CN108931413A - A kind of quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis - Google Patents
A kind of quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis Download PDFInfo
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- CN108931413A CN108931413A CN201810810362.5A CN201810810362A CN108931413A CN 108931413 A CN108931413 A CN 108931413A CN 201810810362 A CN201810810362 A CN 201810810362A CN 108931413 A CN108931413 A CN 108931413A
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- mycobacterium tuberculosis
- antiacid
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 39
- 238000004043 dyeing Methods 0.000 title claims abstract description 33
- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 23
- 230000001458 anti-acid effect Effects 0.000 title claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 41
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 19
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 17
- 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 abstract description 17
- 235000019441 ethanol Nutrition 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229910001868 water Inorganic materials 0.000 claims abstract description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 10
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 8
- DWCZIOOZPIDHAB-UHFFFAOYSA-L methyl green Chemical compound [Cl-].[Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)[N+](C)(C)C)=C1C=CC(=[N+](C)C)C=C1 DWCZIOOZPIDHAB-UHFFFAOYSA-L 0.000 claims abstract description 5
- 239000011780 sodium chloride Substances 0.000 claims abstract description 5
- 239000001103 potassium chloride Substances 0.000 claims abstract description 4
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 4
- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 claims abstract 3
- 229960003699 evans blue Drugs 0.000 claims abstract 3
- 239000002253 acid Substances 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 150000003839 salts Chemical class 0.000 claims description 8
- 230000003196 chaotropic effect Effects 0.000 claims description 7
- 239000000975 dye Substances 0.000 claims description 7
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 6
- 239000004909 Moisturizer Substances 0.000 claims description 5
- 230000001333 moisturizer Effects 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 3
- 239000000987 azo dye Substances 0.000 claims description 2
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 claims description 2
- 239000007850 fluorescent dye Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 150000007522 mineralic acids Chemical class 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims 1
- HPTULVAFZQXKIS-UHFFFAOYSA-N NC1=C(C=CC=C1)C=CC1=CC=CC=C1.N1=NN=CC=C1 Chemical compound NC1=C(C=CC=C1)C=CC1=CC=CC=C1.N1=NN=CC=C1 HPTULVAFZQXKIS-UHFFFAOYSA-N 0.000 claims 1
- 239000006081 fluorescent whitening agent Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 6
- 238000005457 optimization Methods 0.000 abstract description 3
- 238000009472 formulation Methods 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 239000002131 composite material Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 11
- 241000193830 Bacillus <bacterium> Species 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 206010036790 Productive cough Diseases 0.000 description 5
- 235000011187 glycerol Nutrition 0.000 description 5
- 210000003802 sputum Anatomy 0.000 description 5
- 208000024794 sputum Diseases 0.000 description 5
- 238000011017 operating method Methods 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- 201000008827 tuberculosis Diseases 0.000 description 4
- 210000002421 cell wall Anatomy 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 230000003020 moisturizing effect Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 210000002969 egg yolk Anatomy 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- HZLHRDBTVSZCBS-UVJJDBRNSA-N 4-[(e)-(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline;hydrochloride Chemical compound Cl.C1=CC(=N)C(C)=C\C1=C(C=1C=C(C)C(N)=CC=1)/C1=CC=C(N)C=C1 HZLHRDBTVSZCBS-UVJJDBRNSA-N 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 206010056377 Bone tuberculosis Diseases 0.000 description 1
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 1
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- DYDCUQKUCUHJBH-UWTATZPHSA-N D-Cycloserine Chemical compound N[C@@H]1CONC1=O DYDCUQKUCUHJBH-UWTATZPHSA-N 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000167880 Hirundinidae Species 0.000 description 1
- 244000283207 Indigofera tinctoria Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 241000186366 Mycobacterium bovis Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 208000009360 Osteoarticular Tuberculosis Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000000981 basic dye Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
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- 238000012631 diagnostic technique Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
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- 230000036541 health Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229960003350 isoniazid Drugs 0.000 description 1
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000005554 pickling Methods 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 206010038534 renal tuberculosis Diseases 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
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Classifications
-
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
Abstract
The invention discloses a kind of quick antiacid fluorescent dyeing reagents of modified form mycobacterium tuberculosis, include two kinds of reagents, and A liquid includes pure water, sodium chloride or potassium chloride, dimethyl sulfoxide, glycerol, auramine O.B liquid includes pure water, hydrochloric acid, ethyl alcohol, methyl green or Evans blue.Product of the present invention carries out improvement optimization to auramine O fluorescence colour by optimization of C/C composites, develops a kind of mycobacterium tuberculosis of double-formulation quickly antiacid fluorescent dyeing reagent.Only need the operation of two steps that dyeing can be completed, and dyeing time was reduced to 2 minutes or so by traditional 20 minutes, clinical use is more convenient, quick, greatly improves the detection efficiency of mycobacterium tuberculosis.
Description
Technical field
The invention belongs to biomedical diagnostic techniques fields, and in particular to a kind of modified form mycobacterium tuberculosis is quickly antiacid
Fluorescent dyeing reagent.
Background technique
Pulmonary tuberculosis is a kind of common respiratory system communicable disease, spread speed quickly, if failing to receive in time effective
Diagnosis and treatment, it is easy to cause mycobacterium tuberculosis infection other organs or system, such as bone tuberculosis, enteron aisle tuberculosis, kidney tuberculosis,
Not only bring the larger pain of patient, moreover it is possible to improve treatment difficulty, accelerate disease progression, the extended treatment time increases organ function
Failure risk.With the development that deepens continuously of China's medical level, gradually reinforcement to pulmonary tuberculosis prevention and control dynamics, in addition the common people couple
It is gradually increased in what health care required, under comprehensive function, many tuberculars are timely and effectively treated.But according to doctor
Investigation display is learned, lunger's disease incidence is still high, has become whole world public health problem outstanding.
Mycobacterium tuberculosis be elongated slightly curved bacillus, 1~0.4 μm of size.Mycobacterium bovis then compares tubbiness.Point
The bacteria cell wall lipid content of Ramibacterium is higher, accounts for about the 60% of dry weight, especially has a large amount of mycolic acids to be enclosed in peptide poly-
The outside of sugar layer can influence penetrating for dyestuff.Mycobacterium tuberculosis is in vivo and in vitro through penicillin, seromycin or bacteriolyze enzyme induction
The synthesis of peptide glycan in cell wall can be influenced, isoniazid influences the synthesis of mycolic acid, and macrophage swallows mycobacterium tuberculosis
The effect of lysozyme can destroy peptide glycan afterwards, and can lead to it becomes L-type, be in granular form or Filamentous.
It includes that neat-Nissl Ziehi-Neelsen stain, cultivation and auramine O are glimmering that clinic, which is commonly used in the method detected in conjunction with bacillus,
Light decoration method etc..
1. neat-Nissl Ziehi-Neelsen stain: Ziehi-Neelsen stain is a kind of common, easy and rapidly combines bacillus detection side
Method is of great significance to diagnosis lungy.This method is to make mycolic acid secured with carbolfuchsin in a heated condition
It is combined into compound, handles also nondiscoloration with hydrochloride alcohol.After adding alkaline methylene blue to redye again, mycobacteria remains as red,
And the substance in other bacteriums and background is blue.The method is limited by mycobacterium quantity in sputum, and sensibility is low, omission factor
It is high.And life or death bacterium cannot be distinguished in this method, and Mycobacterium tuberculosis and Nontuberculosis mycobacteria especially cannot be distinguished.
2. isolated culture method: obligate aerobic.Optimum temperature is 37 DEG C, is not grown lower than 30 DEG C.Mycobacterium tuberculosis is thin
The lipid content of cell wall is higher, influences the absorption of nutriment, therefore slow growth.In 1 generation of every division, takes in general culture medium
18~24 hours.There are commonly Roche solid mediums, include yolk, glycerol, potato, inorganic salts etc..Yolk is raw containing lipid
The long factor, can stimulating growth.According to how much bacterium is inoculated with, general 2~4 weeks visible colonies are grown.Bacterium colony is in particle, tubercle or cauliflower
Shape, milky or ecru are opaque.Although cultivation high sensitivity but detection cycle is too long, treatment is not only affected adversely, also significantly
Increase pipe be broadcast to others risk.
3. auramine O fluorescence colour: after dyeing and redye by auramine O, with the fluorescence microscopy microscopy containing ultraviolet source
It looks into, acid-fast bacilli is in glassy yellow, and the substance in other bacteriums and background is in dark yellow, and this method can use low power microscopy, because
This can more rapidly find out mycobacteria.There are three types of reagent, three-step approach operations for this method.First step auramine O dyes 15 minutes, rinses
Complete second step pickling 2 minutes has been rinsed third step and has been redyed 2 minutes.Entire dyeing course probably needs 20 minutes.
Summary of the invention
The present invention develops a kind of double-formulation in view of the shortcomings of the prior art, carry out improvement optimization to auramine O fluorescence colour
The quick antiacid fluorescent dyeing reagent of mycobacterium tuberculosis.Product of the present invention stability is good, two kinds of reagents, only needs two steps to operate and is
Achievable dyeing, and dyeing time was reduced to 2 minutes or so by traditional 20 minutes, clinical use is more convenient, quick, significantly
Improve the detection efficiency of mycobacterium tuberculosis.
Specific technical solution of the present invention is as follows:
The quick antiacid fluorescent dyeing reagent of a kind of modified form mycobacterium tuberculosis, it is characterised in that including two kinds of reagent A liquid and B
Liquid.A liquid includes water, salt, chaotropic agent, moisturizer, fluorescein.B liquid includes water, acid, alcohol, counterstain.
Salt described in A liquid of the present invention is inorganic salts, preferably potassium chloride or sodium chloride.It is preferred that the concentration of alkali is 0.5%-1.0%(W/
V), more preferable 0.9%(W/V).
Chaotropic agent described in A liquid of the present invention is selected from water-miscible organic solvent, preferably dimethyl sulfoxide.The effect of chaotropic agent is dye
The solubility and stability of material.It is preferred that the concentration of chaotropic agent is 15-25%(V/V).
Moisturizer described in A liquid of the present invention is selected from glycerol, propylene glycol, one or more of butanediol, preferably glycerine.Moisturizing
The effect of agent is to increase solution entirety moisturizing degree and diopter, is intensified, so that dyeing is relatively sharp.It is preferred that moisturizing
The concentration of agent is 10-30%(V/V).
Fluorescein described in A liquid of the present invention is selected from acid resistant form fluorescent dye, preferably auramine O, preferred concentration 0.1-0.3%(W/
V).
The acid described in B liquid of the present invention is selected from inorganic acid, preferably hydrochloric acid.Hydrochloric acid plays the decolorization after dyeing, tuberculosis point
Branch bacillus is not decolourized due to antiacid characteristic.It is preferred that the concentration of hydrochloric acid is 0.3-0.5%(V/V).
The alcohol described in B liquid of the present invention is selected from monohydric alcohol, preferred alcohol.Preferred concentration is 70-75%(V/V).
Counterstain described in B liquid of the present invention is selected from green basic dye or blue azo dyes.It is preferred that methyl green or ivens
Indigo plant, more preferable methyl green, preferred concentration 0.5-1%(W/V).
The present invention makes the detection method for needing the dyeing of three steps originally by improveing to traditional auramine O fluorescence colour
It can be realized quick two steps dyeing, and shorten dyeing time, only need go out result within two minutes.Clinical use is more convenient, fast
Victory greatly improves the detection efficiency of tubercle bacillus.
Detailed description of the invention
Fig. 1 is reagent of the present invention to the coloration result of sputum specimen, and Fig. 2 is coloration result of traditional auramine O to sputum specimen.
Fig. 3 is reagent of the present invention to the coloration result of lung tissue paraffin section sample, and Fig. 4 is traditional auramine O to lung tissue
The coloration result of paraffin section sample.
Specific embodiment
Illustrate specific steps of the invention by the following examples, but is not limited by the example.
Term as used in the present invention generally there are those of ordinary skill in the art usually to manage unless otherwise indicated
The meaning of solution.
The present invention is described in further detail combined with specific embodiments below and referring to data.It should be understood that these embodiments are
In order to demonstrate the invention, it rather than limits the scope of the invention in any way.
In the examples below, the various processes and method being not described in detail are conventional methods as known in the art.
1 reagent 1 of embodiment: the preparation of the invention patent reagent
A liquid: auramine O powder is dissolved in solution of the concentration for 0.9%(W/V) sodium chloride, sufficiently dissolves, makes the concentration of auramine O
0.1%(W/V), dimethyl sulfoxide and glycerol are added, the concentration 15%(V/V of dimethyl sulfoxide is made), glycerol concentration 20%
(V/V).
B liquid: first configuring acid alcohol solution, so that concentration of hydrochloric acid is 0.3%(V/V), concentration of alcohol 75%(V/V).Again to acid
Methyl green is added in alcoholic solution, so that concentration is 0.5%(W/V).
Reagent 2: the configuration of traditional auramine O:
A liquid: auramine O powder 0.1g is dissolved in 95% ethyl alcohol of 10ml, and 5% carbolic acid is to 100ml after adding dilution；
B liquid: configuration acid alcohol solution, so that concentration of hydrochloric acid is 3%(V/V), concentration of alcohol 75%(V/V).
C liquid: potassium permanganate powder is dissolved in pure water, makes concentration 0.5%(W/V).
The dyeing of 2 sputum specimen of embodiment
The sputum specimen for choosing the tubercle bacillus positive, is coated on 2 glass slides, uses reagent 1 respectively, and reagent 2 is dyed,.Reagent 1
Operating procedure: after A liquid dyes one minute, after being rinsed with water, B liquid is dyed one minute, mounting microscopically observation after rinsing.Examination
2 operating procedure of agent: A reagent dyeing 15 minutes, after being rinsed with water, B liquid was dyed 2 minutes, after C liquid redyes 1 minute, was added after mounting
Coverslip sops up extra dyestuff with paper, then in fluorescence microscopy microscopic observation.Under blue wave band with 410 nm -440nm
Observation, tubercle bacillus are in green fluorescence.Two kinds of reagents can make tubercle bacillus mark fluorescent as the result is shown, and reagent of the present invention contaminates
The color time is shorter.
The dyeing of 3 lung tissue paraffin section of embodiment
The section preparation for choosing three parts of paraffin embedding is dyed with reagent 1,2 respectively after dewaxing, 1 operating procedure of reagent: A liquid
After dyeing one minute, after being rinsed with water, B liquid is dyed one minute, mounting microscopically observation after rinsing.2 operating procedure of reagent:
A reagent dyeing 15 minutes, after being rinsed with water, B liquid was dyed 2 minutes, and after C liquid redyes 1 minute, coverslip is added after mounting, will be more
Remaining dyestuff is sopped up with paper, then in fluorescence microscopy microscopic observation.It is observed under the blue wave band of 410 nm -440nm, tuberculosis bar
Bacterium is in green fluorescence.Two kinds of reagents can make tubercle bacillus mark fluorescent as the result is shown, and the reagent dyeing time of the present invention is shorter.
Claims (9)
1. a kind of quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis, it is characterised in that including two kinds of reagent A liquid and B
Liquid；A liquid includes water, salt, chaotropic agent, moisturizer, fluorescein；B liquid includes water, acid, alcohol, counterstain；Water described in A liquid is purifying
Water, the salt are inorganic salts, and the chaotropic agent is water-miscible organic solvent, and the moisturizer is water-miscible organic solvent, described
Fluorescein is acid resistant form fluorescent dye；Acid described in B liquid is inorganic acid, and the alcohol monohydric alcohol, the counterstain is green alkalinity dye
Material or blue azo dyes.
2. the quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis as described in claim 1, it is characterised in that A liquid institute
Stating salt is potassium chloride or sodium chloride；Fluorescein is double triazine amino-stilbene type fluorescent whitening agents.
3. the quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis as described in claim 1, it is characterised in that A liquid institute
Stating chaotropic agent is dimethyl sulfoxide.
4. the quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis as described in claim 1, it is characterised in that A liquid institute
Stating moisturizer is glycerol, propylene glycol, one or more of butanediol.
5. the quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis as described in claim 1, it is characterised in that A liquid institute
Stating fluorescein is auramine O.
6. the quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis as described in claim 1, it is characterised in that B liquid institute
Stating acid is hydrochloric acid.
7. the quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis as described in claim 1, it is characterised in that B liquid institute
Stating alcohol is ethyl alcohol.
8. the quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis as described in claim 1, it is characterised in that B liquid institute
It states counterstain and is selected from one or more of methyl green and Evans blue.
9. the quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis as described in claim 1, it is characterised in that include
Two kinds of reagents, A liquid include pure water, sodium chloride or potassium chloride, dimethyl sulfoxide, glycerol, auramine O；B liquid includes pure water, hydrochloric acid, second
Alcohol, methyl green or Evans blue.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810810362.5A CN108931413A (en) | 2018-07-23 | 2018-07-23 | A kind of quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810810362.5A CN108931413A (en) | 2018-07-23 | 2018-07-23 | A kind of quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108931413A true CN108931413A (en) | 2018-12-04 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN117025717A (en) * | 2023-10-08 | 2023-11-10 | 江苏美克医学技术有限公司 | Acid-fast tubercle bacillus fluorescent staining kit and detection method and application thereof |
| CN117589543A (en) * | 2023-10-26 | 2024-02-23 | 珠海贝索生物技术有限公司 | Composition for acid-fast bacteria staining, staining method and kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388520A (en) * | 2014-10-27 | 2015-03-04 | 济南百博生物技术股份有限公司 | Fluorescent acid-fast staining solution and method for detecting acid-fast bacilli by using same |
| CN104568551A (en) * | 2013-10-18 | 2015-04-29 | 苏州微利特生物科技有限公司 | Acid resistant staining method |
| CN106198470A (en) * | 2016-06-30 | 2016-12-07 | 江苏莱芙时代生物科技有限公司 | A kind of fungal detection fluorescence staining liquid and application |
| CN106323925A (en) * | 2016-08-11 | 2017-01-11 | 付微 | Fluorescence dye for detecting fungus and dermatozoon |
| CN107246988A (en) * | 2017-06-06 | 2017-10-13 | 珠海贝索生物技术有限公司 | A kind of acid-resisting bacterium colouring method |
-
2018
- 2018-07-23 CN CN201810810362.5A patent/CN108931413A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104568551A (en) * | 2013-10-18 | 2015-04-29 | 苏州微利特生物科技有限公司 | Acid resistant staining method |
| CN104388520A (en) * | 2014-10-27 | 2015-03-04 | 济南百博生物技术股份有限公司 | Fluorescent acid-fast staining solution and method for detecting acid-fast bacilli by using same |
| CN106198470A (en) * | 2016-06-30 | 2016-12-07 | 江苏莱芙时代生物科技有限公司 | A kind of fungal detection fluorescence staining liquid and application |
| CN106323925A (en) * | 2016-08-11 | 2017-01-11 | 付微 | Fluorescence dye for detecting fungus and dermatozoon |
| CN107246988A (en) * | 2017-06-06 | 2017-10-13 | 珠海贝索生物技术有限公司 | A kind of acid-resisting bacterium colouring method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN117025717A (en) * | 2023-10-08 | 2023-11-10 | 江苏美克医学技术有限公司 | Acid-fast tubercle bacillus fluorescent staining kit and detection method and application thereof |
| CN117025717B (en) * | 2023-10-08 | 2024-03-22 | 江苏美克医学技术有限公司 | Acid-fast tubercle bacillus fluorescent staining kit and detection method and application thereof |
| CN117589543A (en) * | 2023-10-26 | 2024-02-23 | 珠海贝索生物技术有限公司 | Composition for acid-fast bacteria staining, staining method and kit |
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