the sample to be tested with the specific capture magnetic beads, magnetic separation and washing, and obtaining an immune magnetic bead separation; (4) adding demagnetization solution for incubation, magnetic separation, taking the supernatant smear, adding bacterial stain or fungal stain, and detecting and observing the bacterial coloring. This method separates the magnetic beads from the bacteria to be tested and then stains the bacteria for observation, which is relatively complicated. Summary of the invention

[0004] In order to overcome the problem that the immunomagnetic beads need to be separated from the microorganisms and then stained in the prior art immunomagnetic bead detection method for microorganisms, which is complicated to operate, the present invention provides a microorganism inspection method based on fluorescent immunomicrospheres. This method does not need to separate the immunomagnetic beads from the microorganisms during the detection process, can perform direct detection, is easy to operate, and has a short detection time.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A microbiological inspection method based on fluorescent immunomicrospheres comprises the following steps:

[0007] (1) dissolving fluorescent quantum dots in an organic solvent, adding swollen superparamagnetic beads, reacting for a period of time, and then removing the solvent from the mixed solution to obtain fluorescent superparamagnetic beads;

[0008] (2) activating the fluorescent superparamagnetic beads, mixing them with the antibodies of the microorganisms to be detected, incubating them, and then blocking them to obtain fluorescent immunomagnetic beads;

[0009] (3) The sample to be tested is mixed with a bacterial fluorescent stain or a fungal fluorescent stain, and then fluorescent immunomagnetic beads are added for mixing and incubation. After magnetic separation and washing, the sample is added to the well plate as the test solution to observe the fluorescence of the fluorescent immunomagnetic beads that capture the microorganisms.

[0010] The present invention embeds fluorescent quantum dots in superparamagnetic beads to obtain fluorescent superparamagnetic beads, then combines the fluorescent superparamagnetic beads with antibodies of microorganisms to be tested to obtain fluorescent immunomagnetic beads that can specifically identify microorganisms, and dyes the microorganisms in the sample to be tested and then combines with the fluorescent immunomagnetic beads to realize direct detection of fluorescence. If only the fluorescent superparamagnetic beads emit fluorescence values ​​in the test liquid, then the test result is negative; if the test liquid contains both the fluorescence values ​​emitted by the fluorescent superparamagnetic beads and the fluorescence values ​​emitted by the dyed microorganisms, then the test result is positive. Different fluorescent quantum dots and antibodies can be used to obtain different fluorescent immunomagnetic beads, thereby realizing the detection of multiple microorganisms.

[0011] Preferably, the step (1) is to dissolve the fluorescent quantum dots in an organic solvent, add the swollen superparamagnetic beads and shake for 3-4 hours, and then remove the organic solvent by rotary evaporation to obtain the fluorescent superparamagnetic beads.

[0012] After the superparamagnetic beads are swollen with an organic solvent, fluorescent quantum dots can enter the superparamagnetic beads. When the organic reagent is removed, the superparamagnetic beads shrink to obtain superparamagnetic beads embedded with quantum dots.

[0013] Preferably, the superparamagnetic beads in step (1) are carboxyl magnetic beads with a particle size of 10 nm-10 μm.

[0014] After the carboxyl group is activated, it can be coupled to an antibody.

[0015] Preferably, the activation process of the fluorescent superparamagnetic beads in step (2) is to add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the fluorescent superparamagnetic beads in sequence, mix for 10-15 minutes after ultrasonication, and wash with 2-(N-morpholino)ethanesulfonic acid buffer after activation.

[0016] The combined action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide can activate the carboxyl group and enhance the binding effect between the carboxyl group and the antibody.

[0017] Preferably, in step (2), the mass ratio of the fluorescent superparamagnetic beads, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(25-30):(25-30).

[0018] Preferably, the incubation temperature in step (2) is 15-20° C. and the incubation time is 1-3 h.

[0019] Preferably, the blocking process in step (2) is to magnetically separate the fluorescent superparamagnetic beads after incubation with the antibody, remove the supernatant and wash with magnetic bead blocking solution, and then block in the magnetic bead blocking solution at 25° C. for 4 hours.

[0020] The magnetic bead blocking solution can bind to the active groups in the magnetic beads that are not bound to the antibodies and block them, thereby preventing the active groups in the magnetic beads from binding to microorganisms and improving the specific response of the magnetic beads to microorganisms.

[0021] Preferably, in step (3), the bacterial fluorescent dye is acridine orange, and the fungal fluorescent dye is fluorescent brightener 28.

[0022] Preferably, in step (3), the process of mixing and incubating the sample to be tested with the fluorescent immunomagnetic beads is vortexed for 10-15 minutes, followed by magnetic separation and re-suspending with sterile water.

[0023] Preferably, the well plate with the test solution added in step (3) is placed on a magnetic plate and its fluorescence is observed.

[0024] Place the well plate with the test solution on the magnetic plate to allow the fluorescent immune magnetic beads that capture the microorganisms to sink to the bottom of the well plate to prevent the magnetic beads from being suspended in the test solution and affecting the fluorescence detection.

[0025] Therefore, the beneficial effects of the present invention are as follows: the test sample can be tested without culturing it, and magnetic beads are used to achieve specific detection of the test sample. During the process, there is no need to separate the immunomagnetic beads and the microorganisms. The operation is convenient, the detection time is short, and multiple microorganisms can be detected simultaneously. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific implementation methods.

[0027] The MES buffer used in the following examples has a pH of 6.0 and a concentration of 0.025 mol / L; the magnetic bead blocking solution includes 0.1% BSA, 0.1% Tween 20 and 0.1 mol / L Tris-Hcl buffer, and the pH of the magnetic bead blocking solution is 8.0; the preservation solution includes 1% BSA, 2% glycerol, 0.1% PVP-k30 and 0.1% Proclin300; the Escherichia coli strains, Staphylococcus aureus strains and Candida albicans strains were all purchased from Tailin Bio.

[0028] Example 1

[0029] (1) Superparamagnetic beads fluorescent labeling:

[0030] S1, dissolving 10 mg of red fluorescent quantum dots with an emission spectrum of 625 nm in 300 mL of tetrahydrofuran to obtain a red fluorescent dye solution;

[0031] S2, take 3mL red fluorescent dye, 20mL 0.5% sodium citrate in a 250mL round-bottom flask, mix well, add 1mL magnetic beads (solid content 2.5%), place in an oscillator at 18°C ​​for 4h, pipette the reaction solution into a new 250mL round-bottom flask, evaporating at 65°C, evaporating the solvent, wash the inner wall microspheres with distilled water, pour into a 50mL centrifuge tube and centrifuge for 2min at a centrifugal force of 2500rpm, then wash with 95% ethanol 3 times, the separation operation during the washing process is a centrifugal force of 1000rpm and a centrifugal time of 2min, to obtain red fluorescent superparamagnetic beads;

[0032] (2) Preparation of fluorescent immunomagnetic beads:

[0033] S1. Take 1 mg of red fluorescent superparamagnetic beads and place them in a 1.5 mL EP tube. Add 1 mL of MES buffer. Repeat the washing steps of ultrasonic, vortex and magnetic separation for 3 times and resuspend them in 1 mL of MES buffer.

[0034] S2. Add 100 μL of 30 mg / mL EDC and 100 μL of 30 mg / mL NHS to the above EP tube. Ultrasonicate the EP tube for 2 min and place it in a tumbling mixer for 15 min. Repeat the washing with MES buffer by ultrasonication, vortexing, and magnetic separation once, and resuspend it in 1 mL of MES buffer.

[0035] S3, add 100 μg of Escherichia coli polyclonal antibody to the above EP tube, place the EP tube in a flip mixer and mix at 18°C ​​for 2 h, remove the supernatant by magnetic separation, add 1 mL of magnetic bead blocking solution, repeat ultrasonic, vortex and magnetic separation for 3 times, resuspend in 1 mL of magnetic bead blocking solution, and block at 25°C for 4 h;

[0036] S4, washing the completely blocked magnetic beads three times by ultrasonication, vortexing, and magnetic separation using a preservation solution, and resuspending the beads in 1 mL of the preservation solution and storing them at 4° C. to obtain red fluorescent Escherichia coli immunoadsorption magnetic beads;

[0037] (3) Detection of Escherichia coli and control group:

[0038] S1. Take the Escherichia coli strain and dilute it to 1000 cfu / mL with physiological saline, take 1 mL of the bacterial solution and put it in an EP tube, add 3 drops of 0.1% acridine orange fluorescent staining solution, then add 50 μL of the above-mentioned red fluorescent Escherichia coli immunoadsorption magnetic beads, place it on a vortex oscillator to react for 15 minutes, magnetically separate, resuspend it with sterile water to 200 μL, add the resuspended solution to a 48-well plate, use a magnetic plate to absorb the magnetic beads in the detection wells to the bottom of the well plate, place the well plate in a detection instrument, and detect the fluorescence value;

[0039] S2. Take 1 mL of normal saline and put it in an EP tube. Add 3 drops of 0.1% acridine orange fluorescent staining solution, then add 50 μL of the above-mentioned red fluorescent Escherichia coli immunoadsorption magnetic beads, place it on a vortex oscillator to react for 15 minutes, magnetically separate, resuspend it to 200 μL with sterile water, add the resuspended solution to a 48-well plate, use a magnetic plate to absorb the magnetic beads in the detection wells to the bottom of the well plate, place the well plate in the detection instrument, and detect the fluorescence value.

[0040] Example 2

[0041] (1) Superparamagnetic beads fluorescent labeling:

[0042] S1, take 10 mg of green fluorescent quantum dots with an emission spectrum of 530 nm and dissolve them in 570 mL of tetrahydrofuran to obtain a green fluorescent dye solution;

[0043] S2, take 3mL green fluorescent dye, 20mL 0.5% sodium citrate in a 250mL round-bottom flask, mix well, add 1mL magnetic beads (solid content 2.5%), place in an oscillator at 18°C ​​for 4h, pipette the reaction solution into a new 250mL round-bottom flask, evaporating at 65°C, evaporating the solvent, wash the inner wall microspheres with distilled water, pour into a 50mL centrifuge tube and centrifuge for 2min at a centrifugal force of 2500rpm, then wash with 95% ethanol 3 times, the separation operation during the washing process is a centrifugal force of 1000rpm and a centrifugal time of 2min, to obtain green fluorescent superparamagnetic beads;

[0044] (2) Preparation of fluorescent immunomagnetic beads:

[0045] S1. Take 1 mg of green fluorescent superparamagnetic beads and place them in a 1.5 mL EP tube. Add 1 mL of MES buffer. Repeat the washing steps of ultrasonic, vortex and magnetic separation for 3 times and resuspend them in 1 mL of MES buffer.

[0046] S2. Add 100 μL of 30 mg / mL EDC and 100 μL of 30 mg / mL NHS to the above EP tube. Ultrasonicate the EP tube for 2 min and place it in a tumbling mixer for 15 min. Repeat the washing with MES buffer by ultrasonication, vortexing, and magnetic separation once, and resuspend it in 1 mL of MES buffer.

[0047] S3, add 100 μg of Staphylococcus aureus polyclonal antibody to the above EP tube, place the EP tube in a flip mixer and mix at 18°C ​​for 2 h, remove the supernatant by magnetic separation, add 1 mL of magnetic bead blocking solution, repeat ultrasonic, vortex and magnetic separation for 3 times, resuspend in 1 mL of magnetic bead blocking solution, and block at 25°C for 4 h;

[0048] S4, washing the completely blocked magnetic beads three times by ultrasonication, vortexing, and magnetic separation using a preservation solution, and resuspending the beads in 1 mL of the preservation solution and storing them at 4° C. to obtain green fluorescent Staphylococcus aureus immunoadsorption magnetic beads;

[0049] (3) Detection of Staphylococcus aureus and control group:

[0050] S1. Take Staphylococcus aureus and dilute it to 1000 cfu / mL with physiological saline. Take 1 mL of bacterial solution and put it in an EP tube. Add 3 drops of 0.1% acridine orange fluorescent staining solution, then add 50 μL of the above-mentioned green fluorescent Staphylococcus aureus immunoadsorption magnetic beads, place it on a vortex oscillator to react for 15 minutes, magnetically separate, resuspend it with sterile water to 200 μL, add the resuspended solution to a 48-well plate, use a magnetic plate to absorb the magnetic beads in the detection wells to the bottom of the well plate, place the well plate in a detection instrument, and detect the fluorescence value;

[0051] S2. Take 1 mL of normal saline and put it in an EP tube. Add 3 drops of 0.1% acridine orange fluorescent staining solution, then add 50 μL of the above-mentioned green fluorescent Staphylococcus aureus immunoadsorption magnetic beads, place it on a vortex oscillator to react for 15 minutes, magnetically separate, resuspend it to 200 μL with sterile water, add the resuspension to a 48-well plate, use a magnetic plate to absorb the magnetic beads in the detection wells to the bottom of the well plate, place the well plate in the detection instrument, and detect the fluorescence value.

[0052] Example 3

[0053] (1) Superparamagnetic beads fluorescent labeling:

[0054] S1, dissolve 40 mg of blue fluorescent quantum dots with an emission spectrum of 460 nm in 107 mL of tetrahydrofuran to obtain a blue fluorescent dye solution;

[0055] S2, take 3mL blue fluorescent dye, 20mL 0.5% sodium citrate in a 250mL round-bottom flask, mix well, add 1mL magnetic beads (solid content 2.5%), place in an oscillator at 18°C ​​for 4h, pipette the reaction solution into a new 250mL round-bottom flask, evaporating at 65°C, evaporating the solvent, wash the inner wall microspheres with distilled water, pour into a 50mL centrifuge tube and centrifuge for 2min at a centrifugal force of 2500rpm, then wash with 95% ethanol 3 times, the separation operation during the washing process is a centrifugal force of 1000rpm and a centrifugal time of 2min, to obtain blue fluorescent superparamagnetic beads;

[0056] (2) Preparation of fluorescent immunomagnetic beads:

[0057] S1. Take 1 mg of blue fluorescent superparamagnetic beads and place them in a 1.5 mL EP tube. Add 1 mL of MES buffer. Repeat the washing steps of ultrasonic, vortex and magnetic separation for 3 times and resuspend them in 1 mL of MES buffer.

[0058] S2. Add 100 μL of 30 mg / mL EDC and 100 μL of 30 mg / mL NHS to the above EP tube. Ultrasonicate the EP tube for 2 min and place it in a tumbling mixer for 15 min. Repeat the washing with MES buffer by ultrasonication, vortexing, and magnetic separation once, and resuspend it in 1 mL of MES buffer.

[0059] S3, add 100 μg of Candida albicans polyclonal antibody to the above EP tube, place the EP tube in a flip mixer and mix at 18°C ​​for 2 h, remove the supernatant by magnetic separation, add 1 mL of magnetic bead blocking solution, repeat ultrasonic, vortex, and magnetic separation for 3 times, resuspend in 1 mL of magnetic bead blocking solution, and block at 25°C for 4 h;

[0060] S4, washing the completely blocked magnetic beads three times by ultrasonication, vortexing, and magnetic separation using a preservation solution, and resuspending the beads in 1 mL of the preservation solution and storing them at 4° C. to obtain blue fluorescent Candida albicans immunoadsorption magnetic beads;

[0061] (3) Detection of Candida albicans and control group:

[0062] S1. Dilute Candida albicans with physiological saline to 1000 cfu / mL, take 1 mL of bacterial solution and put it in an EP tube, add 3 drops of 0.1% acridine orange fluorescent staining solution, then add 50 μL of the above-mentioned blue fluorescent Candida albicans immunoadsorption magnetic beads, place it on a vortex oscillator to react for 15 minutes, magnetically separate, resuspend it with sterile water to 200 μL, add the resuspended solution to a 48-well plate, use a magnetic plate to absorb the magnetic beads in the detection wells to the bottom of the well plate, place the well plate in a detection instrument, and detect the fluorescence value;

[0063] S2. Take 1 mL of normal saline and put it in an EP tube. Add 3 drops of 0.1% acridine orange fluorescent staining solution, then add 50 μL of the above-mentioned blue fluorescent Candida albicans immunoadsorption magnetic beads, place it on a vortex oscillator to react for 15 minutes, magnetically separate, resuspend it to 200 μL with sterile water, add the resuspended solution to a 48-well plate, use a magnetic plate to absorb the magnetic beads in the detection wells to the bottom of the well plate, place the well plate in the detection instrument, and detect the fluorescence value.

[0064] The test results are shown in the following table.

[0065]

[0066] The present invention can detect whether there is a microorganism to be detected in the test sample through different fluorescent colors. When only the fluorescence of the magnetic beads exists in the fluorescent observation result, the test sample is negative. When the fluorescence of the magnetic beads and the fluorescence of the bacterial staining exist at the same time, the test sample is positive.

[0067] Example 4

[0068] Escherichia coli and Staphylococcus aureus mixed sample detection:

[0069] Escherichia coli and Staphylococcus aureus were diluted to 2000 cfu / mL with physiological saline respectively. Three EP tubes were taken and marked as A, B and C. 0.5 mL of Escherichia coli solution, 0.5 mL of Staphylococcus aureus solution and 3 drops of 0.1% acridine orange fluorescent staining solution were added to tube A, and then 50 μL of red fluorescent Escherichia coli immunoadsorption magnetic beads prepared in step (2) of Example 1 were added. 0.5 mL of Escherichia coli solution, 0.5 mL of Staphylococcus aureus solution and 3 drops of 0.1% acridine orange fluorescent staining solution were added to tube B, and then 50 μL of green fluorescent beads prepared in step (2) of Example 2 were added. To tube C, add 0.5 mL of Escherichia coli solution, 0.5 mL of Staphylococcus aureus solution and 3 drops of 0.1% acridine orange fluorescent staining solution, and then add 50 μL of blue fluorescent Candida albicans immunoadsorption magnetic beads prepared in step (2) of Example 3. Then, place tubes A, B and C on a vortex oscillator for reaction for 15 min, separate by magnetic separation, resuspend to 200 μL with sterile water, add the resuspended solutions of the three tubes to different wells of a 48-well plate, respectively, use a magnetic plate to absorb the magnetic beads in the detection wells to the bottom of the well plate, place the well plate in a detection instrument, and detect the fluorescence value.

[0070] In the wells of the well plate of Example 4, yellow-green fluorescent bacteria and round red fluorescent magnetic beads can be observed in the wells where the resuspension solution of tube A is added, yellow-green fluorescent bacteria and round green fluorescent magnetic beads can be observed in the wells where the resuspension solution of tube B is added, and only round blue fluorescent magnetic beads can be observed in the wells where the resuspension solution of tube C is added, which indicates that the fluorescent immunomagnetic beads of the present invention can specifically respond to the bacteria to be tested and can realize the simultaneous detection of multiple microorganisms.

Claims

1. A microbiological inspection method based on fluorescent immunomicrospheres, characterized in that: The following steps are involved: (1) Dissolving fluorescent quantum dots in an organic solvent, adding swollen superparamagnetic beads, reacting for a period of time, and then removing the solvent from the mixture to obtain fluorescent superparamagnetic beads; (2) Activating the fluorescent superparamagnetic beads, mixing them with the antibodies of the microorganisms to be tested, incubating them, and then blocking them to obtain fluorescent immunomagnetic beads; (3) Mix the sample to be tested with bacterial fluorescent dye or fungal fluorescent dye, then add fluorescent immunomagnetic beads for mixing and incubation. After magnetic separation and washing, add the sample to the well plate as the test solution to observe the fluorescence of the fluorescent immunomagnetic beads that capture microorganisms. The fluorescent quantum dots in step (1) and the bacterial fluorescent dye or fungal fluorescent dye in step (3) have different fluorescent colors.

2. A microbiological inspection method based on fluorescent immunomicrospheres according to claim 1, characterized in that: The step (1) is to dissolve the fluorescent quantum dots in an organic solvent, then add the swollen superparamagnetic beads and shake for 3-4 hours, and then remove the organic solvent by rotary evaporation to obtain the fluorescent superparamagnetic beads.

3. The microbiological inspection method based on fluorescent immunomicrospheres according to claim 1 is characterized in that: The superparamagnetic beads in step (1) are carboxyl magnetic beads with a particle size of 10 nm-10 μm.

4. A microbiological inspection method based on fluorescent immunomicrospheres according to claim 1 or 3, characterized in that: The activation process of the fluorescent superparamagnetic beads in step (2) is to add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the fluorescent superparamagnetic beads in sequence, mix for 10-15 minutes after ultrasonication, and wash with 2-(N-morpholino)ethanesulfonic acid buffer after activation.

5. The microbiological inspection method based on fluorescent immunomicrospheres according to claim 4 is characterized in that: In the step (2), the mass ratio of the fluorescent superparamagnetic beads, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(25-30):(25-30).

6. The microbiological inspection method based on fluorescent immunomicrospheres according to claim 1 is characterized in that: In step (2), the incubation temperature is 15-20° C. and the incubation time is 1-3 h.

7. The microbiological inspection method based on fluorescent immunomicrospheres according to claim 1 is characterized in that: The blocking process in step (2) is to magnetically separate the fluorescent superparamagnetic beads after incubation with the antibody, remove the supernatant and wash with magnetic bead blocking solution, and then block in the magnetic bead blocking solution at 25° C. for 4 hours.

8. The microbiological inspection method based on fluorescent immunomicrospheres according to claim 1 is characterized in that: In step (3), the bacterial fluorescent dye is acridine orange, and the fungal fluorescent dye is a fluorescent brightener 28.

9. The microbiological inspection method based on fluorescent immunomicrospheres according to claim 1 is characterized in that: In step (3), the process of mixing and incubating the sample to be tested with the fluorescent immunomagnetic beads is vortexing for 10-15 minutes, followed by magnetic separation and re-suspending with sterile water.

10. A microbiological inspection method based on fluorescent immunomicrospheres according to claim 1 or 9, characterized in that: The well plate with the test solution added in step (3) is placed on a magnetic plate and its fluorescence is observed.

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