CN120427583A - Application of multiple fungal fluorescent staining solution in the preparation of a rapid identification kit for fungal survival/drug resistance - Google Patents
Application of multiple fungal fluorescent staining solution in the preparation of a rapid identification kit for fungal survival/drug resistanceInfo
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Abstract
本发明公开了一种多重真菌荧光染色液在制备快速鉴别真菌存活/耐药试剂盒中的应用，属于生物检测技术领域。该试剂盒在特异真菌染色同时鉴别真菌死活和高精准的快速耐药鉴别；采用荧光提亮剂标记样品中存在的真菌成分，在荧光显微镜下清楚观察到真菌形态。选择性渗透损伤细胞膜的核酸荧光染料在真菌染色的基础上显示真菌的死活状态，荧光染料渗透进细胞与核酸结合发出荧光。细胞的代谢活性染料对药物作用后的真菌进行快速耐药鉴别。能同时实现真菌死活鉴别和耐药性快速检测，操作简便、检测速度快、结果准确。适用于临床真菌感染的快速辅助诊断和治疗。多重真菌荧光染色液制备药敏检测试剂盒，该试剂盒检测时长仅需要1～2h即可完成。
The present invention discloses the use of a multiple fungal fluorescent staining solution in the preparation of a kit for rapid identification of fungal survival/drug resistance, and belongs to the field of biological detection technology. The kit can simultaneously identify the life and death of fungi and quickly identify drug resistance with high precision during specific fungal staining; a fluorescent brightener is used to mark the fungal components present in the sample, and the fungal morphology can be clearly observed under a fluorescence microscope. Nucleic acid fluorescent dyes that selectively penetrate and damage cell membranes display the life and death status of fungi on the basis of fungal staining. The fluorescent dyes penetrate into cells and combine with nucleic acids to emit fluorescence. Metabolic active dyes of cells perform rapid drug resistance identification of fungi after the action of drugs. It can simultaneously achieve identification of the life and death of fungi and rapid detection of drug resistance, with simple operation, fast detection speed and accurate results. It is suitable for rapid auxiliary diagnosis and treatment of clinical fungal infections. A drug sensitivity detection kit is prepared using multiple fungal fluorescent staining solutions, and the detection time of the kit is only 1 to 2 hours to complete.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to application of a multiplex fungus fluorescent staining solution in preparation of a kit for rapidly identifying fungus survival/drug resistance.
Background
According to estimated global fungal infection conditions, the main causes of the increase of global fungal infection include tuberculosis, slow lung obstruction, tumor and organ transplantation, and the increase of high risk groups such as glucocorticoid treatment. Worldwide new and emergent pathogenic fungal infections and antifungal drug resistance situations are alarming.
The diagnosis and treatment of the fungal infection have become one of the medical focus in recent years due to the characteristics of high morbidity, rapid progress of disease course, high death rate of disease and the like, and the lack of early diagnosis markers of the fungal infection. The gold standard for diagnosing fungal infection is still used for culturing and identifying respiratory tract specimens, but the culturing time is long, so that the clinical application is limited. In addition, the related fungus detection methods include fungus culture, wet-chip microscopy, single fungus fluorescent staining, gram staining, 1, 3-beta-D glucan, galactomannan detection, mass spectrometry, sequencing, polymerase chain reaction (polymerase chain reaction, PCR) and the like. However, the traditional fungi have long culture time, so that the optimal treatment period is missed, wet-film microscopic examination and gram staining detection rate are low, single-fungus fluorescent staining can only be used for qualitative fungi, the dead and drug resistant conditions of the fungi cannot be judged, and the detection cost of 1, 3-beta-D glucan, galactomannan detection, mass spectrum, sequencing, polymerase chain reaction (polymerase chain reaction, PCR) and the like is high, and the dead and drug resistant conditions of the fungi cannot be judged.
For drug resistance detection of fungi, the traditional drug sensitive test needs to be cultured for 24-72 hours, and takes a long time, so that the optimal treatment period is missed. The polymerase chain reaction is costly and can only detect known, single drug resistance genes. Other drug-resistant mechanisms such as over-expression mechanism of drug efflux pump, metabolic pathway change mechanism, etc. cannot be detected.
The diagnostic efficiency of the detection technology is different, and in order to improve the diagnostic accuracy, the early diagnosis and the timely treatment of fungal infection are particularly important by combining various fungal detection technologies.
In summary, how to provide a method for detecting the live state of fungi and the drug resistance of fungi with simple operation and rapid results is one of the urgent problems in the field of treating fungal infections.
Disclosure of Invention
The first object of the invention is to provide an application of a multiplex fungus fluorescent staining solution in preparing a kit for rapidly identifying survival/drug resistance of fungi, wherein the kit can identify the death activity of fungi and high-precision rapid drug resistance identification while specific fungi are stained, and the kit can be highly compatible with beta-polysaccharide on the cell wall of fungi by adopting a fluorescent brightening agent, so that fungus components existing in a sample can be marked, and the fungus morphology can be clearly observed under a fluorescent microscope. The nucleic acid fluorescent dye which selectively permeates and damages cell membranes is used for displaying the dead and alive state of fungi on the basis of fungus staining, namely the dead membrane of the fungi is damaged, and the fluorescent dye permeates into cells to combine with nucleic acid to emit fluorescence. And the metabolically active dye of the cells is utilized to rapidly identify the drug resistance of the fungi after the drug action. Therefore, the method can realize the fast detection of the death and the living of fungi and the drug resistance, and has the advantages of simple operation, fast detection speed, accurate result and the like. Is suitable for rapid auxiliary diagnosis and treatment of clinical fungal infection.
The second aim of the invention is to provide an application of the multiple fungus fluorescent staining solution in preparing a drug sensitive detection kit, wherein the detection duration of the kit can be completed only by 1-2 hours.
The invention is realized by the following technical scheme:
the application of a multiplex fungus fluorescent staining solution in preparing a kit for rapidly identifying fungus survival/drug resistance comprises a solution A and a solution B;
The solution A consists of a fluorescent brightening agent and a nucleic acid fluorescent dye which selectively permeates and damages cell membranes;
the solution B consists of a fluorescent brightening agent and a metabolic active dye of cells;
the fluorescent brightening agent is one or a combination of a plurality of fluorescent brightening agents 28, 31, 71, 85, 113, 134, 220 and 351;
The nucleic acid fluorescent dye capable of selectively penetrating and damaging the cell membrane is one or a combination of more of propidium iodide, ethidium iodide, propidium bromide, 7-amino actinomycin D, SYTOX series of dyes, ethidium bromide dimer, 4', 6-diamidino-2-phenylindole, cyanine dyes and rose bengal B;
The metabolically active dye of the cell is one or a combination of more of methylthiazolyl tetrazolium (MTT) dye, CCK-8 dye, resazurin dye, fluorescein diacetate and FUN-1.
The liquid A is used for identifying the dead or alive of the fungi on the basis of the specific staining of the cell walls of the fungi, and the liquid B is used for rapidly identifying the drug resistance of the fungi on the basis of the specific staining of the cell walls of the fungi.
Preferably, the solution A also comprises background dye, stabilizer, dyeing promoter, solvent and deionized water;
The mass concentration of each raw material in the solution A is 0.01-0.2% of fluorescent brightening agent, 0.01-0.1% of nucleic acid fluorescent dye capable of selectively penetrating and damaging cell membranes, 0.001-0.02% of background dye, 1-15% of stabilizer, 1-15% of dyeing auxiliary, 5-12% of cosolvent and 57.68-92.979% of deionized water.
Preferably, the solution B also comprises background dye, buffer solution, solvent and deionized water;
The mass concentration of each raw material in the solution B is 0.01-0.2% of fluorescent brightening agent, 0.01-0.1% of cell metabolism active dye, 0.001-0.02% of background dye, 10-30% of buffer solution, 1-10% of solvent and 59.68-88.979% of deionized water.
Preferably, the background dye is one or more of evans blue, trypan blue, bromophenol blue, thymol blue, bromphenol blue, safranin, eosin, congo red;
the stabilizer is one or a combination of more of glycerol, polyethylene glycol, tris-HCl, sodium sulfite and potassium citrate;
The dyeing auxiliary is one or a combination of more of ethylene glycol, dimethyl sulfoxide, N-methyl pyrrolidone and dimethylacetamide;
The cosolvent is one or a combination of more of potassium hydroxide and sodium hydroxide.
Preferably, the buffer is one or more of phosphate buffer, tris-HCl buffer, glucose buffer and Hepes buffer;
the solvent is dimethyl sulfoxide or methanol.
Preferably, the preparation method of the solution A in the multiplex fungus fluorescent staining solution comprises the following steps:
s1, selecting a cosolvent according to a corresponding proportion, dissolving the cosolvent in deionized water to form an aqueous solution, and naturally cooling to room temperature for later use;
S2, respectively dissolving the fluorescent brightening agent and the nucleic acid fluorescent dye selectively penetrating and damaging the cell membrane into deionized water to form a fluorescent brightening agent aqueous solution and a nucleic acid fluorescent aqueous solution selectively penetrating and damaging the cell membrane for later use;
s3, mixing the aqueous solutions obtained in the steps S2 and S3 for later use;
S4, mixing the background dye, the stabilizer and the dyeing auxiliary for later use;
And S5, dropwise adding the mixed solution obtained in the step S4 into the mixed solution obtained in the step S3, stirring while adding, mixing and dissolving, naturally cooling, and filtering by using a microporous filter membrane with the thickness of 0.22 mu m to obtain the solution A in the multiplex fungus fluorescent staining solution.
Preferably, the preparation method of the solution B in the multiplex fungus fluorescent staining solution comprises the following steps:
s1, selecting a solvent according to a corresponding proportion, dissolving the solvent in deionized water to form an aqueous solution, and naturally cooling to room temperature for later use;
s2, respectively dissolving the fluorescent brightening agent and the metabolic active dye of the cell in deionized water to form an aqueous solution of the fluorescent brightening agent and an aqueous solution of the metabolic active dye of the cell for later use;
s3, mixing the aqueous solutions obtained in the steps S2 and S3 for later use;
S4, mixing the background dye and the buffer solution for later use;
And S5, dropwise adding the mixed solution obtained in the step S4 into the mixed solution obtained in the step S3, stirring while adding, mixing and dissolving, naturally cooling, and filtering with a microporous filter membrane with the thickness of 0.22 mu m to obtain the solution B in the multiplex fungus fluorescent staining solution.
The preparation process of the solution A and the solution B is to filter the prepared reagent with a microporous filter membrane with the size of 0.22 mu m to remove impurities, microorganisms and the like so as to prolong the service life of the reagent, and the filtered reagent is packaged in plastic dropping bottles according to the required specification, and only the bottle body is required to be pressed for dropping one drop without an additional pipetting tool. The use and the operation are simple, convenient and quick.
Preferably, the method for identifying the survival of the fungi by the liquid A comprises the steps of smearing a sample to be tested, dripping the liquid A, covering the liquid A with a cover glass, and observing under a mirror;
The sample to be measured is any one of a skin sample, a sputum sample, a hydrothorax and ascites sample and a body fluid sample.
The result of the fungus survival detection of the solution A shows that only fungus is left after the impurities such as cells are ablated by the cosolvent, the fungus presents white green fluorescence under a mirror, the form is clear, and the living fungus only presents white green fluorescence because nucleic acid fluorescent dye which is formed by the complete and selective permeation of cell membranes and damages the cell membranes cannot enter. And dead bacteria can selectively permeate nucleic acid fluorescent dye which damages the cell membrane to enter the cell to combine with nucleic acid to emit red fluorescence due to the damage of the cell membrane, and the cell wall of the dead bacteria shows white green fluorescence. The live bacteria only show the morphological characteristics of fungi by white-green fluorescence, while dead bacteria show the nucleic acid state of red inside the bacteria besides the morphological characteristics of white-green fluorescence.
The application of the multiplex fungus fluorescent staining solution in the preparation of the drug sensitive detection kit comprises the following steps:
Mixing a sample to be tested and a drug to be tested, placing the mixture into a culture medium, culturing for 24-48 hours, centrifugally flushing the mixture with sterile physiological saline for 2 times, washing out components of the culture medium, fixing the mixture with 10% formalin to prepare single-cell suspension, and dripping A liquid into the single-cell suspension after smearing and covering the single-cell suspension with a cover glass for observation;
The sample to be measured is any one of a skin sample, a sputum sample, a hydrothorax and ascites sample and a body fluid sample.
And (3) the liquid A is subjected to drug sensitivity detection, and the result is judged that after the detected sample is mixed with the drug to be detected, the drug-resistant sample still survives, the result shows the same viable bacteria, the sample without drug resistance dies, and the result shows the same dead bacteria.
Preferably, the solution B in the multiplex fungus fluorescent staining solution is detected by the following method:
And mixing the sample to be detected, the medicine to be detected and the liquid B in a liquid culture medium, incubating for 1-2 h, centrifuging and enriching, and covering a cover glass on a smear to observe under a microscope.
The result of the solution B shows that the non-drug-resistant fungi can not metabolize the metabolic active dye of the cells under the action of the drug, and only shows white green fluorescence. The drug-resistant fungi are not influenced by drugs to normally metabolize the metabolic active dye of the cells, the active dye enters the cells to form a vacuole structure with a compact structure, the cell wall of the cell is white green, and the cell wall of the cell shows remarkable red fluorescence. The non-drug resistant fungus only shows the morphological characteristics of the fungus, while the drug resistant fungus also shows red fluorescence inside the fungus in addition to the morphological characteristics of the white-green fluorescence.
Compared with the prior art, the invention has at least the following technical effects:
The invention provides application of a multiplex fungus fluorescent staining solution in preparing a kit for rapidly identifying survival/drug resistance of fungi, wherein the kit can identify the death activity of the fungi and high-precision rapid drug resistance identification while specific fungi are stained, and the kit can be highly compatible with beta-polysaccharide on the cell wall of the fungi by adopting a fluorescent brightening agent, so that fungus components existing in a sample are marked, and the fungus morphology can be clearly observed under a fluorescent microscope. The nucleic acid fluorescent dye which selectively permeates and damages cell membranes is used for displaying the dead and alive state of fungi on the basis of fungus staining, namely the dead membrane of the fungi is damaged, and the fluorescent dye permeates into cells to combine with nucleic acid to emit fluorescence. And the metabolically active dye of the cells is utilized to rapidly identify the drug resistance of the fungi after the drug action. Therefore, the method can realize the fast detection of the death and the living of fungi and the drug resistance, and has the advantages of simple operation, fast detection speed, accurate result and the like. Is suitable for rapid auxiliary diagnosis and treatment of clinical fungal infection.
The invention provides application of the multiplex fungus fluorescent staining solution in preparation of a drug sensitive detection kit, and the detection duration of the kit can be completed only by 1-2 hours.
The technical scheme of the application utilizes the fluorescent brightening agent, the nucleic acid fluorescent dye selectively penetrating and damaging cell membranes and the metabolic active dye of cells to realize that the form and the death state of fungi can be observed under a fluorescent microscope, help doctors to quickly make diagnosis, and simultaneously realize that the form and the drug-resistant state of the fungi can be observed under the fluorescent microscope.
The solution A is used for testing the survival of the fungi, and has the advantages that the shape and the death state of the fungi can be observed in time just by simple smear dyeing without culturing, and the method is simple, quick, economical and accurate.
The advantage of using solution B for drug sensitive testing is that the principle of fluorescent dye staining is based on the metabolic activity of the cells. This dye is only detectable by the instrument after conversion to red fluorescence by fungi with membrane integrity and metabolic activity.
The liquid A principle is that after membrane damage mediated by drugs, dye permeates into dead cells and colors the dead cells. Generally, a membrane breaker needs to be added to accelerate the permeation speed of the dye. The whole experiment requires 24-48h for incubation. Whereas the effect of the drug on the metabolic activity of fungi is sensitive to the breakdown of the cell membrane, only 1-2h is required.
Compared with the traditional drug sensitivity method of micro dilution which requires 24-72 hours and the paper diffusion method (K-B method) which requires 24-36 hours, the kit only requires 1 hour, and the drug sensitivity result can be known 1-2 days earlier.
Drawings
FIG. 1 is a graph showing the effect of liquid A on viable bacteria;
FIG. 2 is a graph showing the effect of liquid A on dead bacteria;
FIG. 3 is a graph showing the effect of solution B on drug-resistant bacteria;
FIG. 4 is a graph showing the effect of solution B on non-drug-resistant bacteria.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
Example 1 this example performs screening and validation of fluorescent brighteners.
(1) Since the stabilizer and the dyeing assistant are working fluids, the mother liquor needs to be prepared first.
The stabilizer is shown in Table 1, the dyeing promoter is shown in Table 2, and the solution A is shown in Table 3.
The formulation of the stabilizers and dyeing aids was carried out according to tables 1, 2.
Table 1 stabilizer formulation
Table 2 dyeing auxiliary formula
(2) Liquid a was prepared according to table 3, and since the fluorescent brightening agent was selected in this example, the remaining reagent contents were as shown in table 3, and the fluorescent brightening agents 28, 31, 85, 134 and 220 were prepared as shown in table 3, respectively.
Table 3A liquid formulation
(3) The test was carried out by taking solution A prepared from fluorescent brightening agents 28, 31, 85, 134 and 220, dropping the solution A onto a dandruff smear containing malassezia, covering a cover slip, and observing the solution A under a fluorescent microscope, and the results are shown in Table 4:
TABLE 4 fluorescent brightening agent screening results
(4) Conclusion:
as shown in FIG. 1, the effect diagram of the liquid A on the living bacteria is shown;
FIG. 2 shows the effect of liquid A on dead bacteria.
A. the fluorescent brightening agent 31 and the fluorescent brightening agent 134 have white crystals precipitated to indicate that the stability is not qualified and is not applicable, b, the fluorescent brightening agent 31, 85 and 134 have insufficient brightness and poor contrast effect between the background and the target due to the fact that the background is bright, c, the fluorescent brightening agent 28 has normal effect, but the contrast effect between the background and the target is not outstanding due to poor blue filtering effect of computer imaging due to the fact that the background is blue, d, the fluorescent brightening agent 220 has the appearance, the background, the fluorescent intensity and the imaging effect which are all optimal, and the fluorescent brightening agent 220 is selected as a fungal dyeing fluorescent dye.
Example 2 this example demonstrates the screening and validation of nucleic acid fluorochromes that selectively permeate and damage cell membranes.
The ratio of the other components except that the fluorescent brightening agent 220 was used for selectively permeating the nucleic acid fluorescent dye that damaged the cell membrane was the same as in Table 3 of example 1.
The nucleic acid fluorescent dye selectively penetrating and damaging cell membrane is tested by adopting five dyes of propidium iodide, propidium bromide, 7-amino actinomycin D, 4', 6-diamidino-2-phenyl indole and rose bengal B, the content of the dye is 0.01g, and other components are the same as the table 3 of the example 1.
(2) The test comprises taking A solution prepared from five dyes including propidium iodide, propidium bromide, 7-amino actinomycin D, 4', 6-diamidino-2-phenylindole and rose bengal B, dripping onto dandruff smears containing malassezia, wherein one set of smears is fixed by 10% formalin for 5min for breaking membranes, and then is dead bacteria, the other set of smears is living bacteria, a cover slip and a fluorescent microscope are observed, and the results are shown in Table 5:
TABLE 5 nucleic acid fluorescent dye screening results for selectively permeabilizing and damaging cell membranes
(3) The conclusion is that a, propidium bromide and 4', 6-diamidino-2-phenylindole are separated out, the stability is unqualified, the 4', 6-diamidino-2-phenylindole is inapplicable to living bacteria, B, propidium iodide and 7-amino actinomycin are relatively used, but the brightness is inferior to that of rose bengal B, and the fluorescent dye is inapplicable, c, the appearance, the background, the fluorescence intensity and the imaging effect of the rose bengal B are all optimal, and the rose bengal B is selected as the nucleic acid dyeing fluorescent dye of dead fungi.
Example 3 screening and validation of metabolically active dye of cells in solution B in this example.
Wherein the fluorescent brightening agent, the background dye and the deionized water are consistent with the solution A, and the buffer solution and the solvent are working solutions, so that mother solution is required to be prepared first. Buffer as in table 6, solvent as in table 7, and solution b as in table 8.
Buffers and solvents were formulated according to tables 6, 7
Table 6 buffer formulation
TABLE 7 solvent formulation
(2) The preparation of solution B was carried out according to Table 8, and since the present example is a metabolic active dye for screening cells, the remaining reagent contents were prepared according to Table 8, and MTT dye, CCK-8 dye, FUN-1 dye, and Resazurin dye were prepared according to Table 8, respectively.
Table 8B liquid formulation
(3) The test shows that the prepared B of the MTT dye, the CCK-8 dye, the FUN-1 dye and the Resazurin dye is dripped into the mixed medicine, the liquid culture medium and the specimen (respectively having two groups of drug resistance and drug intolerance), the mixed medicine and the specimen are incubated for 1h, the smear can be observed under a lens after being covered with a cover glass after centrifugal enrichment, and the result is shown in Table 9:
TABLE 9 Metabolic reactive dye screening results for cells
(4) Conclusion:
As shown in fig. 3, the effect of the liquid B on the drug-resistant bacteria is shown;
As shown in fig. 4, the effect of the solution B on the drug-intolerant bacteria is shown.
And if the FUN-1 dye has optimal imaging effect, the FUN-1 dye is selected as a drug-resistant active metabolism dyeing fluorescent dye.
In conclusion, the invention develops a rapid drug resistance identification method and a kit for identifying the dead and alive fungi and high accuracy at the same time of specific fungus staining, and has the advantages of simple operation, high detection speed, accurate results and the like. Is suitable for rapid diagnosis and treatment of clinical fungal infection.
Finally, it should be noted that the above description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The application of the multiple fungus fluorescent staining solution in preparing a kit for rapidly identifying the survival/drug resistance of fungi is characterized in that the multiple fungus fluorescent staining solution consists of solution A and solution B;
The solution A consists of a fluorescent brightening agent and a nucleic acid fluorescent dye which selectively permeates and damages cell membranes;
the solution B consists of a fluorescent brightening agent and a metabolic active dye of cells;
the fluorescent brightening agent is one or a combination of a plurality of fluorescent brightening agents 28, 31, 71, 85, 113, 134, 220 and 351;
The nucleic acid fluorescent dye capable of selectively penetrating and damaging the cell membrane is one or a combination of more of propidium iodide, ethidium iodide, propidium bromide, 7-amino actinomycin D, SYTOX series of dyes, ethidium bromide dimer, 4', 6-diamidino-2-phenylindole, cyanine dyes and rose bengal B;
The metabolic active dye of the cell is one or a combination of more of methylthiazolyl tetrazolium dye, CCK-8 dye, resazurin dye, fluorescein diacetate and FUN-1.
2. The use according to claim 1, wherein the liquid a further comprises a background dye, a stabilizer, a dyeing promoter, a solvent and deionized water;
The mass concentration of each raw material in the solution A is 0.01-0.2% of fluorescent brightening agent, 0.01-0.1% of nucleic acid fluorescent dye capable of selectively penetrating and damaging cell membranes, 0.001-0.02% of background dye, 1-15% of stabilizer, 1-15% of dyeing auxiliary, 5-12% of cosolvent and 57.68-92.979% of deionized water.
3. The use according to claim 1, wherein the solution B further comprises a background dye, a buffer, a solvent and deionized water;
The mass concentration of each raw material in the solution B is 0.01-0.2% of fluorescent brightening agent, 0.01-0.1% of cell metabolism active dye, 0.001-0.02% of background dye, 10-30% of buffer solution, 1-10% of solvent and 59.68-88.979% of deionized water.
4. The use according to claim 2, wherein the background dye is one or more of evans blue, trypan blue, bromophenol blue, thymol blue, bromcreosol blue, safranin, eosin, congo red;
the stabilizer is one or a combination of more of glycerol, polyethylene glycol, tris-HCl, sodium sulfite and potassium citrate;
The dyeing auxiliary is one or a combination of more of ethylene glycol, dimethyl sulfoxide, N-methyl pyrrolidone and dimethylacetamide;
The cosolvent is one or a combination of more of potassium hydroxide and sodium hydroxide.
5. The use according to claim 3, wherein the buffer is one or more of phosphate buffer, tris-HCl buffer, glucose buffer, hepes buffer;
the solvent is dimethyl sulfoxide or methanol.
6. The use according to claim 1, wherein the preparation method of the solution a in the multiplex fungal fluorescent staining solution comprises the following steps:
s1, selecting a cosolvent according to a corresponding proportion, dissolving the cosolvent in deionized water to form an aqueous solution, and naturally cooling to room temperature for later use;
S2, respectively dissolving the fluorescent brightening agent and the nucleic acid fluorescent dye selectively penetrating and damaging the cell membrane into deionized water to form a fluorescent brightening agent aqueous solution and a nucleic acid fluorescent aqueous solution selectively penetrating and damaging the cell membrane for later use;
s3, mixing the aqueous solutions obtained in the steps S2 and S3 for later use;
S4, mixing the background dye, the stabilizer and the dyeing auxiliary for later use;
And S5, dropwise adding the mixed solution obtained in the step S4 into the mixed solution obtained in the step S3, stirring while adding, mixing and dissolving, naturally cooling, and filtering by using a microporous filter membrane with the thickness of 0.22 mu m to obtain the solution A in the multiplex fungus fluorescent staining solution.
7. The use according to claim 1, wherein the preparation method of the solution B in the multiplex fungal fluorescent staining solution comprises the following steps:
s1, selecting a solvent according to a corresponding proportion, dissolving the solvent in deionized water to form an aqueous solution, and naturally cooling to room temperature for later use;
s2, respectively dissolving the fluorescent brightening agent and the metabolic active dye of the cell in deionized water to form an aqueous solution of the fluorescent brightening agent and an aqueous solution of the metabolic active dye of the cell for later use;
s3, mixing the aqueous solutions obtained in the steps S2 and S3 for later use;
S4, mixing the background dye and the buffer solution for later use;
And S5, dropwise adding the mixed solution obtained in the step S4 into the mixed solution obtained in the step S3, stirring while adding, mixing and dissolving, naturally cooling, and filtering with a microporous filter membrane with the thickness of 0.22 mu m to obtain the solution B in the multiplex fungus fluorescent staining solution.
8. The application of the solution A to identifying the survival of fungi according to claim 1, wherein the method comprises the steps of smearing a sample to be tested, dripping the sample A, covering the smear with a cover glass, and observing the smear under a microscope;
The sample to be measured is any one of a skin sample, a sputum sample, a hydrothorax and ascites sample and a body fluid sample.
9. The application of the multiplex fungus fluorescent staining solution in the preparation of the drug sensitive detection kit is characterized in that the solution A in the multiplex fungus fluorescent staining solution is detected by adopting the following method:
Mixing a sample to be tested and a drug to be tested, placing the mixture into a culture medium, culturing for 24-48 hours, centrifugally flushing the mixture with sterile physiological saline for 2 times, washing out components of the culture medium, fixing the mixture with 10% formalin to prepare single-cell suspension, and dripping A liquid into the single-cell suspension after smearing and covering the single-cell suspension with a cover glass for observation;
The sample to be measured is any one of a skin sample, a sputum sample, a hydrothorax and ascites sample and a body fluid sample.
10. The use according to claim 9, wherein the multiplex fungal fluorescent staining solution B is detected by:
And mixing the sample to be detected, the medicine to be detected and the liquid B in a liquid culture medium, incubating for 1-2 h, centrifuging and enriching, and covering a cover glass on a smear to observe under a microscope.
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