CN113533004A - Multiple fluorescent staining solution and preparation method and use method thereof - Google Patents
Multiple fluorescent staining solution and preparation method and use method thereof Download PDFInfo
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- CN113533004A CN113533004A CN202110872408.8A CN202110872408A CN113533004A CN 113533004 A CN113533004 A CN 113533004A CN 202110872408 A CN202110872408 A CN 202110872408A CN 113533004 A CN113533004 A CN 113533004A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a multiple fluorescence staining solution, a preparation method and a use method thereof, wherein the multiple fluorescence staining solution comprises the following components in each liter: 105.5-365mM of strong base, 0.01-50g of acid, 5-250g of binding protein, 0.01-0.2L of preservative, 0.1-50L of solubilizer, 0.001-20L of film breaking agent, 0.0194-180mg of fluorescent dye and the balance of sterilized distilled water; the invention combines the fungus detection object and the detection objects of other microorganisms except the fungus, only needs one-time smearing, one-time dyeing and one-time color observation to obtain a detection result, the effect is clearer after dyeing, the dyeing boundary of the target object is sharper, thus being very beneficial to the identification of small target objects, in particular to the fluorescent dyeing effect that the bacteria and the fungus in the microorganism have smaller forms, the boundary is clearer and the sharpening is beneficial to the identification of the target objects.
Description
Technical Field
The invention belongs to the technical field of multiple fluorescence detection, and particularly relates to gynecological vaginal microecology detection; in particular to a multiple fluorescence staining solution, a preparation method and a use method thereof.
Background
The existing gynecological vaginal microecological detection method adopts a two-step method to dye samples, namely, the fungus in each sample needs to be detected independently. Each sample needs to be coated with two slides, one slide is stained with fungi, and an ultraviolet fluorescence module is adopted for detection; the other slide is stained with microorganisms except fungi and other visible components, and a blue light fluorescence module is adopted for detection; therefore, the detection method has the disadvantages of long operation time, complicated steps, two fluorescence modules and high requirements on the configuration of the imaging equipment.
In addition, the current similar products have low dyeing definition on target objects, especially have unclear imaging outlines of small target objects (such as bacteria, fungi and the like), and can cause errors (omission and misidentification) in the identification (manual and automatic) process; the accuracy of the identification of the traditional non-staining mode (saline sheet detection) at present can only reach 75-80% at most due to the limitation of the technology.
Disclosure of Invention
Aiming at the problems of the existing gynecological vaginal microecological detection method, the invention provides a multiple fluorescence staining solution.
The invention adopts the following technical scheme: a multiple fluorescent staining solution comprising the following components per liter: 105.5-365mM of strong base, 0.01-50g of acid, 5-250g of binding protein, 0.01-0.2L of preservative, 0.1-50L of solubilizer, 0.001-20L of film breaking agent, 0.0194-180mg of fluorescent dye and the balance of sterilized distilled water.
Further defined, the strong base comprises NaCl100-2HPO40.5-2mM and KH2PO45-158 mM.
Further defined, the binding protein comprises the following components per liter: 2-100g of mouse anti-monoclonal antibody, 1-50g of sheep anti-monoclonal antibody and 1-50g of rabbit anti-monoclonal antibody.
Further defined, the acid is acetic acid.
Further defined, the preservative is a food or pharmaceutical preservative, preferably Proclin 300.
Further defined, the solubilizing agent is an industrial organic solvent, preferably dimethyl sulfoxide.
Further, the membrane breaking agent is a surfactant for regulating the fluidity of biological membrane molecules, and preferably one of glycerol, glycerol or tween 20.
Further defined, the fluorescent stain comprises the following components per liter: 0.01-10mg of Hochest, 0.001-10mg of 4, 4' -bis (2-benzoxazolyl) stilbene, 0.001-10mg of acridine orange, 0.001-50mg of carboxyfluorescein diacetate succinimide ester, 0.001-10mg of Sybr Green I, 0.001-10mg of Sybr Green II, 0.0003-40mg of cell membrane fluorescent probe, 0.001-10mg of fluorescein isothiocyanate, 0.001-10mg of phycoerythrin, 0.001-10mg of polyamphetamine-chlorophyll-protein complex, 0.001-10mg of propidium iodide and 0.001-10mg of allophycocyanin.
The invention has the beneficial effects that: the invention combines the fungus detection object and other microorganism detection objects except the fungus together, so that only one time of smearing and one time of dyeing is needed, and only one time of color observation is needed to obtain a detection result, the effect is clearer after dyeing, the dyeing boundary of the target object is sharper, thus the invention is very beneficial to the identification of small target objects, especially bacteria and fungi in the microorganism have fluorescent dyeing effects of smaller form, clear boundary and sharper color, is beneficial to the identification of the target objects, is more beneficial to the implementation of artificial intelligent identification, realizes the automation of gynecological vaginal microecological detection, has short operation time by adopting the multiple fluorescent dyeing liquid, has simple operation steps, improves the experimental efficiency, and reduces the configuration requirement on detection equipment;
the multiple fluorescent staining solution provided by the invention can carry out fluorescent staining or marking on all the established microorganisms and histiocytes, completely presents the micro-ecological condition of the detection environment, and is beneficial to the judgment of clinical detection results.
The invention also discloses a preparation method of the multiple fluorescent staining solution, which comprises the following steps:
mixing strong base, acetic acid and sterilized distilled water to prepare a salt mother solution, stirring for 20-35 minutes, filtering by a 0.22 mu m water system membrane, and storing at 2-5 ℃ in a dark place;
preparing binding protein mother liquor, filtering with a 0.22 mu m water system membrane, subpackaging at-20 to-17 ℃ and storing in a dark place;
preparing mother liquor of fluorescent staining agent, filtering with 0.22 μm lipid soluble membrane, and storing at 2-5 deg.C in dark place;
mixing the salt mother liquor, the conjugated protein mother liquor, the fluorescent staining agent mother liquor, the preservative, the solubilizer and the membrane breaking agent, diluting with sterilized distilled water as a diluent to obtain the multiple fluorescent staining solution, and storing at room temperature in a dark place.
The invention has the beneficial effects that: the preparation method is simple, the chelation can be realized only by stirring at normal temperature, and the chelating agent is only stored at normal temperature and has friendly storage condition.
The invention also discloses a using method of the multiple fluorescent staining solution, which comprises the following steps:
submerging the sampling end of the sampling swab by using normal saline and stirring for 1-2 minutes;
smearing a single-layer sample film on a glass slide at the sampling end of the sampling swab;
after the sample is solidified, adding multiple fluorescent staining solutions to completely cover the sample, and flushing the sample after 30-45 seconds;
and airing the washed sample, and placing the sample under a microscope with a blue light fluorescence module for observation.
The invention has the beneficial effects that: the use method is simple, the operability is strong, and factors influencing the detection result are reduced, so that the accuracy of the detection result is high, and the use amount of the physiological saline can be reduced by adopting the use method.
Drawings
FIG. 1 is a microscopic staining pattern of pathogen spores after staining a sample with the multiplex fluorescent staining solution prepared in example 2;
FIG. 2 is a staining pattern of microscopic pathogen spores after staining a sample with a conventional fluorescent staining solution;
FIG. 3 is a microscopic staining pattern of clue cells of pathogens after staining a sample with the multiplex fluorescent staining solution prepared in example 2;
FIG. 4 is a staining chart of a pathogen clue cell under a microscope after a sample is stained by using a conventional fluorescent staining solution;
FIG. 5 is a microscopic staining pattern of epithelial cells, leukocytes, and Lactobacillus after staining a sample with the multiplex fluorescent staining solution prepared in example 2;
FIG. 6 is a staining pattern of Gardner-infected epithelial cells under a microscope after staining a sample with the multiplex fluorescent staining solution prepared in example 2;
FIG. 7 is a staining pattern of funicular fungal spores under a microscope after staining a sample with the multiplex fluorescent staining solution prepared in example 2;
FIG. 8 is a drawing showing the microscopic hyphal staining pattern of a specimen stained with the multiple fluorescent staining solutions prepared in example 2;
FIG. 9 is a staining pattern of Trichomonas microscopically after staining a sample with the multiple fluorescent staining solution prepared in example 2;
FIG. 10 is a microscopic image of a specimen stained with the multiplex fluorescent staining solution prepared in example 2 in example 5;
FIGS. 11-14 are multiple local staining patterns under a microscope after staining a sample with the multiplex fluorescent staining solution prepared in example 2 in example 5;
FIGS. 15-18 are several partial staining patterns of microscopic pathogenic thread cells after staining a sample with the multiplex fluorescent staining solution prepared in example 2 in example 5;
FIG. 19 is a microscopic image of a specimen after staining with a conventional fluorescent staining solution;
FIGS. 20-23 are multiple partial stain maps of microscopic pathogen spores and other typing components after staining a sample with a prior art fluorescent stain;
FIGS. 24-26 are multiple partial staining patterns of microscopic pathogen cord cells after staining a sample with a conventional fluorescent staining solution.
Detailed Description
Example 1
The preparation of the multiple fluorescent staining solution comprises the following steps:
s1: weighing or measuring the following substances: NaCl100 mM, KCl 1mM, Na2HPO4 0.5mM、KH2PO45mM, mouse anti-vaginal Gardner monoclonal antibody A/B/C1 g, rabbit anti-Candida albicans monoclonal antibody A/B1 g, mouse anti-Neisseria gonorrhoeae monoclonal antibody A/B1 g, mouse anti-vaginal Trichomonas vaginalis monoclonal antibody A/B1 g, mouse anti-herpes simplex virus (type I/II) monoclonal antibody A/B1 g, Proclin 3000.01L, DMSO 0.1L, Tween 200.001L, Hochest 0.01mg, 4' -bis (2-benzoxazolyl) stilbene 0.001mg, acridine orange 0.001mg, carboxyfluorescein diacetate succinimidyl ester 0.001mg, Sybr Green I0.001 mg, Sybr Green II 0.001mg, cell membrane fluorescent probe (DiI)0.0001mg, cell membrane fluorescent probe (DiO)0.0001mg, fluorescent probe (R) 0.0001mg, fluorescein isothiocyanate 0.001mg, phycoerythrin 0.001mg, 0.001mg of polydatin-chlorophyll-protein complex, 0.001mg of propidium iodide, 0.001mg of allophycocyanin and 0.01g of acetic acid.
S2: preparing a salt mother solution: mixing NaCl, KCl and Na2HPO4、KH2PO4Acetic acid, taking sterilized distilled water as a solvent, mixing, stirring and stirring for 30 minutes, filtering by a 0.22 mu m water system membrane to obtain 100X salt mother liquor, and storing at 4 ℃ in a dark place;
s3: preparing a binding protein mother liquor: mixing and stirring a mouse anti-vaginal Gardner monoclonal antibody A/B/C, a rabbit anti-Candida albicans monoclonal antibody A/B, a mouse anti-Neisseria gonorrhoeae monoclonal antibody A/B, a mouse anti-trichomonas vaginalis monoclonal antibody A/B and a mouse anti-herpes simplex virus (I/II type) monoclonal antibody A/B for 30 minutes by using sterilized distilled water as a solvent, filtering by using a 0.22 mu m water system membrane to obtain 100X binding protein mother liquor, and storing in a dark place at the temperature of-20 ℃;
s4: preparing a fluorescent dye mother solution: hochest, fluorescent whitening agent, acridine orange, CFSE, Sybr Green I, Sybr Green II, cell membrane fluorescent probe (DiI, DiO, DiR), Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), polymethacrylxanthin-chlorophyll-protein complex (PerCP), Propidium Iodide (PI), Allophycocyanin (APC), stirring for 30 minutes after mixing with DMSO as solvent, filtering with 0.22 μm lipid soluble system membrane to obtain 1000X fluorescent dye mother liquor, and storing at 4 ℃ in a dark place;
s5: mixing 100X salt mother liquor, 100X binding protein mother liquor, 1000X fluorescence staining agent mother liquor, Proclin300, DMSO and Tween 20 with sterilized distilled water as diluent, filtering with 0.22 μm lipid soluble membrane to obtain multiple fluorescence staining solution, and storing at room temperature in dark place.
Example 2
The preparation of the multiple fluorescent staining solution comprises the following steps:
s1: weighing or measuring the following substances: NaCl 135mM, KCl 2.7mM, Na2HPO4 1.5mM、KH2PO48mM, mouse anti-vaginal Gardner monoclonal antibody A/B/C5 g, rabbit anti-Candida albicans monoclonal antibody A/B10 g, mouse anti-Neisseria gonorrhoeae monoclonal antibody A/B7.5 g, mouse anti-vaginal Trichomonas vaginalis monoclonal antibody A/B12 g, mouse anti-herpes simplex virus (type I/II) monoclonal antibody A/B6.7 g, Proclin 3000.05L, DMSO 2.5L, Tween 200.01L, Hochest 0.05mg, 4' -bis (2-benzoxazolyl) stilbene 10mg, acridine orange 10mg, carboxyfluorescein diacetate succinimidyl ester 50mg, Sybr Green I10 mg, Sybr Green II 10mg, cell membrane fluorescent probe (DiI)10mg, cell membrane fluorescent probe (DiO)10mg, cell membrane fluorescent probe (DiR)10mg, fluorescein isothiocyanate 10mg, phycoerythrin 10mg, 10mg of polydatin-chlorophyll-protein complex, 10mg of propidium iodide, 10mg of allophycocyanin and 50g of acetic acid;
s2: preparing a salt mother solution: mixing NaCl, KCl and Na2HPO4、KH2PO4Acetic acid, taking sterilized distilled water as a solvent, mixing, stirring and stirring for 35 minutes, filtering by a 0.22 mu m water system membrane to obtain 100X salt mother liquor, and storing at 4 ℃ in a dark place;
s3: preparing a binding protein mother liquor: mixing and stirring a mouse anti-vaginal Gardner monoclonal antibody A/B/C, a rabbit anti-Candida albicans monoclonal antibody A/B, a mouse anti-Neisseria gonorrhoeae monoclonal antibody A/B, a mouse anti-trichomonas vaginalis monoclonal antibody A/B and a mouse anti-herpes simplex virus (I/II type) monoclonal antibody A/B for 35 minutes by using sterilized distilled water as a solvent, filtering by using a 0.22 mu m water system membrane to obtain 100X binding protein mother liquor, and storing in a dark place at the temperature of-20 ℃;
s4: preparing a fluorescent dye mother solution: hochest, fluorescent whitening agent, acridine orange, CFSE, Sybr Green I, Sybr Green II, cell membrane fluorescent probe (DiI, DiO, DiR), Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), polymethacrylxanthin-chlorophyll-protein complex (PerCP), Propidium Iodide (PI), Allophycocyanin (APC), stirring for 35 minutes after mixing with DMSO as solvent, filtering with 0.22 lipolysis system membrane to obtain 1000X fluorescent dye mother liquor, and storing at 4 ℃ in a dark place;
s5: mixing 100X salt mother liquor, 100X binding protein mother liquor, 1000X fluorescence staining agent mother liquor, Proclin300, DMSO and Tween 20 with sterilized distilled water as diluent, filtering with 0.22 μm lipid soluble membrane to obtain multiple fluorescence staining solution, and storing at room temperature in dark place.
Example 3
The preparation of the multiple fluorescent staining solution comprises the following steps:
s1: weighing or measuring the following substances: NaCl 200mM, KCl 5mM, Na2HPO4 2mM、KH2PO4158mM, mouse anti-vaginal Gardner monoclonal antibody A/B/C50 g, rabbit anti-Candida albicans monoclonal antibody A/B50 g, mouse anti-Neisseria gonorrhoeae monoclonal antibody A/B50 g, mouse anti-Trichomonas vaginalis monoclonal antibody A/B50 g, mouse anti-herpes simplex virus (type I/II) monoclonal antibody A/B50 g, Proclin 3000.2L, DMSO 50L, Tween 2020L, Hochest 10mg, 4' -bis (2-benzoxazolyl) stilbene 0.02mg, acridine orange 0.02mg, carboxyfluorescein diacetate succinimidyl ester 0.05mg, Sybr Green I0.01 mg, Sybr Green II 0.01mg, fluorescent probe (DiI)0.001mg, fluorescent probe (DiO)0.001mg, cell membrane fluorescent probe (DiR)0.001mg, fluorescein isothiocyanate 0.01mg, phycoerythrin 0.01mg, 0.01mg of polydatin-chlorophyll-protein complex, 0.01mg of propidium iodide, 0.01mg of allophycocyanin and 0.5g of acetic acid;
s2: preparing a salt mother solution: mixing NaCl, KCl and Na2HPO4、KH2PO4Acetic acid, taking sterilized distilled water as a solvent, mixing, stirring and stirring for 20 minutes, filtering by a 0.22 mu m water system membrane to obtain 100X salt mother liquor, and storing at 4 ℃ in a dark place;
s3: preparing a binding protein mother liquor: mixing and stirring a mouse anti-vaginal Gardner monoclonal antibody A/B/C, a rabbit anti-Candida albicans monoclonal antibody A/B, a mouse anti-Neisseria gonorrhoeae monoclonal antibody A/B, a mouse anti-trichomonas vaginalis monoclonal antibody A/B and a mouse anti-herpes simplex virus (I/II type) monoclonal antibody A/B by taking sterilized distilled water as a solvent for 20 minutes, filtering a 0.22 mu m water system membrane to obtain 100X binding protein mother liquor, and storing the binding protein mother liquor at the temperature of minus 20 ℃ in a dark place;
s4: preparing a fluorescent dye mother solution: hochest, fluorescent whitening agent, acridine orange, CFSE, Sybr Green I, Sybr Green II, cell membrane fluorescent probe (DiI, DiO, DiR), Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), polymethacrylxanthin-chlorophyll-protein complex (PerCP), Propidium Iodide (PI), Allophycocyanin (APC), stirring for 20 minutes after mixing with DMSO as solvent, filtering with 0.22 μm lipid soluble system membrane to obtain 1000X fluorescent dye mother liquor, and storing at 4 ℃ in a dark place;
s5: mixing 100X salt mother liquor, 100X binding protein mother liquor, 1000X fluorescence staining agent mother liquor, Proclin300, DMSO and Tween 20 with sterilized distilled water as diluent, filtering with 0.22 μm lipid soluble membrane to obtain multiple fluorescence staining solution, and storing at room temperature in dark place.
Example 4
The patient who is examined before in this embodiment is used for the next operation.
This example discloses a method for using the multiple fluorescent staining solution prepared in example 2, comprising the following steps:
h1: adding appropriate amount of physiological saline into a sampling swab (such as a vaginal sampling swab), stirring and mixing uniformly when the sampling swab just submerges a swab sampling end, lightly placing the swab sampling end on a glass slide, and smearing a single-layer sample membrane;
h2: drying at room temperature or baking at 60 deg.C, fixing the sample (methanol for 2min or baking for 3-4 times), adding one drop (about 50 μ L) of the solution prepared in example 2, covering the whole sample layer with bamboo stick, dyeing at room temperature for 30s, and washing with distilled water;
h3: airing the slide at room temperature, placing the slide under a microscope, and observing under a blue light fluorescence module; the staining results are shown in fig. 1, fig. 3, fig. 5, fig. 6, fig. 7, fig. 8 and fig. 9.
Comparative example 1
The results of the tests on samples from the same patient in example 4 were shown in FIGS. 2 and 4, using the reagents of the two-step test available on the market.
Since the disease caused by the pathogen spore and the pathogen clue cell accounts for more than 90% of gynecological vaginal inflammation, the detection effects of the two diseases are mainly compared, wherein the pathogen spore is fungus, and the pathogen clue cell is a squamous epithelial cell infected by bacteria, and the bacteria are mainly gardnerella.
As can be seen from the comparison between FIG. 1 and FIG. 2, the pathogen spore in FIG. 1 is stained red, as indicated by the arrow in FIG. 1, and has clear boundary and high brightness, and is easy to identify and distinguish; in FIG. 2, the pathogen spores and other typical components are stained green (indicated by arrows in FIG. 2), with blurred borders and low brightness, and are not easily identified and distinguished;
as can be seen by comparing FIG. 3 with FIG. 4, the pathogenic thread cells in FIG. 3 are stained red, and the brightness is obviously different from that of the background purple mixed bacteria, so that the pathogenic thread cells are easy to identify and distinguish; in FIG. 4, the clue cells of the pathogen are stained yellow-green, and the background infectious microbes are also stained completely, so that the pathogen is not easy to identify and distinguish.
In FIG. 5, arrow No. 1 indicates epithelial cells, arrow No. 2 indicates leukocytes, and arrow No. 3 indicates Lactobacillus;
FIG. 6 shows the arrows indicating Gardner-infected epithelial cells; FIG. 7 is a schematic representation of a thread cell fungal spore with arrows;
the arrows in FIG. 8 indicate hyphae, and the arrows in FIG. 9 indicate trichomonas.
Example 5
The results of examining the samples collected from the healthy persons and patients using the multiplex fluorescent staining solution prepared in example 1 by the method used in example 4 are shown in FIGS. 10 to 18, and it is clear from FIG. 10 (sample of healthy person) that the epithelial cells and leukocytes are stained; normal lactobacillus is dyed into obvious red, which is beneficial to the clinical judgment of a microecological system; the background is clean and the miscellaneous signal is low.
FIGS. 11-14 (fungal infection patient samples) show the staining of pathogen spores, which are easily identified and identified in FIG. 11-14 (the arrows indicate red spores) with high brightness; FIGS. 15-18 (patients with bacterial infection) show the staining of the pathogenic clue cells, and it can be seen from FIGS. 15-18 that the pathogenic clue cells are stained red, and the brightness is clearly distinguished from the background mixed bacteria, so that the cells are easy to identify and distinguish.
Comparative example 2
The results of the detection of the samples from the same patients corresponding to example 5 by using the two-step detection reagents on the market are shown in fig. 19-26, and it can be seen from fig. 19 that epithelial cells, leukocytes and lactobacilli are all dyed green, and no color distinction is made, which is not favorable for clinical observation and distinction judgment; the background mixed bacteria are also completely stained, and the interference signal is strong.
FIGS. 20-23 show the staining of pathogen spores and other typing components, and it can be seen from FIGS. 20-23 that the pathogen spores and other typing components are stained green and have low brightness and are not easily identified and distinguished (the arrows in the figures refer to green spores and are not easily identified).
FIGS. 24-26 show the staining of the clue cells of pathogens, and it can be seen from FIGS. 24-26 that the clue cells of pathogens are stained yellow-green, and the background bacteria are also stained completely, and are not easily identified and distinguished.
Therefore, the multiple fluorescent staining solution provided by the invention not only can finish the detection of bacteria and fungi at one time, but also can increase the brightness of the fungi and the bacteria after being stained, has clear boundaries and is beneficial to clinical judgment.
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 the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A multiple fluorescent staining solution comprising, per liter: 105.5-365mM of strong base, 0.01-50g of acid, 5-250g of binding protein, 0.01-0.2L of preservative, 0.1-50L of solubilizer, 0.001-20L of film breaking agent, 0.0194-180mg of fluorescent dye and the balance of sterilized distilled water.
2. The multiple fluorescent staining solution of claim 1, wherein the strong base comprises NaCl100-200mM, KCl 1-5mM, Na2HPO40.5-2mM and KH2PO45-158 mM.
3. The multiple fluorescence staining solution of claim 1, wherein the binding protein comprises the following components per liter: 2-100g of mouse anti-monoclonal antibody, 1-50g of sheep anti-monoclonal antibody and 1-50g of rabbit anti-monoclonal antibody.
4. The multiple fluorescence staining solution of claim 1, wherein the acid is acetic acid.
5. The multiple fluorescent staining solution of claim 1, wherein the preservative is a food or pharmaceutical preservative, preferably Proclin 300.
6. The multiple fluorescent staining solution of claim 1, wherein the solubilizing agent is an industrial organic solvent, preferably dimethyl sulfoxide.
7. The multiple fluorescence staining solution of claim 1, wherein the membrane-breaking agent is a surfactant for adjusting the fluidity of the biological membrane molecules, preferably one of glycerol, glycerol or tween 20.
8. The multiple fluorescent staining solution of claim 1, wherein the fluorescent staining agent comprises the following components per liter: 0.01-10mg of Hochest, 0.001-10mg of 4, 4' -bis (2-benzoxazolyl) stilbene, 0.001-10mg of acridine orange, 0.001-50mg of carboxyfluorescein diacetate succinimide ester, 0.001-10mg of Sybr Green I, 0.001-10mg of Sybr Green II, 0.0003-40mg of cell membrane fluorescent probe, 0.001-10mg of fluorescein isothiocyanate, 0.001-10mg of phycoerythrin, 0.001-10mg of polyamphetamine-chlorophyll-protein complex, 0.001-10mg of propidium iodide and 0.001-10mg of allophycocyanin.
9. A method for preparing the multiple fluorescent staining solution of claim 1, comprising the steps of:
mixing strong base, acetic acid and sterilized distilled water to prepare a salt mother solution, stirring for 20-35 minutes, filtering by a 0.22 mu m water system membrane, and storing at 2-5 ℃ in a dark place;
preparing binding protein mother liquor, filtering with a 0.22 mu m water system membrane, subpackaging at-20 to-17 ℃ and storing in a dark place;
preparing mother liquor of fluorescent staining agent, filtering with 0.22 μm lipid soluble membrane, and storing at 2-5 deg.C in dark place;
mixing the salt mother liquor, the conjugated protein mother liquor, the fluorescent staining agent mother liquor, the preservative, the solubilizer and the membrane breaking agent, diluting with sterilized distilled water as a diluent to obtain the multiple fluorescent staining solution, and storing at room temperature in a dark place.
10. A method of using the multiple fluorescent staining solution of claim 1, comprising the steps of:
submerging the sampling end of the sampling swab by using normal saline and stirring for 1-2 minutes;
smearing a single-layer sample film on a glass slide at the sampling end of the sampling swab;
after the sample is solidified, adding multiple fluorescent staining solutions to completely cover the sample, and flushing the sample after 30-45 seconds;
and airing the washed sample, and placing the sample under a microscope with a blue light fluorescence module for observation.
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