CN104596827A - Kit and staining method for doubly staining mycobacterium tuberculosis and antigen of mycobacterium tuberculosis - Google Patents

Kit and staining method for doubly staining mycobacterium tuberculosis and antigen of mycobacterium tuberculosis Download PDF

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CN104596827A
CN104596827A CN201510025852.0A CN201510025852A CN104596827A CN 104596827 A CN104596827 A CN 104596827A CN 201510025852 A CN201510025852 A CN 201510025852A CN 104596827 A CN104596827 A CN 104596827A
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stained
dyeing
staining
mycobacterium tuberculosis
kit
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CN104596827B (en
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车南颖
张海青
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Beijing Chest Hospital
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Beijing Chest Hospital
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Abstract

The invention discloses a kit and a staining method for staining mycobacterium tuberculosis and the expression protein of the mycobacterium tuberculosis. The method for staining the mycobacterium tuberculosis and the expression protein of the mycobacterium tuberculosis comprises the following steps: (1) carrying out primary antibody incubation and secondary antibody incubation to a to-be-tested sample containing mycobacterium tuberculosis to obtain an antibody binding product; and (2) carrying out DAB (diaminobenzidine) staining solution staining, carbolfuchsin staining solution staining and hematoxylin staining solution staining to the antibody binding product sequentially, so as to obtain stained mycobacterium tuberculosis and the stained expression protein. Experimental results show that a novel kit for the tuberculotic pathology diagnosis is developed, and the kit can detect the mycobacterium tuberculosis and the expression protein in one tissue specimen section simultaneously, so as to improve the tuberculotic pathological diagnosis sensibility. The kit can be used conveniently and simply, is suitable for the conventional detection for the pathology department and has good clinical promotion value.

Description

A kind of kit for Much's bacillus and antigen double staining thereof and method thereof
Technical field
The present invention relates to biological technical field, particularly relate to a kind of kit for Much's bacillus and antigen double staining thereof and method thereof.
Background technology
Tuberculosis is the communicable disease of serious threat human health, the whole world has the population of 1/3 to infect Much's bacillus (Mycobacterium tuberculosis, MTB), have 2,000,000 people to die from tuberculosis every year, its mortality ratio only ranked second lower than acquired immune deficiency syndrome (AIDS) in communicable disease.The goldstandard of current diagnosis of tuberculosis is phlegm bacteriological detection.But Sputum smears positive rate is low, Sputum culturing needs the time of one month just can go out result, and therefore the most frequently used phlegm bacteriological detection means cannot meet the demand of clinical definite.In addition, be also applied in diagnosis of tuberculosis based on immunologic ELISPOT and based on the new technology such as PCR of detection of nucleic acids in recent years.But ELISPOT technology cannot differentiate latent infection person and real tuberculosis patient, therefore very limited at the Chinese diagnostic value that MTB infection rate is very high.And multiple round pcr is due to high to the requirement of equipment, technician, and the reasons such as testing cost height cannot spread to medical institutions at different levels.Pathological diagnosis is the important channel that tuberculosis is made a definite diagnosis, especially for the tuberculosis patient of phlegm bacteriology feminine gender and the diagnosis of the outer tubercular of lung.Tuberculosis Histopathological Characteristics is granulomatous inflammation, visible epithelioid cell, Langhans giant cell and caseous necrosis etc. in pathology.Because other are infectious or noninfectious disease also may occur similar pathological change, make a definite diagnosis the existence that tuberculosis also needs to detect MTB in tissue specimen clinically.Current most popular MTB detection method is luxuriant Nissl (Ziehl-Neelsen) decoration method, but it is low to there is susceptibility, and finding MTB the shortcoming such as to waste time and energy.
Summary of the invention
An object of the present invention is to provide the tissue section strain method detected for Much's bacillus.
The tissue section strain method detected for Much's bacillus provided by the invention, comprises the steps:
1) by histotomy to be detected and anti-MTB antigen-antibody solution reaction, an anti-binding section is obtained;
2) by a described anti-binding section and two anti-solution reactions, two anti-binding sections are obtained;
3) with DAB dyeing liquor, described two anti-binding sections are dyeed, obtain DAB stained;
4) with carbolic acid azaleine dyeing liquor, described DAB stained is dyeed, obtain carbolic acid azaleine stained;
5) with haematoxylin dyeing liquid, described carbolic acid azaleine stained is dyeed, obtain stained, realize tissue section strain.
In said method, described anti-MTB antigen-antibody is anti-MTB antigen polyclonal antibody;
The amino acid sequence of described MTB antigen is sequence 1 in sequence table;
Two of described two anti-solution resist for goat anti-rabbit igg antibody; The goat anti-rabbit igg antibody of the concrete HRP mark of described goat anti-rabbit igg antibody.Two anti-solution adopt goat-anti rabbit-HRP two to resist, and step neoplastic product.
In said method, step 1) in, the temperature of described reaction is 23-28 DEG C, and the time of described reaction is 0.5-2h;
Step 2) in, the temperature of described reaction is 23-28 DEG C, and the time of described reaction is 10-20min.
Step 3) in, described dyeing time is 1-10min, dyeing temperature is 23-28 DEG C;
Step 4) in, described dyeing time is 20-90min, dyeing temperature is 23-28 DEG C;
Step 5) in, described dyeing time is 0.5-2min, dyeing temperature is 23-28 DEG C.
In said method, in step 1)-2) between, also comprise the steps: the described anti-binding section of washing;
In step 2)-3) between, also comprise the steps: the described two anti-binding sections of washing;
In step 3)-4) between, also comprise the steps: to wash described DAB stained;
In step 4)-5) between, also comprise the steps: first with differentiation liquid decolour described carbolic acid azaleine stained obtain decolouring after carbolic acid azaleine stained, then wash carbolic acid azaleine stained after described decolouring; The concrete concentration of cleansing solution that described washing adopts is 0.01M, pH value is 7.4 PBS or water;
In step 5) in, after described dyeing obtains stained, also comprise the steps: first to break up liquid to decolour described stained, obtain the section of decolouring poststaining, then wash the section of described decolouring poststaining; The concrete concentration of cleansing solution that described washing adopts is 0.01M, pH value is 7.4 PBS or water.
In said method, described bleaching time is 30 seconds-60 seconds.
In said method, described histotomy to be detected is paraffin-embedded histotomy to be detected.
In said method, the step 1 in described method) front, also comprise the steps:
A, described paraffin-embedded histotomy to be detected is carried out dewaxing aquation, cut into slices after obtaining dewaxing aquation;
B, by cut into slices after described dewaxing aquation concentration be 0.01M, pH value be 6.0 the reparation of citrate buffer solution mesohigh, obtain repairing rear section;
The pressure of described high pressure is 80-100 kPa;
Described repair time is 90s, and the temperature of described reparation is 100 DEG C;
Another object of the present invention is to provide a kind of kit of the tissue section strain for Much's bacillus detection.
Kit provided by the invention, comprises DAB dyeing liquor, carbolic acid azaleine dyeing liquor and haematoxylin dyeing liquid.
Mentioned reagent box also comprises the colouring method be documented on readable carrier, and described colouring method is above-mentioned method.
Described readable carrier is specially instructions.
Above-mentioned kit is following 1) or 2) application at least one is also the scope of protection of the invention:
1) preparation detects or application in auxiliary detection or diagnosis or auxiliary diagnosis Much's bacillus product;
2) whether characterization or assistant identification sample to be tested be containing the application in Much's bacillus product.
0.01mol/L, pH value are the sodium citrate buffer solution of 6.0: by 2.41g sodium citrate (Na 3c 6h 5o 72H 2and 0.38g citric acid (C O) 6h 8o 7h 2o) be dissolved in 1L distilled water.
The Anti-TNF-α liquid solution of anti-MTB antigen: by MTB antigen (amino acid sequence is sequence 1) immunize New Zealand white rabbits, collects rabbit anteserum, utilizes proteinG purifying to obtain the Anti-TNF-α liquid solution of anti-MTB antigen.
DAB dyeing liquor: first configure 20 × DAB dyeing mother liquor: by 0.1g diaminobenzidine (3,3 '-diaminobenzidine, DAB) pour in 10ml distilled water, obtain mixed liquor, add in mixed liquor 3-5 drip 10M HCl to mixed liquor become light brown then DAB dissolve completely, packing, is stored in-20 DEG C.
Use 0.1M PB damping fluid (29g Na before use 2hPO 412H 2o and 3g NaH 2pO 42H 2o is dissolved in 1L distilled water) being dyeed by 20 × DAB after mother liquor is diluted to 1 × working fluid adds H 2o 2to final concentration 0.03%.；
Carbolic acid azaleine dyeing liquor: first configure 10ml basic fuchsin ethanol saturated solution (1g basic fuchsin being dissolved in 10ml absolute ethyl alcohol) and configuration 90ml 5% carbolic acid solution (5g phenol is dissolved in distilled water), then these two kinds of solution mixing are carbolfuchsin liquid.
Differentiation liquid: add 3ml concentrated hydrochloric acid and 70ml absolute ethyl alcohol in 27ml distilled water, mixing is differentiation liquid.
Haematoxylin dyeing liquid: 5g haematoxylin is dissolved in 50ml absolute ethyl alcohol, obtains haematoxylin anhydrous alcohol solution; Again 44g aluminium potassium sulfate is put into 1L distilled water heating for dissolving, obtain aluminum potassium sulfate solution; Again by haematoxylin anhydrous alcohol solution and aluminum potassium sulfate solution mixing, obtain mixed liquor; Be cooled to about 91 DEG C Deng stirring after mixed liquor boiling, slowly add 2.5g mercury oxide, being placed in cold water after dissolving cools, obtain reaction product, next day filters reaction product, collects filtrate, in filtrate, add 4g citric acid again, stir evenly and be haematoxylin dyeing liquid.
Concentration is 0.01M, pH value is 7.4PBS formula: 2.9g Na 2hPO 412H 2o, 0.3g NaH 2pO 42H 2o and 9gNaCl is dissolved in 1L distilled water, and adjust ph is 7.4.
Experiment of the present invention proves, this invention exploits a kind of kit for Much's bacillus and antigen double staining thereof, this kit can be implemented in the albumen simultaneously detecting MTB and expression thereof in a tissue specimen slice, thus improves pathological diagnosis susceptibility lungy.This kit using method is easy, is applicable to pathology department and carries out conventional sense, has good clinical generalization value.
Accompanying drawing explanation
Fig. 1 carries out according to 4 kinds of coloured differently methods the result that dyes in tuberculosis histotomy.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, tuberculosis tissue specimen colouring method
One, the configuration of reagent:
0.01mol/L, pH value are the sodium citrate buffer solution of 6.0: by 2.41g sodium citrate (Na 3c 6h 5o 72H 2and 0.38g citric acid (C O) 6h 8o 7h 2o) be dissolved in 1L distilled water.
Concentration is 0.01M, pH value is 7.4PBS formula: 2.9g Na 2hPO 412H 2o, 0.3g NaH 2pO 42H 2o and 9gNaCl is dissolved in 1L distilled water, and adjust ph is 7.4.
The polyclonal antibody of anti-MTB antigen: by MTB antigen (amino acid sequence is sequence 1) immunize New Zealand white rabbits, collects rabbit anteserum, utilizes proteinG purifying to obtain the polyclonal antibody of anti-MTB antigen.
DAB dyeing liquor: first configure 20 × DAB dyeing mother liquor: by 0.1g diaminobenzidine (3,3 '-diaminobenzidine, DAB) pour in 10ml distilled water, obtain mixed liquor, add in mixed liquor 3-5 drip 10M HCl to mixed liquor become light brown then DAB dissolve completely, packing, is stored in-20 DEG C.
Use 0.1M PB damping fluid (29g Na before use 2hPO 412H 2o and 3g NaH 2pO 42H 2o is dissolved in 1L distilled water) being dyeed by 20 × DAB after mother liquor is diluted to 1 × working fluid adds H 2o 2to final concentration 0.03%.；
Carbolic acid azaleine dyeing liquor: first configure 10ml basic fuchsin ethanol saturated solution (1g basic fuchsin being dissolved in 10ml absolute ethyl alcohol) and configuration 90ml 5% carbolic acid solution (5g phenol is dissolved in distilled water), then these two kinds of solution mixing are carbolfuchsin liquid.
Differentiation liquid: add 3ml concentrated hydrochloric acid and 70ml absolute ethyl alcohol in 27ml distilled water, mixing is differentiation liquid.
Haematoxylin dyeing liquid: 5g haematoxylin is dissolved in 50ml absolute ethyl alcohol, obtains haematoxylin anhydrous alcohol solution; Again 44g aluminium potassium sulfate is put into 1L distilled water heating for dissolving, obtain aluminum potassium sulfate solution; Again by haematoxylin anhydrous alcohol solution and aluminum potassium sulfate solution mixing, obtain mixed liquor; Be cooled to about 91 DEG C Deng stirring after mixed liquor boiling, slowly add 2.5g mercury oxide, being placed in cold water after dissolving cools, obtain reaction product, next day filters reaction product, collects filtrate, in filtrate, add 4g citric acid again, stir evenly and be haematoxylin dyeing liquid.
Two, colouring method
For obtaining optimum dyeing effect, anti-ly to hatch in antigen retrieval, the dyeing of carbolic acid azaleine, primary antibodie and two, the various combination mode of the step such as haematoxylin dyeing, specific as follows:
1, colouring method 1
1) paraffin organization (tuberculosis tissue specimen) section that preparation 4 μm is thick, conventional dimethylbenzene dewaxing (each 10min/ time of dimethylbenzene, 2 times), (100% with ethanol for graded ethanol aquation, 90% ethanol water, 50% ethanol water, 50% ethanol water, each 8min), cut into slices after obtaining dewaxing aquation;
2) concentration be 0.01M, pH value be 6.0 citrate buffer solution mesohigh (80 kPas) repair 90s, obtain repairing rear section, specific as follows:
By 1000ml 0.01M citrate buffer pH6.0 in pressure cooker, heating is until boiling; Section after dewaxing aquation is placed on stainless steel section frame, put into the damping fluid seethed with excitement, cover pot cover, buckle pressure valve, continue to be heated to jet, after 80 kPas of maintenance 90s, pressure cooker leaves thermal source, is cooled to room temperature (after slightly cold, can at accelerating cooling under water tap), take out section, obtain repairing rear section;
First cut into slices twice with after distilled water flushing reparation, rinse twice, each 3 minutes with the PBS that 0.01M, pH value are 7.4 afterwards;
3) to above-mentioned 2) upper dropping anti-MTB antigen Anti-TNF-α liquid solution of cutting into slices after the reparation that obtains, cover the upper tissue to be measured of section, room temperature (25 DEG C) hatches 1h, obtains an anti-binding section; PH value is 7.4 concentration is that 0.01MPBS washes 3 times, washes 5min at every turn;
4) to above-mentioned 3) in the goat anti-rabbit igg antibody solution of the upper HRP of the dropping mark of an anti-binding section after washing (goat-anti rabbit-HRP two resists, step neoplastic product), cover the upper tissue to be measured of section, incubated at room 15min, obtain two anti-binding sections; PH value is 7.4,0.01M PBS washes 3 times, washes 5min at every turn;
5) to above-mentioned 4) in two anti-bindings sections after washing are upper drips DAB dyeing liquor, cover the upper tissue to be measured of section, incubated at room 3min, obtains DAB stained; Wash 5min with water, cessation reaction;
6) to above-mentioned 5) in DAB stained after washing drips carbolic acid azaleine dyeing liquor, cover the upper tissue to be measured of section, incubated at room 1h; Obtain carbolic acid azaleine stained; Wash 5min with water, cessation reaction;
7) to above-mentioned 6) in through washing after carbolic acid azaleine stained on drip differentiation liquid a moment to no longer decolour (bleaching time is 30S); Cut into slices after obtaining the process of differentiation liquid; Rinse with water and wash 1 time;
8) by above-mentioned 7) in after differentiation liquid process after washing section immerse in haematoxylin dyeing liquid the 1min that dyes, obtain haematoxylin dyeing section; Rinse with water and wash 1 time; Be placed in differentiation liquid again to rinse (bleaching time is 60S), then the 5min that is soaked in water, obtain stained.
9) by above-mentioned 8) after the stained conventional gradients dehydration of alcohol that obtains, dimethylbenzene are transparent, neutral gum mounting, microscopy.
2, colouring method 2
1) prepare 4 μm of thick paraffin tissue sections, conventional dimethylbenzene dewaxing, graded ethanol aquation (with 1 of colouring method 1) are identical).
2) drip carbolic acid azaleine dyeing liquor, cover tissue to be measured, incubated at room 1h (with 6 of colouring method 1) is identical);
3) drip differentiation liquid a moment to no longer decolouring, with water rinse wash 1 time (with 7 of colouring method 1) identical);
4) concentration be 0.01M, pH value be 6.0 citrate buffer solution mesohigh repair 90s (with 2 of colouring method 1) identical);
5) drip the polyclonal antibody of anti-MTB antigen, cover tissue to be measured, incubated at room 1h; 0.01M PBS (2.9gNa 2hPO 412H 2o, 0.3g NaH 2pO 42H 2o and 9gNaCl is dissolved in 1L distilled water) wash 3 times, wash 5min (with 3 of colouring method 1) identical) at every turn;
6) drip goat-anti rabbit-HRP two anti-(purchased from neoformation advanced in years), cover tissue to be measured, incubated at room 15min.0.01MPBS washes 3 times, washes 5min (with 4 of colouring method 1) identical at every turn);
7) drip DAB dyeing liquor, cover tissue to be measured, incubated at room 3min.Wash 5min with water, cessation reaction (with 5 of colouring method 1) is identical);
8) contaminate 1min in immersion haematoxylin dyeing liquid, rinse with water and wash 1 time; Rinse in differentiation liquid, then the 5min that is soaked in water (with 8 of colouring method 1) is identical).
9) conventional gradients dehydration of alcohol, dimethylbenzene transparent after, neutral gum mounting, microscopy.
3, colouring method 3
1) prepare 4 μm of thick paraffin tissue sections, conventional dimethylbenzene dewaxing, graded ethanol aquation (with 1 of colouring method 1) are identical).
2) concentration be 0.01M, pH value be 6.0 citrate buffer solution mesohigh repair 90s (with 2 of colouring method 1) identical);
3) drip the polyclonal antibody of anti-MTB antigen, cover tissue to be measured, incubated at room 1h; 0.01M PBS (2.9gNa 2hPO 412H 2o, 0.3g NaH 2pO 42H 2o and 9gNaCl is dissolved in 1L distilled water) wash 3 times, wash 5min (with 3 of colouring method 1) identical) at every turn;
4) drip goat-anti rabbit-HRP two anti-(purchased from neoformation advanced in years), cover tissue to be measured, incubated at room 15min.0.01M PBS washes 3 times, washes 5min (with 4 of colouring method 1) identical at every turn);
5) drip DAB dyeing liquor, cover tissue to be measured, incubated at room 3min.Wash 5min with water, cessation reaction (with 5 of colouring method 1) is identical);
6) contaminate 1min in immersion haematoxylin dyeing liquid, rinse with water and wash 1 time; Rinse in differentiation liquid, then the 5min that is soaked in water (with 8 of colouring method 1) is identical).
7) drip carbolic acid azaleine dyeing liquor, cover tissue to be measured, incubated at room 1h (with 6 of colouring method 1) is identical);
8) drip differentiation liquid a moment to no longer decolouring, with water rinse wash 1 time (with 7 of colouring method 1) identical);
9) conventional gradients dehydration of alcohol, dimethylbenzene transparent after, neutral gum mounting, microscopy.
4, colouring method 4
1) prepare 4 μm of thick paraffin tissue sections, conventional dimethylbenzene dewaxing, graded ethanol aquation (with 1 of colouring method 1) are identical).
2) concentration be 0.01M, pH value be 6.0 citrate buffer solution mesohigh repair 90s (with 2 of colouring method 1) identical);
3) drip the polyclonal antibody of anti-MTB antigen, cover tissue to be measured, incubated at room 1h; 0.01M PBS (2.9gNa 2hPO 412H 2o, 0.3g NaH 2pO 42H 2o and 9gNaCl is dissolved in 1L distilled water) wash 3 times, wash 5min (with 3 of colouring method 1) identical) at every turn;
4) drip carbolic acid azaleine dyeing liquor, cover tissue to be measured, incubated at room 1h (with 6 of colouring method 1) is identical);
5) drip differentiation liquid extremely no longer to decolour a moment, rinse with water and wash 1 time;
6) drip goat-anti rabbit-HRP two anti-(purchased from neoformation advanced in years), cover tissue to be measured, incubated at room 15min.0.01M PBS washes 3 times, washes 5min (with 4 of colouring method 1) identical at every turn);
7) drip DAB dyeing liquor, cover tissue to be measured, incubated at room 3min.Wash 5min with water, cessation reaction (with 5 of colouring method 1) is identical);
8) contaminate 1min in immersion haematoxylin dyeing liquid, rinse with water and wash 1 time; Rinse in differentiation liquid, then the 5min that is soaked in water (with 8 of colouring method 1) is identical).
9) conventional gradients dehydration of alcohol, dimethylbenzene transparent after, neutral gum mounting, microscopy.
Under different amplification, observe the coloration result of above-mentioned 4 kinds of methods with optical microscope, as shown in Figure 1, A, D, G, J are the coloration results obtained under 4 times of object lens; B, E, H, K are the coloration results obtained under 40 times of object lens; C, F, I, L are the coloration results obtained under 100 times of oily mirrors.A, B, C figure carries out according to colouring method 1 result that dyes, and result shows, and MTB thalline, MTB expressing protein, also has nuclear targeting to be all perfectly clear.D, E, F figure carries out according to colouring method 2 result that dyes, and result shows, and MTB expressing protein and nuclear targeting are perfectly clear, but the dyeing of MTB thalline is for negative.G, H, I figure carries out according to colouring method 3 result that dyes, and result shows, and the dyeing of MTB thalline, MTB expressing protein is clear, but nuclear targeting be feminine gender.J, K, L figure carries out according to colouring method 4 result that dyes, display MTB thalline and nuclear targeting clear, but the dyeing of MTB expressing protein is very weak.Colouring method 1 enough obtains optimum dyeing effect, the clear dyeing effect of MTB thalline, MTB expressing protein and histocyte core can be realized, the expression of MTB albumen clearly can be seen under high power lens (× 40 object lens), can clear differentiation MTB thalline under oily mirror (× 100 object lens).
Therefore, above-mentioned DAB dyeing liquor, carbolic acid azaleine dyeing liquor, haematoxylin dyeing liquid can as the key components of kit, and this kit can also comprise the instructions recording said method.This kit may be used for dyeing tuberculosis tissue specimen or MTB thalline or MTB phage surface protein or the tuberculosis histocyte core that dyes.
Embodiment 2, tuberculosis tissue specimen colouring method
In order to explore the clinical value of kit prepared by embodiment 1, the tuberculosis tissue specimen that have collected 212 routine clinical definites (is provided by attached BJ Chest Science Hospital of the Capital University of Medical Sciences, and patient knows the inside story) and 63 example contrast diseased tissue sample (the non-tuberculosis tissue specimens of clinical definite, thered is provided by attached BJ Chest Science Hospital of the Capital University of Medical Sciences, and patient knows the inside story).
Above-mentioned sample is tested according to the colouring method 1 shown in embodiment 1;
Adopt traditional luxuriant Nissl's staining for contrast simultaneously.
The luxuriant Nissl's staining method of tradition:
1) paraffin tissue sections that preparation 4 μm is thick, conventional dimethylbenzene dewaxing, graded ethanol aquation.
2) drip carbolic acid azaleine dyeing liquor, cover tissue to be measured, incubated at room 1h;
3) drip differentiation liquid extremely no longer to decolour a moment, rinse with water and wash 1 time;
4) drip 0.1% methylene blue solution (0.1g methylenum careuleum is dissolved in 100ml distilled water), cover tissue to be measured, incubated at room 20 seconds, rinse with water and wash 1 time;
5) conventional gradients dehydration of alcohol, dimethylbenzene transparent after, neutral gum mounting, microscopy.
Calculate stained positive rate, result is as shown in table 1, with to be stained positive rate be 65.6% (139 examples are positive) of the colouring method 1 of embodiment 1 in 212 routine tuberculosis tissue specimens, be significantly higher than the positive rate 34.4% (73 examples are positive) (p<0.001) of luxuriant Nissl's staining.All not there is positive findings in two kinds of methods in contrast diseased tissue sample, and specificity is all very good.
Table 1 is the Sensitivity and Specificity of 2 kinds of colouring methods
Therefore, the kit of embodiment 1 and method can be used for qualification or assistant identification sample to be tested whether containing Much's bacillus and expressing protein thereof or detection or auxiliary detection or diagnosis or auxiliary diagnosis tuberculosis, and concrete grammar is as follows:
Dye to sample to be tested according to the method for embodiment 1 with the kit of embodiment 1, if sample to be tested can dye, then sample to be tested is or candidate is sample containing Much's bacillus; If sample to be tested can not dye, then sample to be tested be not or candidate for containing the sample of Much's bacillus.

Claims (10)

1., for the tissue section strain method that Much's bacillus detects, comprise the steps:
1) by histotomy to be detected and anti-MTB antigen-antibody solution reaction, an anti-binding section is obtained;
2) by a described anti-binding section and two anti-solution reactions, two anti-binding sections are obtained;
3) with DAB dyeing liquor, described two anti-binding sections are dyeed, obtain DAB stained;
4) with carbolic acid azaleine dyeing liquor, described DAB stained is dyeed, obtain carbolic acid azaleine stained;
5) with haematoxylin dyeing liquid, described carbolic acid azaleine stained is dyeed, obtain stained, realize tissue section strain.
2. method according to claim 1, is characterized in that:
Described anti-MTB antigen-antibody is anti-MTB antigen Anti-TNF-α liquid solution;
The amino acid sequence of described MTB antigen is sequence 1 in sequence table;
Described two resist for goat anti-rabbit igg antibody; The goat anti-rabbit igg antibody of the concrete HRP mark of described goat anti-rabbit igg antibody.
3. method according to claim 1 and 2, is characterized in that:
Step 1) in, the temperature of described reaction is 23-28 DEG C, and the time of described reaction is 0.5-2h;
Step 2) in, the temperature of described reaction is 23-28 DEG C, and the time of described reaction is 10-20min.
Step 3) in, described dyeing time is 1-10min, dyeing temperature is 23-28 DEG C;
Step 4) in, described dyeing time is 20-90min, dyeing temperature is 23-28 DEG C;
Step 5) in, described dyeing time is 0.5-2min, dyeing temperature is 23-28 DEG C.
4., according to described method arbitrary in claim 1-3, it is characterized in that:
In step 1)-2) between, also comprise the steps: the described anti-binding section of washing; The cleansing solution that described washing adopts is specially that concentration is 0.01M, pH value is the PBS of 7.4;
In step 2)-3) between, also comprise the steps: the described two anti-binding sections of washing; The cleansing solution that described washing adopts is specially that concentration is 0.01M, pH value is the PBS of 7.4;
In step 3)-4) between, also comprise the steps: to wash described DAB stained; The concrete concentration of cleansing solution that described washing adopts is 0.01M, pH value is 7.4 PBS or water;
In step 4)-5) between, also comprise the steps: first with differentiation liquid decolour described carbolic acid azaleine stained obtain decolouring after carbolic acid azaleine stained, then wash carbolic acid azaleine stained after described decolouring; The concrete concentration of cleansing solution that described washing adopts is 0.01M, pH value is 7.4 PBS or water;
In step 5) in, after described dyeing obtains stained, also comprise the steps: first to break up liquid to decolour described stained, obtain the section of decolouring poststaining, then wash the section of described decolouring poststaining; The concrete concentration of cleansing solution that described washing adopts is 0.01M, pH value is 7.4 PBS or water.
5. method according to claim 4, is characterized in that:
Described bleaching time is 30 seconds-60 seconds.
6. the method according to claim 1-5, is characterized in that:
Described histotomy to be detected is paraffin-embedded histotomy to be detected.
7. method according to claim 6, is characterized in that:
Step 1 in described method) front, also comprise the steps:
A, described paraffin-embedded histotomy to be detected is carried out dewaxing aquation, cut into slices after obtaining dewaxing aquation;
B, by cut into slices after described dewaxing aquation concentration be 0.01M, pH value be 6.0 the reparation of citrate buffer solution mesohigh, obtain repairing rear section;
The pressure of described high pressure is 80-100 kPa;
Described repair time is 90s, and the temperature of described reparation is 100 DEG C.
8., for a kit for the tissue section strain of Much's bacillus detection, comprise DAB dyeing liquor, carbolic acid azaleine dyeing liquor and haematoxylin dyeing liquid.
9. method according to claim 8, is characterized in that: described kit also comprises the colouring method be documented on readable carrier, and described colouring method is arbitrary described method in claim 1-7.
10. the kit described in claim 8 or 9 is following 1) or 2) application at least one:
1) preparation detects or application in auxiliary detection or diagnosis or auxiliary diagnosis Much's bacillus product;
2) whether characterization or assistant identification sample to be tested be containing the application in Much's bacillus product.
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CN107367607A (en) * 2017-05-25 2017-11-21 长沙金域医学检验所有限公司 A kind of Pathologic specimen section SABC operating method and its system
CN110501489A (en) * 2019-08-27 2019-11-26 武汉顺可达生物科技有限公司 A kind of application of tuberculosis immunity group kit in the diagnosis of tuberculosis pathological tissues
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CN112129610B (en) * 2020-09-22 2021-07-16 无锡市第二人民医院 Bacterial capsule staining method and application thereof

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