Kit for vaginal microorganism fluorescence detection and preparation method thereof

Abstract

The invention discloses a kit for vaginal microorganism fluorescence detection and a preparation method thereof, and belongs to the technical field of pathogenic microorganism fluorescence detection in medical clinical examination. The kit provided by the invention comprises a liquid A and a liquid B, the solution A comprises a fluorescent staining agent, a bacteriostatic agent, an anti-fluorescence decline agent, a counterstaining agent and purified water; the solution B comprises fluorescein, a buffer solution, a preservative, a stabilizer and purified water. According to the application, the liquid A and the liquid B are combined into the kit, pathogens in vaginal secretion can be marked, and the inflammation type can be judged more comprehensively and accurately, so that the whole kit is more accurate and sensitive.

Description

Technical Field

[0001] The present application belongs to the technical field of fluorescence detection of pathogenic microorganisms in medical clinical tests, and in particular to a kit for fluorescence detection of vaginal microorganisms and a preparation method thereof. Background Art

[0002] Infections caused by vaginal microbial proliferation are clinically divided into bacterial vaginosis, vulvovaginal candidiasis, trichomonas vaginitis, aerobic bacterial vaginitis, and cytolytic vaginosis. Women with vaginitis will affect the quality of sperm during pregnancy, and even lead to female infertility; even during pregnancy, it is easy to cause intrauterine infection, birth canal infection, and other links to infect the fetus and newborn, causing miscarriage, premature birth, congenital malformations, mental retardation and other serious consequences. It can also induce other diseases, such as genital infection, pelvic inflammatory disease, periarthritis of the shoulder, sexual intercourse pain, etc. It can be seen that the harm of female vaginitis is quite large.

[0003] Rapid and accurate diagnosis of inflammatory diseases through vaginal secretions is an important prerequisite for clinical treatment. Routine examination of vaginal secretions is currently the most commonly used clinical test item. In outpatient testing, wet slides soaked in 0.9% sodium chloride solution are usually used for direct microscopic examination, which is relatively fast and convenient. However, the wet slide microscopic examination method is for the examiner to observe with the help of a microscope, which has certain subjective factors, and the specimens are directly thrown away after observation, which is not conducive to future traceability. Some hospitals also use fully automatic machines to analyze routine items of secretions, but the machines mostly analyze molds and trichomonas based on the size of cells or bacteria, resulting in a high rate of missed detection and misdiagnosis. Dry chemical enzyme method is also used in the examination of vaginal pathogens, but its timeliness is not as good as traditional wet slides, and there are also many interference factors. The Gram staining method, which is currently more recognized, is relatively accurate, but time-consuming, and has a certain relationship with the operating techniques of each inspector, and is not suitable for promotion in outpatient testing. Therefore, rapid and accurate vaginal secretion detection methods have also received more and more attention, especially in outpatient testing. Summary of the invention

[0004] In order to overcome the above-mentioned defects in the prior art, the present application provides a kit for vaginal microorganism fluorescence detection and a preparation method thereof, which can not only realize the labeling and identification of microorganisms and cells, but also is easy to operate and has high sensitivity and accuracy.

[0005] In order to achieve the above-mentioned invention object, this application provides the following technical solutions: In one aspect, the present application provides a kit for fluorescent detection of vaginal microorganisms, the kit comprising liquid A and liquid B; The A solution includes components with the following concentrations: Fluorescent dye 0.005~1%, antibacterial agent 0.01~30%, anti-fluorescence decay agent 0.02~20%, counterstain 0.002~10%, and the balance is purified water; The B solution includes components with the following concentrations: Fluorescein 0.001~0.8%, buffer solution 0.05~20%, preservative 0.05~20%, stabilizer 0.001~3%, and purified water as the balance.

[0006] Optionally, the concentration of the fluorescent dye is independently selected from any value of 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, or any range between the two; The concentration of the antibacterial agent is independently selected from any value among 0.01%, 0.02%, 0.05%, 0.1%, 1%, 5%, 10%, 20%, 30%, or any range between the two; The concentration of the anti-fluorescence decay agent is independently selected from any value of 0.02%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, or any range between the two; The concentration of the counterstain is independently selected from any value among 0.002%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, or any range between the two; The concentration of the fluorescein is independently selected from any value among 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or any range between two values; The concentration of the buffer solution is independently selected from any value of 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, or any range between the two; The concentration of the preservative is independently selected from any value among 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, or any range between the two; The concentration of the stabilizer is independently selected from any value of 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, or any range therebetween.

[0007] Optionally, the fluorescent dye includes at least one of a fluorescent brightener 28, fluorescein isothiocyanate, and a peridinin-chlorophyll-protein complex.

[0008] Optionally, the antibacterial agent includes at least one of dimethyl sulfoxide, Proclin 300, and potassium hydroxide.

[0009] Optionally, the anti-fluorescence decay agent includes at least one of glycerol, Tween, and gallate.

[0010] Optionally, the counterstain includes at least one of methyl green and Evans blue.

[0011] In the present application, the fluorescent dye in liquid A interacts with the counterstain to accurately and clearly mark the fungi.

[0012] Optionally, the fluorescein includes at least one of acridine orange and rhodamine.

[0013] Optionally, the buffer solution includes at least one of a phosphate buffer solution and a sodium acetate buffer solution.

[0014] Optionally, the pH of the buffer solution is 4.

[0015] Optionally, the phosphate buffer is composed of components with the following concentrations: 0.05-20% disodium hydrogen phosphate and 0.05-15% citric acid.

[0016] Optionally, the preservative includes at least one of Proclin 300 and NP-40.

[0017] Optionally, the concentration of the preservative is 0.05-10%.

[0018] Optionally, the stabilizer includes at least one of EDTA and dithiothreitol.

[0019] Optionally, the concentration of the stabilizer is 0.001-1%.

[0020] In the present application, the components in liquid B cooperate with each other to accurately and clearly mark cells, bacteria, Trichomonas, etc.

[0021] In a second aspect, the present application provides a method for preparing the above-mentioned kit, comprising the following steps: Liquid A and liquid B are combined to obtain the kit for fluorescent detection of vaginal microorganisms.

[0022] Compared with the prior art, this application has the following beneficial effects: The kit provided in the present application includes liquid A and liquid B, wherein the fluorescent dye (such as fluorescent brightener 28) in liquid A interacts with the counterdye (with Evans blue) and the like to accurately and clearly mark fungi; the components in liquid B cooperate with each other to mark cells, bacteria, Trichomonas, etc., and the combination of liquid A and liquid B can mark pathogens in vaginal secretions, and can more comprehensively and accurately judge the type of vaginal inflammation, thereby making the entire kit more accurate and sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a staining image of vaginal Candida mycelium after staining with Liquid A in Example 1 of the present application; Figure 2 This is a staining diagram of vaginal Candida spores and blastospores stained with Liquid A in Example 1 of the present application; Figure 3 This is a staining image after staining vaginal Candida using Liquid A in Example 3 of the present application; Figure 4 This is a clue cell staining image after staining a bacterial vaginitis sample with the B solution in Example 1 of the present application; Figure 5 This is a staining diagram of a Trichomonas vaginitis sample stained with Solution B in Example 1 of the present application. DETAILED DESCRIPTION

[0025] The present application is further described below in conjunction with specific embodiments. The following are only a few embodiments of the present application, and are not intended to limit the present application in any form. Although the present application discloses the following preferred embodiments, they are not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the above disclosed technical content to make some changes or modifications are equivalent to equivalent implementation cases and are within the scope of the technical solution.

[0026] Unless otherwise specified, the raw materials in the examples of the present application were purchased from commercial sources and used directly without any special treatment.

[0027] Unless otherwise specified, the analysis methods in the examples all adopt conventional settings and conventional analysis methods of instruments or equipment.

[0028] Example 1 The vaginal microorganism fluorescence detection reagent of this embodiment comprises liquid A: 0.1% fluorescent brightener 28, 5% dimethyl sulfoxide (DMSO), 10% potassium hydroxide, 3% Tween, 10% glycerol, 2% Proclin 300 and 0.2% Evans blue; liquid B: 0.05% acridine orange, 6% Proclin 300, 0.2% EDTA and buffer solution.

[0029] The vaginal microorganism fluorescence detection kit A liquid comprises the following steps: (1) Dissolve 50 g of potassium hydroxide powder in pure water and adjust the volume to 100 mL to obtain a 50% potassium hydroxide solution; (2) Dissolve 0.5 g of fluorescent brightener 28 in 50% potassium hydroxide solution and adjust the volume to 100 mL to obtain a mixed solution containing 0.5% fluorescent brightener 28 and 50% potassium hydroxide; (3) Dissolve 1 g of Evans blue powder in DMSO and adjust the volume to 25 mL to obtain a mixed solution containing 4% Evans blue and DMSO; (4) Measure 3 mL of Tween, 10 mL of glycerol, and 2 mL of Proclin 300 in a beaker, dissolve them in 50 mL of pure water, add 20 mL of a mixed solution of fluorescent brightener and potassium hydroxide, and 5 mL of a mixed solution of Evans blue and DMSO into the beaker, mix the solution evenly, and dilute to 100 mL to obtain Liquid A of the Vaginal Microbial Fluorescence Detection Kit; The vaginal microorganism fluorescence detection kit liquid B comprises the following steps: (1) Dissolve 1 g of EDTA powder in pure water and dilute to 50 mL to obtain a 2% EDTA solution; (2) Dissolve 0.5 g of acridine orange powder in 2% EDTA solution to obtain a mixed solution containing 0.5% acridine orange and 2% EDTA; (3) Dissolve 5.47 g of solid sodium hydrogen phosphate and 6.46 g of solid citric acid in pure water and adjust the volume to 500 mL to obtain a buffer solution of pH 4.0; (4) Take 10 mL of each component and 90 mL of buffer solution according to the concentration, mix them evenly, and obtain liquid B of the vaginal microorganism fluorescence detection kit.

[0030] Example 2 Compared with Example 1, the fluorescent whitening agent 28 in liquid A is replaced with fluorescein isothiocyanate, 3 g of fluorescein isothiocyanate is dissolved in pure water and the volume is fixed to 100 mL to obtain a 3% fluorescein isothiocyanate mother solution. The other steps and raw materials are performed in the same manner as in Example 1, and the components are mixed to obtain a vaginal microorganism fluorescence detection kit.

[0031] Example 3 Compared with Example 1, Tween is removed from the components of Solution A, and the other steps and raw materials are carried out in the same manner as in Example 1. The components are mixed to obtain a vaginal microorganism fluorescence detection kit.

[0032] Comparative Example 1 Compared with Example 1, the pH of solution B was adjusted to 4.4, and the other steps and raw materials were the same as those in Example 1; the pH 4.4 buffer solution was prepared as follows: Weigh 6.26 g of solid disodium hydrogen phosphate and 5.87 g of solid citric acid, dissolve in pure water, and dilute to 500 mL to obtain a buffer solution with a pH of 4.4.

[0033] Comparative Example 2 Compared with Example 1, the pH of solution B was adjusted to 4.8, and the other steps and raw materials were the same as those in Example 1; the pH 4.8 buffer solution was prepared as follows: Weigh 6.99 g of disodium hydrogen phosphate solid and 5.33 g of citric acid solid, dissolve in pure water, and dilute to 500 mL to obtain a buffer solution with a pH of 4.8.

[0034] Comparative Example 3 Compared with Example 1, the pH of solution B was adjusted to 5.0, and the other steps and raw materials were the same as those in Example 1; the pH 5.0 buffer solution was prepared as follows: Weigh 6.99 g of disodium hydrogen phosphate solid and 5.33 g of citric acid solid, dissolve in pure water, and dilute to 500 mL to obtain a buffer solution with a pH of 5.0.

[0035] Comparative Example 4 Compared with Example 1, EDTA is removed from the components of liquid B, and the other steps and raw materials are carried out in the same manner as in Example 1. The components are mixed to obtain a vaginal microorganism fluorescence detection kit.

[0036] Test Example 1 Normal samples and confirmed vaginal Candida vaginitis, bacterial vaginitis, and Trichomonas vaginitis samples were selected, and the vaginal secretion fluorescence detection kit prepared in Examples 1 to 3 and Comparative Examples 1 to 4 was used to stain the selected clinical samples of vaginal secretions. The staining process is as follows: Take a number of clean glass slides, apply the clinical samples of vaginal secretions to the two marked positions on the glass slides, respectively, use the vaginal secretion fluorescence detection kit prepared in Examples 1 to 3 and Comparative Examples 1 to 4 to add a drop of liquid A and a drop of liquid B respectively, cover with a cover glass, absorb the excess staining liquid with absorbent paper, use our company's digital biological microscope with U (365nm) band and B (470nm) band excitation light to automatically focus and automatically take pictures of the smears, and observe the color and morphology of cells and microorganisms through the pictures. The test results are shown in Table 1 and Figures 1 to 5 shown.

[0037] Table 1 Grouping Vaginal Candida vaginitis sample Bacterial vaginosis sample Trichomonas vaginitis sample Example 1 Spores and hyphae are well stained, and the morphology is clear and easy to identify. Clue cells are well stained and have clear morphology and are easy to identify The color and shape of Trichomonas are clear and easy to identify Example 2 Spores and hyphae are well stained, and the morphology is clear and easy to identify. Clue cells are well stained and have clear morphology and are easy to identify The color and shape of Trichomonas are clear and easy to identify Example 3 The staining of spores and hyphae is weak, the morphology is clear and easy to identify, and there are fewer colored spores and hyphae Clue cells are well stained and have clear morphology and are easy to identify The color and shape of Trichomonas are clear and easy to identify Comparative Example 1 Spores and hyphae are well stained, and the morphology is clear and easy to identify. Clue cells are weakly stained and have a vague morphology The shape of Trichomonas is clear but the color is not clear enough Comparative Example 2 Spores and hyphae are well stained, and the morphology is clear and easy to identify. Clue cells are weakly stained and have a vague morphology The shape of Trichomonas is clear but the color is not clear enough Comparative Example 3 Spores and hyphae are well stained, and the morphology is clear and easy to identify. Clue cells are weakly stained and have a vague morphology The shape of Trichomonas is clear but the color is fuzzy Comparative Example 4 Spores and hyphae are well stained, and the morphology is clear and easy to identify. Clue cells are weakly stained, but have clear morphology and are easy to identify. Trichomonas are clearly shaped but less pigmented It can be seen from the data in Table 1 that after the vaginal secretion samples are stained using the vaginal microorganism fluorescence detection kit of the present invention, the staining effect of fungi, clue cells and Trichomonas is obvious, and the morphology is clear and easy to identify. It can be seen from Example 3 and Comparative Example 4 that the use of Tween in liquid A and EDTA in liquid B can greatly improve the recognition of cells and microorganisms by the kit, resulting in better staining effect, clearer morphology, and higher positive detection rate.

[0038] Figure 1 , Figure 2 This is a staining result diagram of the hyphae and spores (blastospores) of vaginal Candida in Example 1 of the present invention. It can be seen that the hyphae and spores are blue fluorescent and have clear and recognizable shapes, indicating that the kit can well label the hyphae and spores (blastospores) of fungi; Figure 3 This is a staining result diagram of vaginal Candida in Example 3 of the present invention. It can be seen that this example can also identify fungi and the morphology can be well identified, but the clarity is not enough; Figure 4 This is a staining image of clue cells after bacterial vaginosis samples were stained in Example 1 of the present invention. The cell nucleus is yellow, and the cytoplasm is red due to the attachment of Gardnerella, indicating that this kit can well mark Gardnerella and can be well identified through clue cells; Figure 5 This is a staining result diagram of a Trichomonas vaginitis sample according to Example 1 of the present invention. From this diagram, we can clearly see the pear-shaped, red Trichomonas body with a yellow nucleus at 1 / 3, indicating that the kit can well mark Trichomonas.

[0039] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A kit for fluorescent detection of vaginal microorganisms, characterized in that: The kit comprises liquid A and liquid B; The A solution includes components with the following concentrations: Fluorescent dye 0.005~1%, antibacterial agent 0.01~30%, anti-fluorescence decay agent 0.02~20%, counterstain 0.002~10%, and the balance is purified water; The B solution includes components with the following concentrations: Fluorescein 0.001~0.8%, buffer solution 0.05~20%, preservative 0.05~20%, stabilizer 0.001~3%, and purified water as the balance.

2. The kit according to claim 1, characterized in that The fluorescent dye comprises at least one of a fluorescent whitening agent 28, fluorescein isothiocyanate, and a peridinin-chlorophyll-protein complex.

3. The kit according to claim 1 or 2, characterized in that The antibacterial agent includes at least one of dimethyl sulfoxide, Proclin 300 and potassium hydroxide.

4. The kit according to claim 3, characterized in that The anti-fluorescence decay agent includes at least one of glycerol, Tween and gallate.

5. The kit according to claim 4, characterized in that The counterstain includes at least one of methyl green and Evans blue.

6. The kit according to claim 5, characterized in that The fluorescein includes at least one of acridine orange and rhodamine.

7. The kit according to claim 6, characterized in that The buffer solution includes at least one of a phosphate buffer solution and a sodium acetate buffer solution; Preferably, the pH of the buffer solution is 4; Preferably, the phosphate buffer is composed of components with the following concentrations: 0.05-20% disodium hydrogen phosphate and 0.05-15% citric acid.

8. The kit according to claim 7, characterized in that The preservative includes at least one of Proclin 300 and NP-40; Preferably, the concentration of the preservative is 0.05-10%.

9. The kit according to claim 8, characterized in that The stabilizer includes at least one of EDTA and dithiothreitol; Preferably, the concentration of the stabilizer is 0.001-1%.

10. The method for preparing the kit according to any one of claims 1 to 9, characterized in that: The steps include: Liquid A and liquid B are combined to obtain the kit for fluorescent detection of vaginal microorganisms.

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