A highly identifiable fungal fluorescent staining solution

Abstract

The invention discloses a highly identifiable fungal fluorescent staining liquid, characterized in that it is made of the following components by mass percentage: fluorescent whitening agent 0.3-1.5%, background dye 0.05-0.25%, penetrant 3-13%, anti-fluorescence decay agent 1.5-4.5%, surfactant 0.01-0.05%, stabilizer 14-32%, sodium camphorsulfonate 0.1-0.4%, tea polyphenols 0.3-0.6%, nicotinamide adenine dinucleotide 0.1-0.3%, betaine 0.01-0.03%, water-soluble cyclodextrin polymer 0.01-0.03%, N, N-bis (2-hydroxyethyl) 2-aminoethanesulfonic acid sodium 0.01-0.04%, and the balance is deionized water. The invention also discloses a preparation method of the highly identifiable fungal fluorescent staining liquid. The high-recognition fungal fluorescent staining liquid disclosed by the present invention has high recognition and sensitivity, good staining effect on fungi, good long-term storage stability, convenient use, sufficient applicability, and excellent accuracy and efficiency in interpreting staining results.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical diagnosis, and in particular relates to a highly identifiable fungal fluorescent staining solution. Background Art

[0002] In recent years, with the development of society, people's pace of life has gradually accelerated, the pressure from life and work has become greater and greater, and the number of people with low immunity has increased year by year, so that fungal infections have become increasingly prominent. In particular, due to the continuous emergence of people infected with immunodeficiency viruses, fungi that were originally non-pathogenic have become pathogenic fungi. Therefore, fungal infections have become an increasingly serious problem.

[0003] Fungal detection is particularly important for clinical diagnosis and treatment. With the development of fungal detection technology, more and more methods can be applied to the detection and identification of fungi. Common detection methods include fluorescent staining, Parker ink staining, lactic acid phenol cotton blue staining, periodic acid Schiff (PAS) staining, etc. However, Parker ink staining, lactic acid phenol cotton blue staining, periodic acid Schiff (PAS) staining, etc., have the disadvantages of complicated steps and long reaction time. Fluorescent staining is based on the principle that fluorescent brighteners bind to polysaccharides on the fungal cell wall, but cannot bind to tissue cells, and stimulate fluorescence under UV irradiation, which distinguishes tissue cells from fungi with huge contrast. Fluorescent staining has high affinity and the reaction time only takes tens of seconds, which greatly improves the sensitivity and efficiency of clinical fungal detection. However, the existing fluorescent staining solution has the disadvantages of low applicability, poor staining effect on fungi, fast quenching of fluorescence effect after staining, and instability of the staining solution for long-term storage, which makes the fluorescence intensity after staining low. In addition, the fluorescent staining solutions on the market have more or less defects such as poor recognition, high production costs, cumbersome usage steps, inability to preserve and archive the tested samples for a long time, and easy detachment during the tissue section staining process.

[0004] In order to solve the above problems, Chinese invention patent application CN109297790A relates to a method for preparing a fluorescent staining solution for detecting fungi, which is composed of 280.001-0.003% fluorescent whitening agent, 5-15% potassium hydroxide, 3-6% dimethyl sulfoxide, 2-5% sodium chloride, 5-15% glycerol, 0.01-0.03% Evans blue, and the balance is purified water. The mycelium and spore structure of the fluorescent staining solution provided by the invention are clearly visible, easy to identify fungi, with high sensitivity, improving the accuracy and efficiency of the interpretation of the staining results, high detection rate, and wide application range. However, the recognition, specificity and long-term storage stability of the fluorescent staining solution for fungi need to be further improved.

[0005] Therefore, the development of a high-recognition fungal fluorescent staining solution with high recognition and sensitivity, good staining effect on fungi, good long-term storage stability, easy use, sufficient applicability, and excellent accuracy and efficiency in interpreting staining results meets market demand, has broad market value and application prospects, and is of great significance to promoting the development of the field of biomedical diagnosis. Summary of the invention

[0006] The main purpose of the present invention is to provide a highly identifiable fungal fluorescent staining liquid with high recognition and sensitivity, good staining effect on fungi, good long-term storage stability, convenient use, sufficient applicability, and excellent accuracy and efficiency in interpreting staining results. At the same time, the present invention also provides a preparation method of the highly identifiable fungal fluorescent staining liquid, which has simple process, simple operation, low energy consumption, is suitable for industrial production and application, and has significant economic benefits.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is: a highly identifiable fungal fluorescent staining solution, characterized in that it is made of the following components in percentage by mass: 0.3-1.5% fluorescent whitening agent, 0.05-0.25% background dye, 3-13% penetrant, 1.5-4.5% anti-fluorescence decay agent, 0.01-0.05% surfactant, 14-32% stabilizer, 0.1-0.4% sodium camphorsulfonate, 0.3-0.6% tea polyphenols, 0.1-0.3% nicotinamide adenine dinucleotide, 0.01-0.03% betaine, 0.01-0.03% water-soluble cyclodextrin polymer, 0.01-0.03% sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate, and the balance is deionized water.

[0008] Preferably, the fluorescent whitening agent is a mixture of fluorescent whitening agent 28, fluorescent whitening agent 31, and fluorescent whitening agent 220 in a mass ratio of 1:(1-3):(0.8-1.2).

[0009] Preferably, the background dye is at least one of Evans blue, bromocresol blue and bromophenol blue.

[0010] Preferably, the penetrant is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidone.

[0011] Preferably, the anti-fluorescence decay agent is a mixture of ascorbic acid, potassium phytate, and propyl gallate in a mass ratio of 1:(0.8-1.2):(2-4).

[0012] Preferably, the surfactant is Tween 20.

[0013] Preferably, the dissolution agent is at least one of triethylamine, sodium ethoxide, and potassium tert-butyl alcohol.

[0014] Preferably, the stabilizer is a mixture of water-soluble polymerized cyclodextrin and glycerol in a mass ratio of 1:(3-5).

[0015] Preferably, the water-soluble polymerized cyclodextrin has a CAS number of 25655-42-9, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number: S871829.

[0016] Another object of the present invention is to provide a method for preparing the highly identifiable fungal fluorescent staining solution, characterized in that it comprises the following steps: uniformly mixing the components according to mass percentage to obtain the highly identifiable fungal fluorescent staining solution.

[0017] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0018] (1) The highly identifiable fungal fluorescent staining solution provided by the present invention only requires the components to be evenly mixed, does not require complicated processes and special equipment, requires little capital investment, has low labor intensity, has a good working environment, has a wide supply of raw materials, is easy to operate, has high preparation efficiency, is easy to mass produce, and has significant economic benefits.

[0019] (2) The highly identifiable fungal fluorescent staining solution provided by the present invention is made of the following components in percentage by mass: 0.3-1.5% fluorescent whitening agent, 0.05-0.25% background dye, 3-13% penetrant, 1.5-4.5% anti-fluorescence decay agent, 0.01-0.05% surfactant, 14-32% stabilizer, 0.1-0.4% sodium camphorsulfonate, 0.3-0.6% tea polyphenols, 0.1-0.3% nicotinamide adenine dinucleotide, 0.01-0.03% betaine, 0.01-0.03% water-soluble cyclodextrin polymer, 0.01-0.03% sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate, and the balance is deionized water. The components work together to make the prepared fungal fluorescent staining solution have high recognition and sensitivity, good staining effect on fungi, good long-term storage stability, easy use, sufficient applicability, and excellent accuracy and efficiency in interpreting staining results.

[0020] (3) The highly identifiable fungal fluorescent staining solution provided by the present invention, the addition of water-soluble cyclodextrin polymer and cosolvent can promote the dissolution of the fluorescent whitening agent in water, and at the same time help the fluorescent whitening agent penetrate into the sample, so that the reagent can be placed stably for a long time, so that the detection method that originally required multiple steps can achieve fast one-step staining, and shorten the staining time. Clinical use is more convenient and quick, greatly improving the detection efficiency of fungi.

[0021] (4) The highly identifiable fungal fluorescent staining liquid provided by the present invention, wherein the dissolution promoter is at least one of triethylamine, sodium ethoxide, and potassium tert-butyl alcohol; the dissolution promoter has the effect of dissolving keratin and permeabilizing tissues. The use of these organic bases instead of traditional inorganic strong bases can effectively ensure the stability of the fluorescent whitening agent, improve the staining effect and long-term storage stability.

[0022] (5) The highly identifiable fungal fluorescent staining solution provided by the present invention comprises sodium camphorsulfonate, tea polyphenols and nicotinamide adenine dinucleotide, which act synergistically to effectively improve the fluorescence stability, thereby improving the accuracy and efficiency of the interpretation of the staining results and improving the staining effect. DETAILED DESCRIPTION

[0023] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0024] Example 1

[0025] A highly identifiable fungal fluorescent staining solution, characterized in that it is made of the following components in percentage by mass: 0.3% fluorescent whitening agent, 0.05% background dye, 3% penetrant, 1.5% anti-fluorescence decay agent, 0.01% surfactant, 14% stabilizer, 0.1% sodium camphorsulfonate, 0.3% tea polyphenols, 0.1% nicotinamide adenine dinucleotide, 0.01% betaine, 0.01% water-soluble cyclodextrin polymer, 0.01% sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate, and the balance is deionized water.

[0026] The fluorescent whitening agent is a mixture of fluorescent whitening agent 28, fluorescent whitening agent 31, and fluorescent whitening agent 220 in a mass ratio of 1:1:0.8.

[0027] The background dye is Evans blue; the penetrant is N,N-dimethylformamide; the anti-fluorescence decay agent is a mixture of ascorbic acid, potassium phytate, and propyl gallate in a mass ratio of 1:0.8:2; the surfactant is Tween 20; and the chaotropic agent is triethylamine.

[0028] The stabilizer is a mixture of water-soluble polymerized cyclodextrin and glycerol in a mass ratio of 1:3; the CAS number of the water-soluble polymerized cyclodextrin is 25655-42-9, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number: S871829.

[0029] A method for preparing the highly identifiable fungal fluorescent staining liquid is characterized by comprising the following steps: uniformly mixing the components according to mass percentage to obtain the highly identifiable fungal fluorescent staining liquid.

[0030] Example 2

[0031] A highly identifiable fungal fluorescent staining solution, characterized in that it is made of the following components in percentage by mass: 0.6% fluorescent whitening agent, 0.1% background dye, 5% penetrant, 2.5% anti-fluorescence decay agent, 0.02% surfactant, 19% stabilizer, 0.2% sodium camphorsulfonate, 0.4% tea polyphenols, 0.15% nicotinamide adenine dinucleotide, 0.015% betaine, 0.015% water-soluble cyclodextrin polymer, 0.02% sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate, and the balance is deionized water.

[0032] The fluorescent whitening agent is a mixture of fluorescent whitening agent 28, fluorescent whitening agent 31, and fluorescent whitening agent 220 in a mass ratio of 1:1.5:0.9; the background dye is bromocresol blue; the penetrant is dimethyl sulfoxide; and the anti-fluorescence decay agent is a mixture of ascorbic acid, potassium phytate, and propyl gallate in a mass ratio of 1:0.9:2.5.

[0033] The surfactant is Tween 20; the dissolution agent is sodium ethoxide; the stabilizer is a mixture of water-soluble polymerized cyclodextrin and glycerol in a mass ratio of 1:3.5; the CAS number of the water-soluble polymerized cyclodextrin is 25655-42-9, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number: S871829.

[0034] A method for preparing the highly identifiable fungal fluorescent staining liquid is characterized by comprising the following steps: uniformly mixing the components according to mass percentage to obtain the highly identifiable fungal fluorescent staining liquid.

[0035] Example 3

[0036] A highly identifiable fungal fluorescent staining solution, characterized in that it is made of the following components in percentage by mass: 1.2% fluorescent whitening agent, 0.15% background dye, 9% penetrant, 3% anti-fluorescence decay agent, 0.035% surfactant, 26% stabilizer, 0.25% sodium camphorsulfonate, 0.45% tea polyphenols, 0.2% nicotinamide adenine dinucleotide, 0.02% betaine, 0.02% water-soluble cyclodextrin polymer, 0.025% sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate, and the balance is deionized water.

[0037] The fluorescent whitening agent is a mixture of fluorescent whitening agent 28, fluorescent whitening agent 31 and fluorescent whitening agent 220 in a mass ratio of 1:2:1; the background dye is bromophenol blue; and the penetrant is N-methylpyrrolidone.

[0038] The anti-fluorescence decay agent is a mixture of ascorbic acid, potassium phytate, and propyl gallate in a mass ratio of 1:1:3; the surfactant is Tween 20; the dissolution agent is potassium tert-butyl alcohol; the stabilizer is a mixture of water-soluble polymerized cyclodextrin and glycerol in a mass ratio of 1:4; the CAS number of the water-soluble polymerized cyclodextrin is 25655-42-9, purchased from Shanghai McLean Biochemical Technology Co., Ltd., product number: S871829.

[0039] A method for preparing the highly identifiable fungal fluorescent staining liquid is characterized by comprising the following steps: uniformly mixing the components according to mass percentage to obtain the highly identifiable fungal fluorescent staining liquid.

[0040] Example 4

[0041] A highly identifiable fungal fluorescent staining solution, characterized in that it is made of the following components in percentage by mass: 1.3% fluorescent whitening agent, 0.22% background dye, 11% penetrant, 4.1% anti-fluorescence decay agent, 0.04% surfactant, 30% stabilizer, 0.35% sodium camphorsulfonate, 0.55% tea polyphenols, 0.22% nicotinamide adenine dinucleotide, 0.025% betaine, 0.025% water-soluble cyclodextrin polymer, 0.035% sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate, and the balance is deionized water.

[0042] The fluorescent whitening agent is a mixture of fluorescent whitening agent 28, fluorescent whitening agent 31, and fluorescent whitening agent 220 in a mass ratio of 1:2.5:1.1; the background dye is a mixture of Evans blue, bromocresol blue, and bromophenol blue in a mass ratio of 1:2:3.

[0043] The penetrant is a mixture of N,N-dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidone in a mass ratio of 1:3:5; the anti-fluorescence decay agent is a mixture of ascorbic acid, potassium phytate, and propyl gallate in a mass ratio of 1:1.1:3.5; the surfactant is Tween 20; and the chaotropic agent is a mixture of triethylamine, sodium ethoxide, and potassium tert-butyl alcohol in a mass ratio of 1:3:5.

[0044] The stabilizer is a mixture of water-soluble polymerized cyclodextrin and glycerol in a mass ratio of 1:4.5; the CAS number of the water-soluble polymerized cyclodextrin is 25655-42-9, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number: S871829.

[0045] A method for preparing the highly identifiable fungal fluorescent staining liquid is characterized by comprising the following steps: uniformly mixing the components according to mass percentage to obtain the highly identifiable fungal fluorescent staining liquid.

[0046] Example 5

[0047] A highly identifiable fungal fluorescent staining solution, characterized in that it is made of the following components in percentage by mass: 1.5% fluorescent whitening agent, 0.25% background dye, 13% penetrant, 4.5% anti-fluorescence decay agent, 0.05% surfactant, 32% stabilizer, 0.4% sodium camphorsulfonate, 0.6% tea polyphenols, 0.3% nicotinamide adenine dinucleotide, 0.03% betaine, 0.03% water-soluble cyclodextrin polymer, 0.04% sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate, and the balance is deionized water.

[0048] The fluorescent whitening agent is a mixture of fluorescent whitening agent 28, fluorescent whitening agent 31, and fluorescent whitening agent 220 in a mass ratio of 1:3:1.2; the background dye is Evans blue; the penetrant is N-methylpyrrolidone; and the anti-fluorescence decay agent is a mixture of ascorbic acid, potassium phytate, and propyl gallate in a mass ratio of 1:1.2:4.

[0049] The surfactant is Tween 20; the solubilizing agent is potassium tert-butyl alcohol; the stabilizer is a mixture of water-soluble polymerized cyclodextrin and glycerol in a mass ratio of 1:5; the CAS number of the water-soluble polymerized cyclodextrin is 25655-42-9, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number: S871829.

[0050] A method for preparing the highly identifiable fungal fluorescent staining liquid is characterized by comprising the following steps: uniformly mixing the components according to mass percentage to obtain the highly identifiable fungal fluorescent staining liquid.

[0051] Comparative Example 1

[0052] This example provides a highly identifiable fungal fluorescent staining solution, the formulation and preparation method of which are substantially the same as those of Example 1, except that nicotinamide adenine dinucleotide is not added.

[0053] Comparative Example 2

[0054] This example provides a highly identifiable fungal fluorescent staining solution, the formula and preparation method of which are basically the same as those of Example 1, except that no water-soluble cyclodextrin polymer and sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate are added.

[0055] In order to further illustrate the beneficial technical effects of the high-density polyurethane foam involved in the embodiments of the present invention, the highly identifiable fungal fluorescent staining liquid prepared in the above embodiments 1-5 and comparative examples 1-2 was subjected to relevant performance experiments, and the experimental results are shown in Table 1.

[0056] The test method is as follows: turn on the ultra-clean workbench UV lamp for disinfection and sterilization half an hour in advance, and clean and disinfect the operator's clothes and hands; take out the prepared culture medium and number it according to the gynecological sample; according to the corresponding number, open the plate and drop a drop of sterilized saline on it, dip the sampling swab in saline and evenly spread it on the plate, cover the culture dish cover, and put the sampling swab back into the original tube; invert the culture dish and culture it in a 33℃ biochemical incubator for 24 to 72 hours, during which time observe the growth of the colony in the culture dish. Spread the sample evenly in the middle of the slide, add fungal fluorescent staining solution, cover with a coverslip, and examine under a microscope. Statistical calculations were performed on sensitivity, specificity, accuracy, and missed detection rate.

[0057] Table 1

[0058] project Sensitivity Specificity Accuracy Missed detection rate unit ％ ％ ％ ％ Example 1 75.16 99.59 92.98 8.33 Example 2 76.02 99.71 93.55 8.01 Example 3 76.89 99.78 94.02 7.69 Example 4 77.54 99.84 94.67 7.21 Example 5 78.22 99.90 95.11 6.98 Comparative Example 1 70.30 99.04 90.32 9.78 Comparative Example 2 71.05 89.89 90.10 9.32

[0059] It can be seen from the above data results that the high-recognition fungal fluorescent staining solution disclosed in the embodiment of the present invention has higher sensitivity, specificity and accuracy than the control example, and lower missed detection rate; the addition of nicotinamide adenine dinucleotide, water-soluble cyclodextrin polymer and sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate is beneficial to improving the above performance.

[0060] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A highly identifiable fungal fluorescent staining solution, characterized in that: The invention is prepared from the following components in percentage by mass: 0.3-1.5% of fluorescent whitening agent, 0.05-0.25% of background dye, 3-13% of penetrant, 1.5-4.5% of anti-fluorescence decay agent, 0.01-0.05% of surfactant, 14-32% of stabilizer, 0.1-0.4% of sodium camphorsulfonate, 0.3-0.6% of tea polyphenols, 0.1-0.3% of nicotinamide adenine dinucleotide, 0.01-0.03% of betaine, 0.01-0.03% of water-soluble cyclodextrin polymer, 0.01-0.03% of sodium N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate, and the balance is deionized water; The fluorescent whitening agent is a mixture of fluorescent whitening agent 28, fluorescent whitening agent 31, and fluorescent whitening agent 220 in a mass ratio of 1:(1-3):(0.8-1.2); The background dye is at least one of Evans blue, bromocresol blue and bromophenol blue; The penetrant is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidone; The anti-fluorescence decay agent is a mixture of ascorbic acid, potassium phytate, and propyl gallate in a mass ratio of 1:(0.8-1.2):(2-4); The surfactant is Tween 20; The dissolution agent is at least one of triethylamine, sodium ethoxide and potassium tert-butyl alcohol; The stabilizer is a mixture of water-soluble polymerized cyclodextrin and glycerol in a mass ratio of 1:(3-5).

2. The highly identifiable fungal fluorescent staining solution according to claim 1, characterized in that: The water-soluble polymerized cyclodextrin has a CAS number of 25655-42-9 and was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with a product number of S871829.

3. A method for preparing the highly identifiable fungal fluorescent staining solution according to any one of claims 1-2, characterized in that: The method comprises the following steps: uniformly mixing the components according to mass percentage to obtain a highly identifiable fungal fluorescent staining liquid.