Fungus multi-fluorescent staining preparation as well as preparation method and application thereof

Abstract

The invention provides a fungus multi-fluorescent dyeing preparation and a preparation method and application thereof.The dyeing preparation comprises a solution A and a solution B. The solution A is mainly prepared from, by mass, 0.6-1 part of CYP51 fluorescent dye, 5-10 parts of fluorescent whitening agent, 3-4 parts of potassium hydroxide, 0.2-1 part of dimethyl sulfoxide and a proper amount of glycerin; the liquid B is mainly prepared from the following components in parts by mass: 0.05 to 0.2 part of ethidium bromide, 0.05 to 0.2 part of acridine orange, 0.5 to 2 parts of calcium fluorescent white, 7 to 10 parts of propidium iodide, 0.3 to 1 part of dimethyl sulfoxide and a proper amount of glycerol. According to the fluorescent staining preparation, the staining accuracy is improved, and dead bacteria and drug-resistant bacteria in fungi can be better stained.

Description

Technical Field

[0001] This invention relates to the field of fungal staining, and more specifically, to a fungal multiplex fluorescent staining agent, its preparation method, and its application. Background Technology

[0002] Fungal infections are characterized by high infection rates, high mortality rates, low diagnosis rates, and low treatment rates. Globally, up to 1 billion people are affected by fungal infections annually, with 310 million suffering from fungal infections. Fatal fungal infections can reach 11.5 million, and the number of deaths from fungal infections exceeds 1.5 million.

[0003] Fungal infections are mainly divided into superficial fungal infections (skin, mucous membranes, secretions) and invasive fungal infections (affecting internal organs, subcutaneous tissues, muscles, etc.). In recent years, there has been a sharp increase in patients with immunodeficiency or impairment due to bone marrow transplantation, immunosuppressant use, the AIDS epidemic, radiotherapy and chemotherapy for tumors, deep vein nutrition, and invasive catheterization for mechanical ventilation; as well as a sharp increase in patients with gastrointestinal flora imbalance due to the use of broad-spectrum antibiotics and complex gastrointestinal surgeries. Candida, Cryptococcus neoformans, and Aspergillus are the most common pathogenic fungi, and the annual incidence of invasive fungal infections is as high as one in 100,000 or even higher. The mortality rate of Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans is even higher, exceeding 20% ​​and even reaching 90%.

[0004] To better detect fungi, the technique of fungal staining directly determines the accuracy of the detection. However, the current fluorescent color reagents have poor safety and accuracy, which affects their widespread application.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a fungal multiplex fluorescent staining agent, its preparation method, and its application. This fungal multiplex fluorescent staining agent improves the accuracy of staining through the specific combination of solution A and solution B, and can achieve better staining of dead fungi and drug-resistant fungi. Existing staining agents cannot achieve good staining of dead fungi.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: This invention provides a fungal multiplex fluorescent staining preparation, mainly comprising solution A and solution B: Solution A is mainly prepared from the following components: by mass parts, 0.6-1 parts of CYP51 fluorescent dye, 5-10 parts of fluorescent whitening agent, 3-4 parts of potassium hydroxide, 0.2-1 parts of dimethyl sulfoxide, and an appropriate amount of glycerol; Solution B is mainly prepared from the following components: by mass parts, ethidium bromide 0.05-0.2 parts, acridine orange 0.05-0.2 parts, calcium fluorescein 0.5-2 parts, propidium iodide 7-10 parts, dimethyl sulfoxide 0.3-1 parts, and glycerol as appropriate amount.

[0008] Preferably, as a further feasible option, the A solution is mainly prepared from the following components: by mass parts, 0.7-0.9 parts of CYP51 fluorescent dye, 7-8 parts of fluorescent whitening agent, 3.2-3.8 parts of potassium hydroxide, 0.5-0.8 parts of dimethyl sulfoxide (DMSO), and an appropriate amount of glycerol.

[0009] Preferably, as a further feasible option, the B solution is mainly prepared from the following components: by mass parts, ethidium bromide 0.08-0.15 parts, acridine orange 0.08-0.15 parts, calcium fluorescent white 0.7-1.5 parts, propidium iodide 8-9 parts, dimethyl sulfoxide 0.4-0.8 parts, and an appropriate amount of glycerol.

[0010] Preferably, as a further feasible option, the CYP51 fluorescent dye is formed by mixing Evans blue with glycerol.

[0011] In solution A, the role of CYP51 fluorescent dye is because fungal cell walls contain chitin and CYP51 protein inside the cells. The CYP51 protein of fungi can be stained by CYP51 protein fluorescent dye.

[0012] The application of CYP51 fluorescent dyes in staining drug-resistant bacteria (especially drug-resistant fungi) mainly involves assessing the resistance of pathogens to azole antifungal drugs by detecting the activity or expression level of CYP51 enzymes.

[0013] CYP51 (sterol 14α-demethylase) is a member of the cytochrome P450 family and participates in a key step in ergosterol synthesis in fungal cell membranes. Azole drugs (such as fluconazole and itraconazole) inhibit CYP51, blocking ergosterol synthesis and leading to fungal death. Drug resistance can develop in fungi through the following pathways: CYP51 gene mutations: leading to changes in enzyme structure and reduced drug affinity; CYP51 overexpression: increasing enzyme production to counteract drug inhibition; and efflux pump activation: reducing intracellular drug concentration. Therefore, this invention uses CYP51 fluorescent dye because of its wide range of staining applications.

[0014] Specifically, the CYP51 fluorescent dye of the present invention is a mixture of Evans blue and glycerol. Because the molecular weight of Evans blue is similar to that of plasma albumin, it can be used for cell staining to distinguish between live and dead cells. Live bacteria are stained blue-green and dead bacteria are stained orange-red. Therefore, this dye was specially selected.

[0015] The purpose of adding fluorescent brighteners is to enhance the fluorescence contrast of target structures, for example, by accumulating on the surface of specific cellular components (such as proteins or nucleic acids) through non-covalent binding or electrostatic interactions, thereby increasing signal intensity and achieving a "complementary color effect." Fluorescent brighteners specifically bind to chitin in the cell wall of fungi, allowing for clear observation of fungal morphological characteristics under a fluorescence microscope, making them suitable for staining live fungi.

[0016] Dimethyl sulfoxide (DMSO) promotes dye penetration by disrupting the hydrogen bond network of the cell membrane lipid bilayer. Its penetration capacity depends on its concentration; and it promotes dye penetration through the disruption of fungi. The high polarity of DMSO facilitates dye penetration by disrupting the hydrogen bond network of the cell membrane lipid bilayer. The dosage of DMSO is highly critical; experiments have shown that too high a dosage will destroy the fungi, while too low a dosage will prevent dye penetration. Therefore, this invention optimizes the optimal dosage of DMSO.

[0017] In summary, of the three substances mentioned above, Evans blue is used for live staining to distinguish between live and dead cells, as dead cells will be stained. However, in fungal fluorescent staining, it may be used for background staining or as a counterstain. Fluorescent brighteners bind to chitin and cellulose in the fungal cell wall, emitting fluorescence. Dimethyl sulfoxide is typically used as a solvent to help the dye penetrate the cell wall. The three substances work together to achieve better results.

[0018] Of course, special attention needs to be paid to the dosage ratio. A relatively larger amount of fluorescent whitening agent is needed, while Evans blue may require a lower amount to avoid masking the fluorescence signal. Too little dimethyl sulfoxide (DMSO) may affect staining penetration, while too much may be toxic to cells or affect fluorescence. Experiments have shown that excessively high or low dosages can lead to background interference or unsatisfactory staining results, so adjusting the ratio is crucial. The solubility and compatibility of each reagent also need to be considered; for example, DMSO is compatible with Evans blue and fluorescent whitening agents, and it's best to control it at a suitable pH value, ideally 8±1. The type of fungus and its cell wall structure also need to be considered, as different fungi may have different dye-binding abilities, resulting in slight variations in the required dosage.

[0019] In solution B, ethidium bromide is added to produce orange fluorescence upon embedding double-stranded DNA / RNA (fluorescence enhancement approximately 50-fold after binding), but it cannot penetrate intact cell membranes (suitable for labeling dead or fixed cells). Acridine orange is added to penetrate living cell membranes, binding to double-stranded DNA (green fluorescence, 525 nm) and to single-stranded RNA or acidic lysosomes (red fluorescence, 650 nm) depending on the pH environment. This is used to distinguish between live and dead cells and to detect lysosomal activity.

[0020] Dimethyl sulfoxide (DMSO) has a similar effect to solution A, namely, enhancing dye solubility and cell membrane permeability.

[0021] When determining the ratio of ethidium bromide and acridine orange in dimethyl sulfoxide, it is important to consider the dye solubility, staining effect (such as fluorescence intensity and background signal), cytotoxicity, and the potential interactions that may occur when the dyes are used simultaneously, requiring adjustments to the ratio to avoid interference.

[0022] Therefore, the ratio of ethidium bromide to acridine orange requires careful attention, otherwise it will affect the staining effect. If the amount of ethidium bromide is too high, it may mask the signal of acridine orange, or the two may compete for binding to nucleic acids, leading to inaccurate results. Therefore, it is necessary to optimize the experiment to find the optimal ratio of the two to achieve the best staining effect and specificity.

[0023] In addition, solution B also contains calcium fluorescein and propidium iodide. Calcium fluorescein binds to β-glucan and chitin in the fungal cell wall, emitting a blue-white fluorescence to mark the morphological outlines of all fungi (whether alive or dead). Propidium iodide penetrates only the damaged cell membrane of dead fungi, embedding into double-stranded nucleic acids (DNA / RNA) and emitting red fluorescence to mark dead fungi. Controlling the appropriate dosage of both together achieves a more obvious staining effect on dead fungi. Too little dosage may not achieve the desired staining effect, while too much may have the opposite effect. Therefore, the dosage of both needs to be controlled within an appropriate range.

[0024] In specific staining applications, ethidium bromide and acridine orange are used as fluorescent dyes, combined with calcium fluorescent white and propidium iodide, with dimethyl sulfoxide as the solvent. It is crucial that each component be controlled within a suitable dosage range. Ethidium bromide (EB) is commonly used for nucleic acid staining because it can insert into the DNA double helix structure and emit fluorescence. Adding this component improves penetration efficiency and reduces background signal. Acridine orange (AO) can simultaneously stain DNA and RNA, distinguishing between live and dead cells, and is used for real-time monitoring of RNA in live cells because it fluoresces in different colors when binding nucleic acids at different pH levels. Calcium fluorescent white improves solubility and enhances the resolution of fungal structures. Propidium iodide increases the staining speed of dead cells and reduces the false staining rate. Dimethyl sulfoxide is typically used as a solvent to help the dye penetrate the cell membrane, addressing the aggregation problem of calcium fluorescent white and promoting the rapid penetration of dyes such as acridine orange and propidium iodide into cells or tissues. It also improves the penetration efficiency of ethidium bromide into fixed cells or tissue sections, especially suitable for thick samples, extending the preservation time of stained samples and reducing non-specific binding. In summary, each component in solution B plays a different role, but they need to be mixed in the right proportions to achieve good results. This combined effect enhances the fluorescence performance.

[0025] In summary, the fluorescent staining formulation of this invention, through optimal ratio and control of the permeation-enhancing effect of DMSO, enables multidimensional detection of fungi, taking into account fungal morphology, viability, and metabolic activity, making it more suitable for accurate and rapid clinical diagnosis.

[0026] This invention also provides a method for preparing a fungal multiplex fluorescent staining agent, comprising the following steps: Preparation of Solution A: Dissolve the fluorescent whitening agent in distilled water, and at the same time dissolve the CYP51 fluorescent dye in distilled water. After mixing the two, add potassium hydroxide, dimethyl sulfoxide and glycerol and mix. Prepare solution B: Dissolve ethidium bromide in distilled water, dissolve acridine orange in distilled water, mix the two together, and then add calcium fluorescein, propidium iodide, dimethyl sulfoxide, and glycerol.

[0027] The fungal multiplex fluorescent staining preparation of the present invention has a wide range of applications in fungal staining.

[0028] Preferably, as a further feasible option, the application method specifically includes the following steps: First, add solution A to the sample, let it stand for a while, and then add solution B.

[0029] Preferably, as a further feasible approach, solution A is first added to the sample and allowed to stand for 2-5 minutes, then solution B is added and allowed to stand before observation under a microscope.

[0030] Preferably, as a further feasible option, the solution B is added and then allowed to stand for 2-5 minutes.

[0031] The preparation method of the fluorescent staining agent of the present invention is simple to operate, with close connection between the operation steps, and has wide application. Attached Figure Description

[0032] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings.

[0033] Figure 1 This is a staining result diagram of the fungal multiplex fluorescent staining preparation of Example 1 of the present invention; Figure 2 This is a diagram showing the fungal-specific results of Experiment Example 2 of the present invention; Figure 3 This is a graph showing the fungal repeatability results of Experiment Example 2 of the present invention; Figure 4 This is a graph showing the fungal repeatability test results of Experiment Example 2 of the present invention; Figure 5This is a diagram showing the staining results of the fungal multiplex fluorescent staining preparation of Comparative Example 1 of the present invention; Figure 6 This is a diagram showing the staining results of the fungal multiplex fluorescent staining preparation of Comparative Example 2 of the present invention; Figure 7 This is a diagram showing the staining results of the fungal multiplex fluorescent staining preparation of Comparative Example 3 of the present invention; Figure 8-9 This is a diagram showing the staining results of the fungal multiplex fluorescent staining preparation of Comparative Example 4 of the present invention. Detailed Implementation

[0034] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0035] Example 1 Solution A is prepared as follows (by weight percentage): 0.6% CYP51 fluorescent dye Evans Blue, 10% fluorescent whitening agent, 3% potassium hydroxide, 1% dimethyl sulfoxide, appropriate amount of glycerol, and the remainder is distilled water. (1) Weigh the amount of fluorescent whitening agent according to the formula using a precision analytical balance and dissolve it in deionized water.

[0036] (2) Preparation of Evans Blue: Weigh the amount of Evans Blue according to the formula, add an appropriate amount of glycerin, and add pure distilled water to dissolve it completely.

[0037] (3) Weigh out the amount of potassium hydroxide in the formula and dissolve it in deionized water. At 20°C, slowly add glycerol and dimethyl sulfoxide in the formula proportion while stirring until completely dissolved.

[0038] (4) Mix the solutions from steps (1) and (2), and while stirring continuously, mix them with the solution from step (3). After stirring continuously, add the corresponding amount of distilled water to the formula amount and seal the container.

[0039] (5) Let stand at room temperature away from light for 24 hours, then repackage.

[0040] Solution B is prepared as follows (by weight percentage): ethidium bromide 0.2%, acridine orange 0.05%, calcium fluorescent white 0.5%, propidium iodide 7%, dimethyl sulfoxide 1%, glycerol as needed, and the remainder is distilled water; (1) Weigh the prescribed amount of ethidium bromide using a precision analytical balance and dissolve it in deionized water. Similarly, weigh the prescribed amounts of acridine orange, calcium fluorescein, and propidium iodide, and dissolve them in deionized water respectively.

[0041] (2) After mixing the four solutions from the above steps, slowly add glycerol and dimethyl sulfoxide in the formula proportions at 20°C while stirring, and add distilled water to the formula amount until completely dissolved.

[0042] (3) Let stand at room temperature away from light for 24 hours, then repackage.

[0043] (4) Packaging specifications: 50 servings / box; 100 servings / box; 200 servings / box.

[0044] (5) Packaging appearance: The label is clearly printed, there is no leakage, and the components inside the box are complete; liquid A is purple-blue and liquid B is orange-yellow.

[0045] Example 2 The specific operating steps are the same as in Example 1, except that solution A is prepared as follows: by weight percentage, CYP51 fluorescent dye Evans blue 1%, fluorescent whitening agent 5%, potassium hydroxide 4%, dimethyl sulfoxide 0.2%, glycerol as needed, and the remainder is distilled water. Solution B is prepared as follows: by weight percentage, ethidium bromide 0.05%, acridine orange 0.2%, calcium fluorescent white 2%, propidium iodide 10%, dimethyl sulfoxide 0.3%, glycerol as needed, and the remainder is distilled water.

[0046] Example 3 The specific operating steps are the same as in Example 1, except that solution A is prepared as follows: by weight percentage, CYP51 fluorescent dye Evans blue 0.7%, fluorescent whitening agent 8%, potassium hydroxide 3.2%, dimethyl sulfoxide 0.8%, glycerol as needed, and the remainder is distilled water. Solution B is prepared as follows: by weight percentage, ethidium bromide 0.08%, acridine orange 0.15%, calcium fluorescent white 0.7%, propidium iodide 9%, dimethyl sulfoxide 0.8%, glycerol as needed, and the remainder is distilled water.

[0047] Example 4 The specific operating steps are the same as in Example 1, except that solution A is prepared as follows: by weight percentage, CYP51 fluorescent dye Evans blue 0.9%, fluorescent whitening agent 7%, potassium hydroxide 3.8%, dimethyl sulfoxide 0.5%, glycerol as needed, and the remainder is distilled water. Solution B is prepared as follows: by weight percentage, ethidium bromide 0.15%, acridine orange 0.08%, calcium fluorescent white 1.5%, propidium iodide 8%, dimethyl sulfoxide 0.4%, glycerol as needed, and the remainder is distilled water.

[0048] Experimental Example 1 Applicable instruments: Microscope selection; fluorescence microscope with UV and B band fluorescence modules.

[0049] The prepared and dispensed solutions A and B from Example 1 were then used to conduct the experiment according to the following steps: 1. Using standard strains (live fungi), select standard strain sample CMCC(F)98001 and place it on a glass slide to prepare one sample detection area. First, add one drop of solution A to the sample, let it stand for 2-5 minutes, then add one drop of solution B and let it stand for 2-5 minutes. 2. Using standard strains (dead bacteria), select the dead bacteria of standard strain sample CMCC(F)98001 and place them on a glass slide to prepare one sample detection area. First, add one drop of solution A to the sample, let it stand for 2-5 minutes, then add one drop of solution B and let it stand for 2-5 minutes. 3. Using standard strains (drug-resistant bacteria), select standard strain sample BNCC263246 and place it on a glass slide to prepare one sample detection area. First, add one drop of solution A to the sample, let it stand for 2-5 minutes, then add one drop of solution B and let it stand for 2-5 minutes. 4. Microscopic observation: Blot away excess staining solution with filter paper, and observe the stained slide under a fluorescence microscope. (The results are as follows...) Figure 1 As shown.

[0050] Through experiments; from Figure 1 The results show that rapid fluorescent staining can accurately identify live, dead, and drug-resistant fungi; and the hyphae and spores of surviving fungi are clearly visible in blue-green. The hyphae and spores of dead fungi are pink; the internal structure of drug-resistant fungi shows obvious clump staining.

[0051] Examples 2-4 also yielded the same experimental results after the above experiments.

[0052] The results show that the fluorescent staining formulation of this invention provides better rapid fluorescent staining of fungi, more accurate identification, and is safer and more controllable. The staining formulation of this invention is a safe, reliable, stable, rapid, and accurate fluorescent staining diagnostic reagent.

[0053] Comparative Example 1 The specific operating steps are the same as in Example 1, except that calcium fluorescent white is not added to solution B.

[0054] Prepare and dispense solutions A and B for Comparative Example 1, and perform the experiment according to the following steps: Using standard strains (live fungi), the standard strain sample CMCC(F)98001 was selected and experimented according to the method in Experiment Example 1. The specific experimental results are as follows: Figure 5 As shown, it was found that without the addition of calcium fluorescent white, it could not cooperate well with other components to achieve good counterstaining of the strain, resulting in problems such as large background interference and low image contrast.

[0055] Comparative Example 2 The specific operating steps are the same as in Example 1, except that the amount of propidium iodide in solution B is 5%.

[0056] Prepare and dispense solutions A and B for Comparative Example 2, and perform the experiment according to the following steps: Using standard strains (live fungi), the standard strain sample CMCC(F)98001 was selected and experimented according to the method in Experiment Example 1. The specific experimental results are as follows: Figure 6 As shown, when propidium iodide is not added within an appropriate dosage range, a good staining effect cannot be achieved. Figure 6 The staining effect was poor, which shows the importance of adding an appropriate amount of propidium iodide.

[0057] Comparative Example 3 The specific operating steps are the same as in Example 1, except that the amount of dimethyl sulfoxide added to solution B is 0.1%.

[0058] Prepare and dispense solutions A and B for Comparative Example 3, and perform the experiment according to the following steps: Using standard strains (live fungi), the standard strain sample CMCC(F)98001 was selected and experimented according to the method in Experiment Example 1. The specific experimental results are as follows: Figure 7 As shown, when the amount of dimethyl sulfoxide is too small, it affects the staining permeability, thus revealing... Figure 7 The staining effect was unsatisfactory.

[0059] Comparative Example 4 The staining solution of Example 1 in patent CN106198470A was used for staining.

[0060] Prepare and dispense solutions A and B for Comparative Example 4, and perform the experiment according to the following steps: Using standard strains (live fungi), the standard strain sample CMCC(F)98001 was selected and experimented according to the method in Experiment Example 1. The specific experimental results are as follows: Figure 8-9 As shown in the accompanying drawings, the staining results also demonstrate that the staining effect of the staining solution in the embodiments of the present invention is not as good as that of the present invention.

[0061] Experiment Example 2 A fluorescence microscope equipped with UV and B band fluorescence modules was selected. Specificity, stability, and repeatability tests were conducted in an accelerated laboratory using solutions A and B prepared in Example 1. (1) Specificity test The staining solution was added separately to fungal, bacterial, and host cell samples for testing. Under a fluorescence microscope, only fungi showed complete and clear fluorescent morphology. For example... Figure 2 As shown.

[0062] (2) Stability test The prepared and dispensed solutions A and B were placed under accelerated conditions (60℃) for 7 days. Visual inspection revealed no stratification or precipitation in solutions A and B; no other abnormalities were observed. Subsequent staining with CMCC(F)98001 on the same fungal specimen showed no attenuation of fluorescence signal, and the staining effect was clear and intact; the stability and reproducibility were excellent. Figure 3 As shown.

[0063] (3) Repeatability test Three experienced staff members were selected to perform fluorescent staining on 100 specimens of fungal infection (using standard strain sample CMCC(F)98001). The specimens were prepared and aliquoted with solutions A and B, and observed under a fluorescence microscope according to standard operating procedures. The results showed that the morphology was intact and clear; the positive rate was 100%. Figure 4 As shown.

[0064] Although the invention has been illustrated and described with reference to specific embodiments, it should be understood that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be included in the appended claims.

Claims

1. A fungal multiplex fluorescent staining agent, characterized in that, It mainly includes solution A and solution B: Solution A is mainly prepared from the following components: by mass parts, 0.6-1 parts of CYP51 fluorescent dye, 5-10 parts of fluorescent whitening agent, 3-4 parts of potassium hydroxide, 0.2-1 parts of dimethyl sulfoxide, and an appropriate amount of glycerol; Solution B is mainly prepared from the following components: by mass parts, ethidium bromide 0.05-0.2 parts, acridine orange 0.05-0.2 parts, calcium fluorescein 0.5-2 parts, propidium iodide 7-10 parts, dimethyl sulfoxide 0.3-1 parts, and glycerol as appropriate amount.

2. The fungal multiplex fluorescent staining preparation according to claim 1, characterized in that, Solution A is mainly prepared from the following components: by mass parts, 0.7-0.9 parts of CYP51 fluorescent dye, 7-8 parts of fluorescent whitening agent, 3.2-3.8 parts of potassium hydroxide, 0.5-0.8 parts of dimethyl sulfoxide, and an appropriate amount of glycerol.

3. The fungal multiplex fluorescent staining preparation according to claim 1, characterized in that, Solution B is mainly prepared from the following components: by mass parts, ethidium bromide 0.08-0.15 parts, acridine orange 0.08-0.15 parts, calcium fluorescein 0.7-1.5 parts, propidium iodide 8-9 parts, dimethyl sulfoxide 0.4-0.8 parts, and glycerol as appropriate amount.

4. The fungal multiplex fluorescent staining preparation according to any one of claims 1-3, characterized in that, The CYP51 fluorescent dye is formed by mixing Evans blue with glycerol.

5. The method for preparing the fungal multiplex fluorescent staining agent according to any one of claims 1-4, characterized in that, Includes the following steps: Preparation of Solution A: Dissolve the fluorescent whitening agent in distilled water, and at the same time dissolve the CYP51 fluorescent dye in distilled water. After mixing the two, add potassium hydroxide, dimethyl sulfoxide and glycerol and mix. Prepare solution B: Dissolve ethidium bromide in distilled water, dissolve acridine orange in distilled water, mix the two together, and then add calcium fluorescein, propidium iodide, dimethyl sulfoxide, and glycerol.

6. The use of the fungal multiplex fluorescent staining preparation according to any one of claims 1-4 in fungal staining.

7. The fungal multiplex fluorescent staining preparation according to claim 6, characterized in that, The application method specifically includes the following steps: First, add solution A to the sample, let it stand for a while, and then add solution B.

8. The application method according to claim 7, characterized in that, First, add solution A to the sample and let it stand for 2-5 minutes. Then, add solution B and let it stand before observing it under a microscope.

9. The application method according to claim 7, characterized in that, After adding solution B, let it stand for 2-5 minutes.

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