Application of multi-fungus fluorescent staining solution in preparation of kit for rapidly identifying survival / drug resistance of fungi

Abstract

The invention discloses application of a multi-fungus fluorescent staining solution in preparation of a kit for rapidly identifying survival / drug resistance of fungi, and belongs to the technical field of biological detection. The kit can be used for identifying the death and activity of fungi and high-precision rapid drug resistance identification while specific fungi staining is carried out; a fluorescence brightening agent is adopted to mark fungal components in a sample, and the fungal form is clearly observed under a fluorescence microscope. The nucleic acid fluorescent dye capable of selectively permeating and damaging the cell membrane displays the dead and living state of fungi on the basis of fungus dyeing, and the fluorescent dye permeates into cells and is combined with nucleic acid to emit fluorescence. Metabolic reactive dyes of the cells are used for carrying out rapid drug resistance identification on fungi subjected to drug action. The method can simultaneously realize fungal death and activity identification and rapid drug resistance detection, and is simple and convenient to operate, high in detection speed and accurate in result. The kit is suitable for rapid auxiliary diagnosis and treatment of clinical fungal infection. When the multi-fungus fluorescent staining solution is used for preparing the drug sensitivity detection kit, the detection time of the kit is only 1-2 hours.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to an application of a multiple fungal fluorescent staining solution in preparing a kit for rapidly identifying fungal survival / drug resistance. Background Art

[0002] Estimates of global fungal infection trends indicate that the primary drivers of this increase include an increase in high-risk populations, such as those suffering from tuberculosis, chronic obstructive pulmonary disease, cancer, organ transplantation, and glucocorticoid therapy. The global prevalence of new and emerging pathogenic fungal infections and antifungal resistance is alarming.

[0003] Fungal infections are characterized by high morbidity, rapid progression, and high mortality rates, coupled with a lack of early diagnostic markers. The diagnosis and treatment of fungal infections have become a major medical concern in recent years. Currently, the gold standard for diagnosing fungal infections remains respiratory specimen culture and identification, but this long culture time limits its clinical application. Other methods for fungal detection include culture, wet mount microscopy, single-plex fungal fluorescent staining, Gram staining, 1,3-β-D glucan and galactomannan assays, mass spectrometry, sequencing, and polymerase chain reaction (PCR). However, traditional fungal culture methods are time-consuming, leading to missed treatment windows; wet mount microscopy and Gram staining have low detection rates; single-plex fungal fluorescent staining can only qualitatively identify the fungus but cannot determine whether it is viable or drug-resistant; and tests such as 1,3-β-D glucan and galactomannan assays, mass spectrometry, sequencing, and polymerase chain reaction (PCR) are costly and also cannot determine whether the fungus is viable or drug-resistant.

[0004] Traditional antimicrobial susceptibility testing (ADA) for fungal resistance requires 24-72 hours of incubation, which is time-consuming and can lead to missing the optimal treatment window. PCR, on the other hand, is costly and can only detect known, single resistance genes. Other resistance mechanisms, such as overexpression of drug efflux pumps and altered metabolic pathways, are undetectable.

[0005] The diagnostic efficacy of the above detection technologies varies. In order to improve the diagnostic accuracy, it is particularly important to combine various fungal detection technologies for early diagnosis and timely treatment of fungal infections.

[0006] In summary, how to provide a method that is easy to operate, produces quick results, and can detect both the living and dead states of fungi and their drug resistance is one of the problems that urgently need to be solved in the field of fungal infection treatment. Summary of the Invention

[0007] The first purpose of the present invention is to provide a multiple fungal fluorescent staining solution for use in the preparation of a rapid fungal survival / resistance identification kit, which can identify the live or dead fungi and quickly identify drug resistance with high accuracy while staining specific fungi. The kit uses a fluorescent brightener that has a high affinity with the β-polysaccharide on the fungal cell wall, thereby marking the fungal components present in the sample, and the fungal morphology can be clearly observed under a fluorescence microscope. The nucleic acid fluorescent dye that selectively penetrates and damages the cell membrane is used to display the live or dead state of the fungi on the basis of fungal staining, that is, the dead membrane of the fungus is damaged, the fluorescent dye penetrates into the cell and combines with the nucleic acid to emit fluorescence. The metabolically active dye of the cell is used to quickly identify the drug resistance of the fungi after the action of the drug. In this way, the live or dead fungi identification and rapid drug resistance detection can be achieved simultaneously, with the advantages of simple operation, fast detection speed, and accurate results. It is suitable for rapid auxiliary diagnosis and treatment of clinical fungal infections.

[0008] The second purpose of the present invention is to provide an application of a multiple fungal fluorescent staining solution in the preparation of a drug sensitivity detection kit, which can complete the detection in only 1 to 2 hours.

[0009] The present invention is achieved through the following technical solutions:

[0010] A use of a multiple fungal fluorescent staining solution in the preparation of a rapid fungal survival / drug resistance identification kit, wherein the multiple fungal fluorescent staining solution is composed of solution A and solution B;

[0011] The solution A is composed of a fluorescent brightener and a nucleic acid fluorescent dye that selectively permeates and damages cell membranes;

[0012] The B solution is composed of a fluorescent brightener and a cell metabolic activity dye;

[0013] The fluorescent brightener is a combination of one or more of fluorescent brightener 28, fluorescent brightener 31, fluorescent brightener 71, fluorescent brightener 85, fluorescent brightener 113, fluorescent brightener 134, fluorescent brightener 220, and fluorescent brightener 351;

[0014] The nucleic acid fluorescent dye that selectively permeates and damages cell membranes is a combination of one or more of propidium iodide, ethidium iodide, propidium bromide, 7-aminoactinomycin D, SYTOX series dyes, ethidium bromide dimer, 4',6-diamidino-2-phenylindole, cyanine dyes, and Rose Bengal B;

[0015] The cell metabolic activity dye is one or more of a combination of methylthiazolyl tetrazolium (MTT) dye, CCK-8 dye, resazurin dye, fluorescein diacetate, and FUN-1.

[0016] The function of the above-mentioned liquid A is to identify the life or death of fungi based on the specific staining of the fungal cell wall; the function of liquid B is to quickly identify the drug resistance of fungi based on the specific staining of the fungal cell wall.

[0017] Preferably, the solution A further comprises a background dye, a stabilizer, a dyeing aid, a dissolution promoter and deionized water;

[0018] The mass concentrations of the raw materials in the solution A are: 0.01-0.2% of a fluorescent brightener, 0.01-0.1% of a nucleic acid fluorescent dye that selectively permeates and damages cell membranes, 0.001-0.02% of a background dye, 1-15% of a stabilizer, 1-15% of a dyeing aid, 5-12% of a dissolution promoter, and 57.68-92.979% of deionized water.

[0019] Preferably, the solution B further comprises background dye, buffer, solvent and deionized water;

[0020] The mass concentrations of the raw materials in the B solution are: 0.01-0.2% of fluorescent brightener, 0.01-0.1% of cell metabolic activity dye, 0.001-0.02% of background dye, 10-30% of buffer, 1-10% of solvent and 59.68-88.979% of deionized water.

[0021] Preferably, the background dye is a combination of one or more of Evans blue, trypan blue, bromophenol blue, thymol blue, bromocresol blue, safranin, eosin, and Congo red;

[0022] The stabilizer is a combination of one or more of glycerol, polyethylene glycol, Tris-HCl, sodium sulfite, and potassium citrate;

[0023] The dyeing auxiliary agent is a combination of one or more of ethylene glycol, dimethyl sulfoxide, N-methylpyrrolidone, and dimethylacetamide;

[0024] The dissolution promoter is a combination of one or more of potassium hydroxide and sodium hydroxide.

[0025] Preferably, the buffer is a combination of one or more of phosphate buffer, Tris-HCl buffer, glucose buffer, and Hepes buffer;

[0026] The solvent is dimethyl sulfoxide or methanol.

[0027] Preferably, the method for preparing solution A in the multiple fungal fluorescent staining solution comprises the following steps:

[0028] S1. Select a co-solvent according to the corresponding ratio and dissolve it in deionized water to form an aqueous solution, naturally cool it to room temperature, and set aside;

[0029] S2, dissolving a fluorescent brightener and a fluorescent dye for selectively permeating nucleic acids that damage cell membranes in deionized water to form a fluorescent brightener aqueous solution and a fluorescent dye for selectively permeating nucleic acids that damage cell membranes, respectively, for use;

[0030] S3, mixing the aqueous solutions obtained in S2 and S3 above for standby use;

[0031] S4, mixing the background dye, stabilizer and dyeing auxiliary for use;

[0032] S5. Add the mixed solution described in S4 dropwise into the mixed solution obtained in S3, stirring while adding. After mixing and dissolving, cool naturally and filter with a 0.22 μm microporous filter membrane to obtain liquid A in the multiple fungal fluorescent staining solution.

[0033] Preferably, the method for preparing solution B in the multiple fungal fluorescent staining solution comprises the following steps:

[0034] S1. Dissolve the selected solvent in deionized water in a corresponding proportion to form an aqueous solution, cool it naturally to room temperature, and set aside;

[0035] S2, dissolving a fluorescent brightener and a cell metabolic active dye in deionized water to form a fluorescent brightener aqueous solution and a cell metabolic active dye aqueous solution, respectively, for use;

[0036] S3, mixing the aqueous solutions obtained in S2 and S3 above for standby use;

[0037] S4, mix the background dye and buffer and set aside;

[0038] S5. Add the mixed solution described in S4 dropwise into the mixed solution obtained in S3, stirring while adding. After mixing and dissolving, cool naturally and filter with a 0.22 μm microporous filter membrane to obtain solution B in the multiple fungal fluorescent staining solution.

[0039] The preparation process for Solution A and Solution B is to filter the prepared reagents through a 0.22μm microporous filter membrane to remove impurities and microorganisms, thereby extending the shelf life of the reagents. The filtered reagents are then packaged into plastic dropper bottles according to the required specifications. To use, simply press the bottle to dispense a drop, without the need for additional pipetting tools. Operation is simple and fast.

[0040] Preferably, the method of using the liquid A to identify the survival of fungi is as follows: smear the sample to be tested, dropwise add the liquid A, cover with a cover glass, and observe under a microscope;

[0041] The sample to be tested is any one of a skin sample, a sputum sample, a pleural effusion sample, and a body fluid sample.

[0042] The above-mentioned fungal survival test in Solution A shows that impurities such as cells are dissolved by the solvent, leaving only the fungi. Under the microscope, the fungi exhibit white-green fluorescence, and their morphology is clearly visible. Live bacteria, due to their intact cell membranes, selectively permeate the damaged nucleic acid fluorescent dye, preventing it from entering, and thus exhibit only white-green fluorescence. In contrast, dead bacteria, due to their damaged cell membranes, selectively permeate the damaged nucleic acid fluorescent dye, allowing it to enter the cells and bind to the nucleic acids, emitting red fluorescence. Simultaneously, their cell walls exhibit white-green fluorescence. Live bacteria exhibit only white-green fluorescence, reflecting the morphological characteristics of fungi, while dead bacteria exhibit red nucleic acid content within their interiors, in addition to the white-green fluorescence.

[0043] A multiple fungal fluorescent staining solution is used in the preparation of a drug sensitivity detection kit, wherein solution A in the multiple fungal fluorescent staining solution is detected using the following method:

[0044] Mix the test sample and the test drug in a culture medium and culture for 24 to 48 hours. Rinse twice with sterile saline by centrifugation to remove the culture medium components. Fix the suspension with 10% formalin to prepare a single-cell suspension. After smearing, add solution A, cover with a coverslip, and observe under a microscope.

[0045] The sample to be tested is any one of a skin sample, a sputum sample, a pleural effusion sample, and a body fluid sample.

[0046] The results of the above-mentioned drug sensitivity test of liquid A are judged as follows: after the test sample is mixed with the drug to be tested, the drug-resistant sample still survives and the result is the same as that of live bacteria, while the non-drug-resistant sample dies and the result is the same as that of dead bacteria.

[0047] Preferably, the B solution in the multiple fungal fluorescent staining solution is detected by the following method:

[0048] The sample to be tested, the drug to be tested, and solution B are mixed and placed in liquid culture medium and incubated for 1 to 2 hours. After centrifugation and enrichment, the smear is covered with a coverslip and can be observed under a microscope.

[0049] The results of Solution B above indicate that non-resistant fungi are unable to metabolize the metabolically active dye under the influence of the drug, and therefore exhibit only white-green fluorescence. However, resistant fungi metabolize the metabolically active dye normally, unaffected by the drug. The dye enters the cell, forming a distinct, compact vacuole structure that exhibits significant red fluorescence, while its cell walls also exhibit white-green fluorescence. Therefore, non-resistant fungi exhibit only white-green fluorescence, reflecting their morphological characteristics, while resistant fungi exhibit both white-green fluorescence and internal red fluorescence.

[0050] Compared with the prior art, the present invention has at least the following technical effects:

[0051] (1) The present invention provides an application of a multiple fungal fluorescent staining solution in the preparation of a kit for rapid identification of fungal survival / drug resistance. The kit can identify the life and death of fungi and quickly identify drug resistance with high accuracy while staining specific fungi. The kit uses a fluorescent brightener that has a high affinity with the β-polysaccharide on the fungal cell wall, thereby marking the fungal components present in the sample, and the fungal morphology can be clearly observed under a fluorescence microscope. A nucleic acid fluorescent dye that selectively penetrates and damages the cell membrane is used to display the life and death status of the fungi on the basis of fungal staining, that is, the dead membrane of the fungus is damaged, and the fluorescent dye penetrates into the cell and combines with the nucleic acid to emit fluorescence. A metabolically active dye of the cell is used to quickly identify the drug resistance of the fungi after the action of the drug. In this way, the identification of the life and death of the fungi and the rapid detection of drug resistance can be achieved simultaneously, with the advantages of simple operation, fast detection speed, and accurate results. It is suitable for rapid auxiliary diagnosis and treatment of clinical fungal infections.

[0052] (2) The present invention provides an application of a multiple fungal fluorescent staining solution in the preparation of a drug sensitivity detection kit, which can complete the detection in only 1 to 2 hours.

[0053] (3) The technical solution of this application utilizes fluorescent brighteners, nucleic acid fluorescent dyes that selectively penetrate and damage cell membranes, and metabolically active dyes of cells to enable the morphology and life and death status of fungi to be observed under a fluorescence microscope, thereby helping doctors to make a quick diagnosis; at the same time, the morphology and drug resistance status of fungi can be observed under a fluorescence microscope.

[0054] The advantage of using liquid A for fungal survival testing is that it does not require cultivation. After a simple smear staining, the morphology of the fungi and its life or death status can be immediately observed. It is simple, fast, economical and accurate.

[0055] The advantage of using solution B for drug susceptibility testing is that the principle of fluorescent dye staining is based on the metabolic activity of cells. This dye can only be detected by the instrument after it is converted into red fluorescence by fungi with intact membranes and metabolic activity.

[0056] Solution A works by allowing the dye to penetrate dead cells and stain them after drug-induced membrane damage. A membrane-breaking agent is typically added to accelerate dye penetration. The entire incubation period requires 24-48 hours. However, the drug's effect on fungal metabolic activity is more sensitive to cell membrane damage, requiring only 1-2 hours.

[0057] No complicated sample processing is required. Compared with the existing drug sensitivity methods, the microdilution method takes 24-72 hours and the disk diffusion method (KB method) takes 24-36 hours, this kit only takes 1 hour, which means that the drug sensitivity results can be known 1-2 days earlier. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1This is the effect of liquid A on live bacteria;

[0059] Figure 2 This is the effect of liquid A on dead bacteria;

[0060] Figure 3 This is a graph showing the effect of solution B on drug-resistant bacteria;

[0061] Figure 4 This is a diagram showing the effect of liquid B on non-resistant bacteria. DETAILED DESCRIPTION

[0062] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0063] Example 1 This example is used to screen and verify fluorescent brightening agents.

[0064] (1) Since the stabilizer and dyeing aid are working solutions, the mother solution needs to be prepared first.

[0065] The stabilizer is shown in Table 1, the dyeing auxiliary is shown in Table 2, and the A solution is shown in Table 3.

[0066] Prepare stabilizers and dyeing aids according to Table 1 and Table 2.

[0067] Table 1 Stabilizer formula

[0068]

[0069] Table 2 Dyeing aid formula

[0070]

[0071] (2) Prepare Solution A according to Table 3. Since this example is to screen fluorescent brighteners, the contents of the remaining reagents are based on Table 3. Fluorescent brighteners 28, 31, 85, 134, and 220 are prepared according to Table 3 respectively.

[0072] Table 3 Liquid A formula

[0073]

[0074] (3) Test: Liquid A prepared with fluorescent brighteners 28, 31, 85, 134, and 220 was added dropwise to a dandruff smear containing Malassezia, covered with a coverslip, and observed under a fluorescence microscope. The results are shown in Table 4:

[0075] Table 4 Fluorescent brightener screening results

[0076]

[0077] (4) Conclusion:

[0078] like Figure 1 Shown is the effect of liquid A on live bacteria;

[0079] like Figure 2 Shown is the effect of liquid A on dead bacteria.

[0080] a. Fluorescent brightener 31 and fluorescent brightener 134 have white crystals precipitated, indicating that their stability is unqualified and they are not applicable. b. Fluorescent brighteners 31, 85, and 134 are not bright enough, and the background is bright, resulting in poor contrast between the background and the target, and are not applicable. c. Fluorescent brightener 28 has a normal effect, but because the background is blue, computer imaging has poor blue filtering effect, resulting in a weak contrast between the background and the target, and is not applicable. d. Fluorescent brightener 220 has the best appearance, background, fluorescence intensity, and imaging effect, so Fluorescent brightener 220 is selected as the fluorescent dye for staining fungi.

[0081] Example 2: This example screens and verifies nucleic acid fluorescent dyes that selectively permeate and damage cell membranes.

[0082] The fluorescent brightener used was fluorescent brightener 220. Except for the nucleic acid fluorescent dye that selectively permeates and damages cell membranes, the proportions of the other components were the same as those in Table 3 of Example 1.

[0083] Preparation: Five fluorescent dyes, including propidium iodide, propidium bromide, 7-aminoactinomycin D, 4',6-diamidino-2-phenylindole, and Rose Bengal B, were used for testing as nucleic acid fluorescent dyes that selectively permeate and damage cell membranes. The content of each dye was 0.01 g. Other components were the same as those in Table 3 of Example 1.

[0084] (2) Test: Liquid A prepared from five dyes, namely propidium iodide, propidium bromide, 7-aminoactinomycin D, 4',6-diamidino-2-phenylindole, and Rose Bengal B, was added dropwise to a dandruff smear containing Malassezia. One group of smears was fixed with 10% formalin for 5 minutes to break the membrane, indicating dead bacteria and the other group alive bacteria. The smears were covered with a coverslip and observed under a fluorescence microscope. The results are shown in Table 5:

[0085] Table 5 Screening results of nucleic acid fluorescent dyes that selectively permeate and damage cell membranes

[0086]

[0087]

[0088] (3) Conclusion: a. Propidium bromide and 4',6-diamidino-2-phenylindole have crystals precipitated, indicating that their stability is unqualified, and 4',6-diamidino-2-phenylindole can also stain living bacteria and is not suitable; b. Propidium iodide and 7-aminoactinomycin are relatively sufficient, but their brightness is not as good as Rose Bengal B and they have certain toxicity, so they are not suitable; c. Rose Bengal B has the best appearance, background, fluorescence intensity, and imaging effect, so it is selected as the fluorescent dye for nucleic acid staining of dead fungi.

[0089] Example 3: This example screens and verifies the metabolic activity dyes of cells in solution B.

[0090] The fluorescent brightener, background dye, and deionized water are the same as those in Solution A. Since the buffer and solvent are working solutions, a stock solution must be prepared first. The buffer is shown in Table 6, the solvent is shown in Table 7, and Solution B is shown in Table 8.

[0091] Prepare buffer and solvent according to Table 6 and Table 7

[0092] Table 6 Buffer formulation

[0093]

[0094]

[0095] Table 7 Solvent formulation

[0096]

[0097] (2) Prepare Solution B according to Table 8. Since this example is to screen the metabolic activity dye of cells, the contents of the remaining reagents are based on Table 8. MTT dye, CCK-8 dye, FUN-1 dye, and resazurin dye are prepared according to Table 8 respectively.

[0098] Table 8 Liquid B formula

[0099]

[0100] (3) Test: Liquid B prepared with MTT dye, CCK-8 dye, FUN-1 dye, and resazurin dye was added dropwise to the mixed drug, liquid culture medium, and specimen (two groups, drug-resistant and drug-inresistant), mixed, and incubated for 1 hour. After centrifugation and enrichment, the smear was covered with a coverslip and observed under a microscope; the results are shown in Table 9:

[0101] Table 9 Cell metabolic activity dye screening results

[0102]

[0103] (4) Conclusion:

[0104] like Figure 3Shown is the effect of solution B on drug-resistant bacteria;

[0105] like Figure 4 Shown is the effect of liquid B on non-resistant bacteria.

[0106] FUN-1 dye has the best imaging effect, so FUN-1 dye is selected as the active metabolic staining fluorescent dye for drug resistance.

[0107] In summary, the present invention has developed a method and kit for rapid identification of fungal viability and drug resistance, which can simultaneously identify dead and alive fungi with specific fungal staining and high accuracy. The method has the advantages of simple operation, fast detection speed, and accurate results, and is suitable for the rapid diagnosis and treatment of clinical fungal infections.

[0108] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Use of a multiple fungal fluorescent staining solution in the preparation of a rapid fungal survival / drug resistance identification kit, characterized in that: The multiple fungal fluorescent staining solution is composed of solution A and solution B; The solution A is composed of a fluorescent brightener and a nucleic acid fluorescent dye that selectively permeates and damages cell membranes; The B solution is composed of a fluorescent brightener and a cell metabolic activity dye; The fluorescent brightener is a combination of one or more of fluorescent brightener 28, fluorescent brightener 31, fluorescent brightener 71, fluorescent brightener 85, fluorescent brightener 113, fluorescent brightener 134, fluorescent brightener 220, and fluorescent brightener 351; The nucleic acid fluorescent dye that selectively permeates and damages cell membranes is a combination of one or more of propidium iodide, ethidium iodide, propidium bromide, 7-aminoactinomycin D, SYTOX series dyes, ethidium bromide dimer, 4',6-diamidino-2-phenylindole, cyanine dyes, and Rose Bengal B; The cell metabolic activity dye is a combination of one or more of methylthiazolyl tetrazolium dye, CCK-8 dye, resazurin dye, fluorescein diacetate, and FUN-1.

2. The use according to claim 1, characterized in that The solution A also includes background dye, stabilizer, dyeing assistant, dissolution agent and deionized water; The mass concentrations of the raw materials in the solution A are: 0.01-0.2% of a fluorescent brightener, 0.01-0.1% of a nucleic acid fluorescent dye that selectively permeates and damages cell membranes, 0.001-0.02% of a background dye, 1-15% of a stabilizer, 1-15% of a dyeing aid, 5-12% of a dissolution promoter, and 57.68-92.979% of deionized water.

3. The use according to claim 1, characterized in that The B solution also includes background dye, buffer, solvent and deionized water; The mass concentrations of the raw materials in the B solution are: 0.01-0.2% of fluorescent brightener, 0.01-0.1% of cell metabolic activity dye, 0.001-0.02% of background dye, 10-30% of buffer, 1-10% of solvent and 59.68-88.979% of deionized water.

4. The use according to claim 2, characterized in that The background dye is a combination of one or more of Evans blue, trypan blue, bromophenol blue, thymol blue, bromocresol blue, safranin, eosin, and Congo red; The stabilizer is a combination of one or more of glycerol, polyethylene glycol, Tris-HCl, sodium sulfite, and potassium citrate; The dyeing auxiliary agent is a combination of one or more of ethylene glycol, dimethyl sulfoxide, N-methylpyrrolidone, and dimethylacetamide; The dissolution promoter is a combination of one or more of potassium hydroxide and sodium hydroxide.

5. The use according to claim 3, characterized in that The buffer is a combination of one or more of phosphate buffer, Tris-HCl buffer, glucose buffer, and Hepes buffer; The solvent is dimethyl sulfoxide or methanol.

6. The use according to claim 1, characterized in that The preparation method of liquid A in the multiple fungal fluorescent staining solution comprises the following steps: S1. Select a co-solvent according to the corresponding ratio and dissolve it in deionized water to form an aqueous solution, naturally cool it to room temperature, and set aside; S2, dissolving a fluorescent brightener and a fluorescent dye for selectively permeating nucleic acids that damage cell membranes in deionized water to form a fluorescent brightener aqueous solution and a fluorescent dye for selectively permeating nucleic acids that damage cell membranes, respectively, for use; S3, mixing the aqueous solutions obtained in S2 and S3 above for standby use; S4, mixing the background dye, stabilizer and dyeing auxiliary for use; S5. Add the mixed solution described in S4 dropwise into the mixed solution obtained in S3, stirring while adding. After mixing and dissolving, cool naturally and filter with a 0.22 μm microporous filter membrane to obtain liquid A in the multiple fungal fluorescent staining solution.

7. The use according to claim 1, characterized in that The preparation method of solution B in the multiple fungal fluorescent staining solution comprises the following steps: S1. Dissolve the selected solvent in deionized water in a corresponding proportion to form an aqueous solution, cool it naturally to room temperature, and set aside; S2, dissolving a fluorescent brightener and a cell metabolic active dye in deionized water to form a fluorescent brightener aqueous solution and a cell metabolic active dye aqueous solution, respectively, for use; S3, mixing the aqueous solutions obtained in S2 and S3 above for standby use; S4, mix the background dye and buffer and set aside; S5. Add the mixed solution described in S4 dropwise into the mixed solution obtained in S3, stirring while adding. After mixing and dissolving, cool naturally and filter with a 0.22 μm microporous filter membrane to obtain solution B in the multiple fungal fluorescent staining solution.

8. The use according to claim 1, characterized in that The method of using Liquid A to identify the survival of fungi is as follows: smear the sample to be tested, add Liquid A dropwise, cover with a cover glass, and observe under a microscope; The sample to be tested is any one of a skin sample, a sputum sample, a pleural effusion sample, and a body fluid sample.

9. Use of a multiple fungal fluorescent staining solution in the preparation of a drug sensitivity detection kit, characterized in that: Liquid A in the multiple fungal fluorescent staining solution is detected using the following method: Mix the test sample and the test drug in a culture medium and culture for 24 to 48 hours. Rinse twice with sterile saline by centrifugation to remove the culture medium components. Fix the suspension with 10% formalin to prepare a single-cell suspension. After smearing, add solution A, cover with a coverslip, and observe under a microscope. The sample to be tested is any one of a skin sample, a sputum sample, a pleural effusion sample, and a body fluid sample.

10. The use according to claim 9, characterized in that Solution B in the multiple fungal fluorescent staining solution is detected using the following method: The sample to be tested, the drug to be tested, and solution B are mixed and placed in liquid culture medium and incubated for 1 to 2 hours. After centrifugation and enrichment, the smear is covered with a coverslip and can be observed under a microscope.

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