CN117025717B - Acid-fast tubercle bacillus fluorescent staining kit and detection method and application thereof - Google Patents
Acid-fast tubercle bacillus fluorescent staining kit and detection method and application thereof Download PDFInfo
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- CN117025717B CN117025717B CN202311286296.3A CN202311286296A CN117025717B CN 117025717 B CN117025717 B CN 117025717B CN 202311286296 A CN202311286296 A CN 202311286296A CN 117025717 B CN117025717 B CN 117025717B
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 38
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 12
- 238000001514 detection method Methods 0.000 title abstract description 37
- 239000007788 liquid Substances 0.000 claims abstract description 49
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 40
- 230000017423 tissue regeneration Effects 0.000 claims abstract description 32
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 25
- 241000779819 Syncarpia glomulifera Species 0.000 claims abstract description 21
- 239000001739 pinus spp. Substances 0.000 claims abstract description 21
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229940043267 rhodamine b Drugs 0.000 claims abstract description 21
- 229940036248 turpentine Drugs 0.000 claims abstract description 21
- 238000010186 staining Methods 0.000 claims abstract description 18
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims abstract description 17
- 239000010931 gold Substances 0.000 claims abstract description 12
- 229910052737 gold Inorganic materials 0.000 claims abstract description 12
- -1 gold amine Chemical class 0.000 claims abstract description 12
- 238000002791 soaking Methods 0.000 claims description 73
- 239000011521 glass Substances 0.000 claims description 17
- 238000011010 flushing procedure Methods 0.000 claims description 16
- 238000007789 sealing Methods 0.000 claims description 16
- 238000004043 dyeing Methods 0.000 claims description 11
- 230000007935 neutral effect Effects 0.000 claims description 11
- KSCQDDRPFHTIRL-UHFFFAOYSA-N auramine O Chemical compound [H+].[Cl-].C1=CC(N(C)C)=CC=C1C(=N)C1=CC=C(N(C)C)C=C1 KSCQDDRPFHTIRL-UHFFFAOYSA-N 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 abstract description 75
- 239000012188 paraffin wax Substances 0.000 abstract description 27
- 230000000694 effects Effects 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 2
- 239000012192 staining solution Substances 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 100
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 40
- 230000000052 comparative effect Effects 0.000 description 16
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 12
- 239000011347 resin Substances 0.000 description 9
- 229920005989 resin Polymers 0.000 description 9
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 6
- 239000008096 xylene Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000000975 dye Substances 0.000 description 4
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 2
- 206010036790 Productive cough Diseases 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229960000907 methylthioninium chloride Drugs 0.000 description 2
- 210000003802 sputum Anatomy 0.000 description 2
- 208000024794 sputum Diseases 0.000 description 2
- 238000007447 staining method Methods 0.000 description 2
- 201000008827 tuberculosis Diseases 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 1
- 206010048612 Hydrothorax Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
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- 238000003771 laboratory diagnosis Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- 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
-
- 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"
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides an acid-fast tubercle bacillus fluorescent staining kit, a detection method and application thereof, belonging to the technical field of medicine and biochemistry; the dewaxing liquid contains turpentine and tissue repair liquid which are independent of each other, and the tissue repair liquid consists of triton 100 and ethylenediamine tetraacetic acid; the staining solution contains rhodamine B solution, gold amine O solution and hydrochloric acid alcohol solution which are independent from each other. The kit solves the problem of poor staining effect of the acid-fast tubercle bacillus in paraffin tissue slice samples, and has higher detection rate of the acid-fast tubercle bacillus.
Description
Technical Field
The invention relates to the technical fields of medicine and biochemistry, in particular to an acid-fast tubercle bacillus fluorescent staining kit, a detection method and application thereof.
Background
Mycobacterium tuberculosis detection positive is a gold standard for clinical diagnosis of tuberculosis. In laboratory diagnosis of tuberculosis, the most commonly used specimen is a sputum specimen, but since tubercle bacillus can infect organs other than the lung, it is necessary to examine an appropriate specimen at a corresponding focus part in clinical diagnosis and treatment. Typical sample types are: sputum, focal tissue, hydrothorax and ascites, urine, cerebrospinal fluid, blood, serum and the like. The current laboratory examination methods of tubercle bacillus include the following four methods: microscopy, isolation and identification, molecular biological detection and immunological detection.
Current dewaxing techniques involve immersing tissue slices in a sequence of xylene solution, absolute ethanol solution, 95% ethanol solution, 85% ethanol solution, distilled water. The conventional dewaxing technology for acid-fast tubercle bacillus staining detection has many defects, such as high xylene toxicity and cancerogenic risk in long-term contact; the dyeing step is complex to operate; acid-fast tubercle bacillus can not be observed after dewaxing, and the detection rate is low.
In the current acid-fast tubercle bacillus staining detection, the staining effect of paraffin tissue sections is poor, and the problem of high omission ratio needs to be solved.
Disclosure of Invention
The invention aims to solve the problems and provide an acid-fast tubercle bacillus fluorescent staining kit which solves the problem of poor acid-fast tubercle bacillus staining effect of paraffin tissue slice samples and improves the detection rate of the acid-fast tubercle bacillus.
The technical scheme of the invention is as follows: the invention provides an acid-fast tubercle bacillus fluorescent staining kit, which comprises dewaxing liquid, staining liquid and sealing liquid which are independent from each other; the dewaxing liquid contains turpentine and tissue repair liquid which are independent of each other, and the tissue repair liquid consists of triton 100 and ethylenediamine tetraacetic acid; the staining solution contains rhodamine B solution, gold amine O solution and hydrochloric acid alcohol solution which are independent from each other.
The invention relates to an action principle of an acid-fast tubercle bacillus fluorescent staining kit: the turpentine can dissolve and remove paraffin on paraffin tissues, then the tissue structure is repaired by a repairing liquid, after dewaxing is finished, the high affinity of the gold amine O solution and rhodamine B for resisting acid bacteria is utilized, acid-fast bacteria in a specimen are dyed by the gold amine O and the rhodamine B under the condition of room temperature, and then the color is removed by hydrochloric acid alcohol solution; in the process, the acid-fast bacteria in the sample can resist the decoloring treatment of acid ethanol, and under the excitation of blue light irradiation of a fluorescence microscope, the acid-fast bacteria present bright golden fluorescence, and other bacteria and substances in the background present dark color, so that the high-efficiency and accurate detection is realized.
Preferably, in the tissue repair liquid, the final volume percentage concentration of the triton 100 is 0.1-5%;
the final concentration of the ethylenediamine tetraacetic acid is 1mmol/L-0.1mol/L.
Preferably, in the tissue repair liquid, the final volume percentage concentration of the triton 100 is 1-3%;
the final concentration of the ethylenediamine tetraacetic acid is 10-100mmol/L.
Preferably, the volume percentage concentration of the rhodamine B solution is 0.05-2%;
the volume percentage concentration of the gold amine O solution is 0.01-0.05%;
the volume percentage concentration of hydrochloric acid in the hydrochloric acid-alcohol solution is 1-5%, and the volume percentage concentration of alcohol in the hydrochloric acid-alcohol solution is 70-80%.
Through the preferable technical scheme, the rhodamine B solution and the gold amine O solution are dyes, the dye precipitation can be observed when the dye concentration is too high, the dyed acid-fast tubercle bacillus is influenced, the dyed fluorescence intensity is influenced when the dye concentration is too low, and the dyed acid-fast tubercle bacillus is inconvenient to observe; too low a concentration of hydrochloric acid alcohol may affect the elution effect, and too high a concentration may cause excessive elution, and thus the due accurate detection cannot be achieved.
Preferably, the encapsulation liquid is a neutral gum.
The invention also provides a method for detecting the acid-fast tubercle bacillus, which comprises the following steps: soaking the tissue slice in turpentine, and then soaking in tissue repair liquid; placing the soaked tissue slice on a glass slide, and then dripping rhodamine B solution on the tissue slice for soaking and flushing; dripping a solution of the gold amine O for soaking and washing; then dropwise adding a hydrochloric acid alcohol solution for soaking and washing; after the glass slide is dried, dripping a sealing liquid into the sample for sealing; the tissue repair liquid consists of triton 100 and ethylenediamine tetraacetic acid.
Preferably, the tissue slice is soaked in turpentine for 5-20min;
soaking in tissue repair liquid for 8-12min.
Preferably, the rhodamine B solution is soaked for 1-2min;
the time for soaking the auramine O solution is 12-18min;
the soaking time of the hydrochloric acid alcohol solution is 2-3min.
The invention also provides application of the acid-fast tubercle bacillus fluorescent staining kit in preparation of an acid-fast tubercle bacillus detection reagent.
The invention also provides application of the acid-fast tubercle bacillus fluorescent staining kit obtained by the preparation method in preparation of the acid-fast tubercle bacillus detection reagent.
Compared with the prior art, the invention has the beneficial effects that: according to the scheme, turpentine and repair liquid are used in dewaxing technology, after paraffin on paraffin tissues is removed, the tissue structure is repaired, the removal effect of the paraffin can be improved by using the turpentine, and then the tissue structure is repaired by using the tissue repair liquid with a specific formula, so that the subsequent dyeing effect can be enhanced.
Drawings
FIG. 1 is a graph showing the positive detection result of example 1 of the present invention;
FIG. 2 is a graph showing the positive detection result of example 2 of the present invention;
FIG. 3 is a graph showing the positive detection result of example 3 of the present invention;
FIG. 4 is a graph showing the positive detection result of example 4 of the present invention;
FIG. 5 is a graph showing the positive detection results of example 5 of the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, wherein it is to be understood that the invention is illustrated in the appended drawings. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
Example 1
120 acid-fast tubercle bacillus positive paraffin section tissue samples are collected, and dewaxing and staining are carried out according to the following steps:
s1, taking a tissue sample of a positive paraffin section of the mycobacterium tuberculosis, soaking the tissue sample in turpentine twice for 10min, and then soaking the tissue sample in a tissue repair liquid for 10min, wherein the tissue repair liquid contains 1% triton 100 and 10mmol/L (ethylenediamine tetraacetic acid) EDTA (pH9.0).
S2, after dewaxing the tissue sample, placing the tissue sample on a glass slide, then dripping 2 drops of 0.1% rhodamine B solution on the tissue slice, covering and soaking for 1min, and flushing with running water; then 2 drops of 0.05% auramine O solution are dripped on the tissue slice for covering and soaking for 15min, and then the tissue slice is washed by running water; then 2 drops of 3% hydrochloric acid alcohol solution are dripped on the tissue slice to cover and soak for 2min, and then the tissue slice is washed by running water and dried.
S3, dropwise adding a drop of neutral resin sealing piece into the sample after the slide is dried, and then observing.
The positive detection results of example 1 are shown in FIG. 1.
Example 2
120 acid-fast tubercle bacillus positive paraffin section tissue samples are collected, and dewaxing and staining are carried out according to the following steps:
s1, taking a tissue sample of a positive paraffin section of the mycobacterium tuberculosis, soaking the tissue sample in turpentine twice for 10min, and then soaking the tissue sample in a tissue repair liquid for 10min, wherein the tissue repair liquid contains 1% triton 100 and 10mmol/L (ethylenediamine tetraacetic acid) EDTA (pH9.0).
S2, after dewaxing the tissue sample, placing the tissue sample on a glass slide, then dripping 1 drop of 0.1% rhodamine B solution on the tissue slice, covering and soaking for 1min, and flushing with running water; then 1 drop of 0.05% auramine O solution is dripped on the tissue slice for covering and soaking for 15min, and then the tissue slice is washed by running water; then 1 drop of 3% hydrochloric acid alcohol solution is dripped on the tissue slice to cover and soak for 2min, and then the tissue slice is washed with running water and dried.
S3, dropwise adding a drop of neutral resin sealing piece into the sample after the slide is dried, and then observing.
The positive detection results of example 2 are shown in FIG. 2.
Example 3
120 acid-fast tubercle bacillus positive paraffin section tissue samples are collected, and dewaxing and staining are carried out according to the following steps:
s1, taking a tissue sample of a positive paraffin section of the mycobacterium tuberculosis, soaking the tissue sample in turpentine twice for 10min, and then soaking the tissue sample in a tissue repair liquid for 10min, wherein the tissue repair liquid contains 1% triton 100 and 10mmol/L (ethylenediamine tetraacetic acid) EDTA (pH9.0).
S2, after dewaxing the tissue sample, placing the tissue sample on a glass slide, then dripping 1 drop of 0.1% rhodamine B solution on the tissue slice, covering and soaking for 1min, and flushing with running water; then 2 drops of 0.05% auramine O solution are dripped on the tissue slice for covering and soaking for 15min, and then the tissue slice is washed by running water; then 2 drops of 3% hydrochloric acid alcohol solution are dripped on the tissue slice to cover and soak for 2min, and then the tissue slice is washed by running water and dried.
S3, dropwise adding a drop of neutral resin sealing piece into the sample after the glass slide is dried, and then observing
The positive detection results of example 3 are shown in FIG. 3.
Example 4
120 acid-fast tubercle bacillus positive paraffin section tissue samples are collected, and dewaxing and staining are carried out according to the following steps:
s1, taking a tissue sample of a positive paraffin section of the mycobacterium tuberculosis, soaking the tissue sample in turpentine twice for 10min, and then soaking the tissue sample in a tissue repair liquid for 10min, wherein the tissue repair liquid contains 1% triton 100 and 1mmol/L (ethylenediamine tetraacetic acid) EDTA (pH9.0).
S2, after dewaxing the tissue sample, placing the tissue sample on a glass slide, then dripping 1 drop of 0.1% rhodamine B solution on the tissue slice, covering and soaking for 1min, and flushing with running water; then 2 drops of 0.05% auramine O solution are dripped on the tissue slice for covering and soaking for 15min, and then the tissue slice is washed by running water; then 1 drop of 3% hydrochloric acid alcohol solution is dripped on the tissue slice to cover and soak for 2min, and then the tissue slice is washed with running water and dried.
S3, dropwise adding a drop of neutral resin sealing piece into the sample after the slide is dried, and then observing.
The positive detection results of example 4 are shown in FIG. 4.
Example 5
120 acid-fast tubercle bacillus positive paraffin section tissue samples are collected, and dewaxing and staining are carried out according to the following steps:
s1, taking a tissue sample of a positive paraffin section of the mycobacterium tuberculosis, soaking the tissue sample in turpentine twice for 10min, and soaking the tissue sample in a tissue repair liquid for 10min, wherein the tissue repair liquid contains 1% triton 100 and 0.1mmol/L (ethylenediamine tetraacetic acid) EDTA (pH9.0).
S2, after dewaxing the tissue sample, placing the tissue sample on a glass slide, then dripping 1 drop of 0.1% rhodamine B solution on the tissue slice, covering and soaking for 1min, and flushing with running water; then 2 drops of 0.05% auramine O solution are dripped on the tissue slice for covering and soaking for 15min, and then the tissue slice is washed by running water; then 2 drops of 3% hydrochloric acid alcohol solution are dripped on the tissue slice to cover and soak for 2min, and then the tissue slice is washed by running water and dried.
S3, dropwise adding a drop of neutral resin sealing piece into the sample after the slide is dried, and then observing.
The positive detection results of example 5 are shown in FIG. 5.
Comparative example 1
The comparative example was dewaxed and dyed by conventional means, operating as follows:
collecting 120 cases of acid-fast bacillus tuberculosis positive paraffin section tissue samples, and sequentially dewaxing and staining according to the following steps:
s1, dewaxing treatment is carried out by adopting the following reagents in sequence, and flushing with running water is carried out after each soaking: (1) soaking in xylene solution for 10min; (2) soaking in xylene solution for 10min; (3) soaking in absolute ethanol solution for 5min; (4) soaking in absolute ethanol solution for 5min; (5) soaking in 95% ethanol solution for 5min; (6) soaking in 95% ethanol solution for 5min; (7) soaking in 85% ethanol solution for 5min; (8) soaking in 85% ethanol solution for 5min; (9) soaking in distilled water for 5min; soaking in distilled water for 5min, and dewaxing.
S2, dyeing treatment is carried out by adopting the following reagents in sequence: (1) dripping an alkaline reddish solution to cover the sample, dyeing at room temperature for 15min, and flushing with running water; (2) dripping acidic alcohol solution to cover the glass slide, decolorizing for 2min, and washing with running water; (3) and (3) dropwise adding methylene blue solution for dyeing for 1min, washing with running water, and drying for detection.
Comparative example 2
Collecting 120 cases of acid-fast bacillus tuberculosis positive paraffin section tissue samples, and sequentially dewaxing and staining according to the following steps:
s1, dewaxing treatment is carried out by adopting the following reagents in sequence, and flushing with running water is carried out after each soaking: (1) soaking in xylene solution for 10min; (2) soaking in xylene solution for 10min; (3) soaking in absolute ethanol solution for 5min; (4) soaking in absolute ethanol solution for 5min; (5) soaking in 95% ethanol solution for 5min; (6) soaking in 95% ethanol solution for 5min; (7) soaking in 85% ethanol solution for 5min; (8) soaking in 85% ethanol solution for 5min; (9) soaking in distilled water for 5min; soaking in distilled water for 5min, and dewaxing.
S2, dyeing treatment is carried out by adopting the following reagents in sequence: (1) dripping 2 drops of 0.1% rhodamine B solution on the tissue slice on the glass slide, covering and soaking for 1min, and flushing with running water; (2) 2 drops of 0.05% of gold amine O solution are dripped to cover and soak for 15min, and then the solution is washed by running water; (3) dripping 2 drops of 3% hydrochloric acid alcohol solution on the slice, covering and soaking for 2min, and flushing with running water; (4) and (5) dropwise adding a drop of neutral resin sealing piece into the sample for observation after the slide is dried.
Comparative example 3
Collecting 120 cases of acid-fast bacillus tuberculosis positive paraffin section tissue samples, and sequentially dewaxing and staining according to the following steps:
s1, dewaxing treatment is carried out by adopting the following reagents in sequence: (1) soaking turpentine for 10min; (2) soaking turpentine for 10min; (3) the tissue repair liquid is soaked for 10min, and the tissue repair liquid contains 1% triton 100 and 10mmol/L (ethylenediamine tetraacetic acid) EDTA (pH9.0).
S2, dyeing treatment is carried out by adopting the following reagents in sequence: (1) dripping an alkaline reddish solution to cover the sample, dyeing at room temperature for 15min, and flushing with running water; (2) dripping acidic alcohol solution to cover the glass slide, decolorizing for 2min, and washing with running water; (3) and (3) dropwise adding methylene blue solution for dyeing for 1min, washing with running water, and drying for detection.
Comparative example 4
120 acid-fast tubercle bacillus positive paraffin section tissue samples are collected, and dewaxing and staining are carried out according to the following steps:
s1, dewaxing: taking a tissue sample of the positive paraffin section of the mycobacterium tuberculosis, mixing turpentine and gasoline in a ratio of 1:1, and then placing the tissue sample into the tissue sample for soaking for 10min for 3 times.
S2, dyeing: after dewaxing a tissue sample, dropwise adding 2 drops of 0.1% rhodamine B solution into a tissue slice on a glass slide, covering and soaking for 1min, and then flushing with running water; then 2 drops of 0.05% of gold amine O solution are added dropwise for covering and soaking for 15min, and then the solution is washed by running water; then 2 drops of 3% hydrochloric acid alcohol solution are dripped on the slices to cover and soak for 2min, and then the slices are washed with running water and dried.
S3, observing: after the slide is dried, a drop of neutral resin sealing piece is dripped into the sample, and then observation is carried out.
Comparative example 5
120 acid-fast tubercle bacillus positive paraffin section tissue samples are collected, and dewaxing and staining are carried out according to the following steps:
s1, taking a tissue sample of a positive paraffin section of the mycobacterium tuberculosis, soaking the tissue sample in turpentine twice for 10min, and then soaking the tissue sample in a tissue repair liquid for 10min, wherein the tissue repair liquid contains 1% triton 100 and 10mmol/L (ethylenediamine tetraacetic acid) EDTA (pH9.0).
S2, after dewaxing the tissue sample, placing the tissue sample on a glass slide, then dripping 2 drops of 0.1% rhodamine B solution on the tissue slice, covering and soaking for 1min, and flushing with running water; then 2 drops of 0.05% auramine O solution are dripped on the tissue slice for covering and soaking for 15min, and then the tissue slice is washed by running water; then 2 drops of 3% hydrochloric acid alcohol solution are dripped on the tissue slice to cover and soak for 2min, and then the tissue slice is washed by running water and dried.
S3, dropwise adding a drop of neutral resin sealing piece into the sample after the glass slide is dried, and then observing
Comparative example 6
120 acid-fast tubercle bacillus positive paraffin section tissue samples are collected, and dewaxing and staining are carried out according to the following steps:
s1, taking a tissue sample of a positive paraffin section of the mycobacterium tuberculosis, soaking the tissue sample in turpentine twice for 10min, and then soaking the tissue sample in a tissue repair liquid for 10min, wherein the tissue repair liquid contains 1% triton 100 and 1mol/L (ethylenediamine tetraacetic acid) EDTA (pH9.0).
S2, after dewaxing the tissue sample, placing the tissue sample on a glass slide, then dripping 2 drops of 0.1% rhodamine B solution on the tissue slice, covering and soaking for 1min, and flushing with running water; then 2 drops of 0.05% auramine O solution are dripped on the tissue slice for covering and soaking for 15min, and then the tissue slice is washed by running water; then 2 drops of 3% hydrochloric acid alcohol solution are dripped on the tissue slice to cover and soak for 2min, and then the tissue slice is washed by running water and dried.
S3, dropwise adding a drop of neutral resin sealing piece into the sample after the slide is dried, and then observing.
Test examples
The results of the tests of examples 1-3 and comparative examples 1-6 are shown in Table 1.
TABLE 1 detection results of acid fast tubercle bacillus positive samples
Conclusion: as can be seen from Table 1, comparative example 1 was tested by the conventional dewaxing staining method, and the detection rate of the positive sample was only 55%; when comparative example 2 only adopts the conventional dewaxing method, the detection rate of the positive sample is only 70%; when comparative example 3 only adopts the traditional staining method, the detection rate of the positive sample is only 74%; when the dewaxing liquid used in comparative example 4 is a mixed solution of turpentine and kerosene, the detection rate of positive samples is only 60%; when the comparative example 5 uses high concentration of triton 100, the detection rate of the positive sample is only 84%; when EDTA was used in a high concentration in comparative example 6, the detection rate of the positive samples was only 86%. As can be seen from the detection rate of the positive samples of the comparative examples 1-3 and the comparative examples 1-6, the kit provided by the invention can obtain more accurate detection results of the acid-fast tubercle bacillus.
The above specific embodiments are provided for illustrative purposes only and are not intended to limit the invention, and modifications, no inventive contribution, will be made to the embodiments by those skilled in the art after having read the present specification, as long as they are within the scope of the patent statutes.
Claims (6)
1. The kit is characterized by comprising dewaxing liquid, staining liquid and sealing liquid which are independent from each other;
the dewaxing liquid contains turpentine and tissue repair liquid which are independent of each other, and the tissue repair liquid consists of triton 100 and ethylenediamine tetraacetic acid;
the dyeing liquid contains rhodamine B solution, gold amine O solution and hydrochloric acid alcohol solution which are independent from each other;
in the tissue repair liquid, the final volume percentage concentration of the triton 100 is 1%;
the final concentration of the ethylenediamine tetraacetic acid is 10mmol/L;
the volume percentage concentration of the rhodamine B solution is 0.1%;
the volume percentage concentration of the gold amine O solution is 0.05%;
the volume percentage concentration of hydrochloric acid in the hydrochloric acid-alcohol solution is 3%.
2. The kit for acid fast tubercle bacillus fluorescent staining according to claim 1 wherein the sealing liquid is a neutral gum.
3. The method for using the acid-fast tubercle bacillus fluorescent staining kit as claimed in claim 1, comprising the following steps:
soaking the acid-fast tubercle bacillus positive tissue slice in turpentine, and then soaking in tissue repair liquid;
placing the soaked tissue slice on a glass slide, and then dripping rhodamine B solution on the tissue slice for soaking and flushing;
dripping a solution of the gold amine O for soaking and washing;
then dropwise adding a hydrochloric acid alcohol solution for soaking and washing;
after the glass slide is dried, dripping a sealing liquid into the sample for sealing;
the tissue repair liquid consists of triton 100 and ethylenediamine tetraacetic acid.
4. The method of claim 3, wherein the tissue slice is soaked in turpentine twice, for 10min each time;
soaking in tissue repair liquid for 8-12min.
5. A method according to claim 3, wherein the rhodamine B solution is immersed for a period of 1 to 2 minutes;
the time for soaking the auramine O solution is 12-18min;
the soaking time of the hydrochloric acid alcohol solution is 2-3min.
6. Use of the acid-fast tubercle bacillus fluorescent staining kit of any of claims 1-2 in the preparation of a reagent for detecting acid-fast tubercle bacillus.
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| CN1298293A (en) * | 1998-04-24 | 2001-06-06 | 宝洁公司 | Cleansing articles for skin and/or hair which also deposits skin care actives |
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| CN109799124A (en) * | 2019-02-15 | 2019-05-24 | 上海皓信生物科技有限公司 | A kind of mycobacterium tuberculosis fluorescence acid-fast stain liquid |
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|---|---|---|---|---|
| US20160032180A1 (en) * | 2012-11-26 | 2016-02-04 | Agienic, Inc. | Antimicrobial Resin Coated Proppants |
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| CN1298293A (en) * | 1998-04-24 | 2001-06-06 | 宝洁公司 | Cleansing articles for skin and/or hair which also deposits skin care actives |
| CN104297038A (en) * | 2014-10-10 | 2015-01-21 | 丁昊 | Mycobacterium tuberculosis fluorescent acid-fast staining dye liquid |
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