CN105717310A - Immuno-fluorescent staining method for detecting mycobacterium tuberculosis in leukocytes and kit - Google Patents
Immuno-fluorescent staining method for detecting mycobacterium tuberculosis in leukocytes and kit Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses a method for rapidly detecting mycobacterium tuberculosis in blood (blood plasma and blood cells) through fluorescent staining and a kit.ESAT-6 and CFP-10 antigens of the mycobacterium tuberculosis in the blood are specifically detected by using monoclonal antibodies of RD-1 zone ESAT-6 and CFP-10 antigens of the mycobacterium and are used for etiologic detection and early-stage and rapid clinical diagnosis of tuberculosis diseases.According to the method, the blood is divided into the blood plasma and the blood cells, the mycobacterium tuberculosis enriched in the blood plasma is separated by using a micro-fluid device based on a membrane, meanwhile a slide is coated with hemocytes (leukocytes), and the mycobacterium tuberculosis in the blood plasma and the leukocytes are detected by using the monoclonal antibodies of the ESAT-6 and CFP-10 antigens and adopting direct and indirect fluorescent staining methods.The method is simple in operation, high in sensitivity and strong in specificity and is a definite tuberculosis etiology diagnosis method, and the kit for detecting the mycobacterium tuberculosis in the blood is developed.In addition, by means of the method, pathogenic mycobacterium tuberculosis infection can be detected, and theoretically non-pathogenic mycobacterium tuberculosis produced in blood due to bacillus calmette guerin vaccine inoculation can be also distinguished.
Description
Technical field
A kind of immunofluorescence dyeing method detecting mycobacterium tuberculosis in leukocyte and test kit.
Background technology
Tuberculosis is the chronic infectious disease caused by single pathogen m tuberculosis infection.In recent years, due to the increase of population mobility, the appearance of drug resistance tuberculosis, the factor such as double infection of tulase and HIV (human immunodeficiency virus), tuberculosis is staged a comeback again, again becomes great public health problem and the social problem of serious harm body health of people.Estimate that the whole world about 2,000,000,000 people infects mycobacterium tuberculosis at present according to the World Health Organization's report, new cases 9,000,000 in 2013.China is high burden country lungy, and tuberculosis patient quantity occupies second place of the world, year number of the infected be about 1,300,000, account for the 14% of whole world number of the infected.Tuberculosis is a kind of the highest disease of heterogeneity, has people quickly to fall ill, becomes active tuberculosis patient, have people then not fall ill throughout one's life after organism infection mycobacterium tuberculosis, the person that becomes latent infection；The tuberculosis patient form of expression is the most varied, and have shows as pulmonary tuberculosis, and have then shows as the outer tuberculosis of different types of lung, and this heterogeneity seriously hinders prevention lungy, treatment and the progress of diagnosis.
The most conventional mycobacterium tuberculosis detection method has a variety of, is respectively arranged with its feature, but often exists and operate length complicated, time-consuming, false positive rate or the more high defect of false negative rate.Existing diagnosis of tuberculosis method, mainly includes conventional bacteria detection technique, immunology detection technology and molecular Biological Detection technology, will be that main progress and trend to various detection techniques is simply summarized with bacteriological detection technology below.
(1) conventional bacteria detection technique
The expectorant mycobacterium tuberculosis positive is Diagnosis of pulmonary goldstandard lungy, and inspection method has Sputum smears method and Sputum culturing method.
1, Sputum smears method is that the sputum sample obtained from patients with respiratory tract carries out smear, then dyes, and microscopy the most under the microscope, if apoplexy due to phlegm has antibacterial to be easily detected, it is also possible to which kind of or any antibacterial explanation be roughly.So sputum smear examination be a kind of simplicity, quick, inexpensive, the conventional inspection method of PRELIMINARY RESULTS can be obtained.But, clinical effectiveness confirms that Sputum smears method positive rate is low, about 30-40%, and needs antibacterial >=10 in sputum specimen4/ mL could detect, and sensitivity is low, and specificity is poor.In order to improve recall rate, there is people to be improved later, utilized Sedimentation smear technique, the major part of mycobacterium tuberculosis contained in about 5mL sputum is concentrated in 0.1mL precipitation, make its sensitivity increase, but still specific problem can not be solved, it is impossible to meet clinical demand.But, owing to it is simple to operate, it is not necessary to expensive equipment and high-caliber experiment condition, the technical staff being suitable for health technology condition low developed area uses, so, although smear method positive rate is the highest, remain the tulase detection method that grass-roots unit commonly uses.
2, Sputum culturing method is to obtain specifically what antibacterial exactly, allowing it grow by artificial method, in order to observe, it is possible to utilize cultivation bacterium to do drug sensitivity test, it is to avoid to use antibiotic blindly, make patient be effectively treated earlier.Sputum culturing more accurately and reliably, but needs the relatively long time, it is generally required to 2-4 is all compared with Sputum smears.Sputum culturing method is the longest and positive rate is low, typically at about 50-60%, is worth limited to clinical early diagnosis, in addition it is also necessary to make the inspections such as rabat, lung CT, bronchoscopy and tuberculosis antibody and assist diagnosis.
3, in Sputum smears and Sputum culturing, the collection of sputum specimen is most important, and the most whether the quality of sputum specimen quality, the censorship factor such as all will directly affect testing result.
To sum up, the shortcoming that conventional bacteria detection method exists following three aspects:
First, originally detect based on patient's sputum sample, the most whether the presence or absence of patient's expectorant, acquisition method whether correct, quality, the censorship factor such as all will directly affect testing result, thus cause bacteriological detection based on sputum sample basis cannot obtain higher recall rate and more preferable specificity, need novel detection method to substitute tradition expectorant bacterium detection so far.
Second, lunger accounts for the 80% of whole tubercular, and the most nearly 50% is the patient that expectorant bacterium is negative, will certainly cause failing to pinpoint a disease in diagnosis of this part population by tradition expectorant bacterium bacteriological detection, needs additive method auxiliary diagnosis；The tubercular of the most about 20% shows as the outer tuberculosis of lung, sputum sample cannot be obtained originally, the most also cannot obtain clear and definite bacteriology's evidence, can only be by the auxiliary diagnosis of other method, or exploratory antituberculosis therapy, up to being suitable for bacterium the moon and the novel fine mycology detection method of the outer tuberculosis of lung.
3rd, the evaluation whether cured for antituberculosis therapy at present, WHO recommends equally with Bacteriological Indexes as criterion, therefore, for the pulmonary tuberculosis patient that expectorant bacterium is positive, negative at last 2 months continuous Phlegm incubation of predetermined treatment duration, is cloudy turning and cures.And for the tuberculosis patient of expectorant bacterium feminine gender, after completing predetermined treatment duration, expectorant bacterium remains as negative patient, it is and cures the full course for the treatment of.But, general expectorant bacterium positive patient treatment the most all cannot obtain sputum sample this, and Endophytic bacteria whether remove unknown；And expectorant bacterium negative patient this show as feminine gender always, it is impossible to really judge whether cure.So, using Phlegm incubation result as the standard for the treatment of effectiveness evaluation, certainly will cause treatment the most thoroughly or cross and control phenomenon.
(2) immunologic test
Nassau etc. utilize elisa at tubercular's Virus monitory to tuberculosis antibody first, thus open new era of tuberculosis immunity detection, and current tuberculosis immunology detection becomes one of tuberculosis auxiliary detection important means.
1, antibody test
Tuberculosis antibody test technology constantly obtains improvement and optimizes.ELISA is to measure tuberculosis antibody research and use one of most technology, can complete relative to its 2-3 h quick, easy of traditional detection method.But the retardance of antibody mediated immunity reaction causes in early days diagnosis of tuberculosis result to be easily generated false negative, common antigen causes simultaneously cross reaction and cannot distinguish between non-tuberculous mycobacteria and all can cause false positive.Therefore, antibody test reagent is not recommended to be applied to the clinical diagnosis of tubercular by WHO.
2, Detection of antigen
After body is infected, first occurring that tuberculosis antigen, the detection to tuberculosis antigen is more suitable for early diagnosis than antibody test the most in theory.Double antibody sandwich method is the common method of current Detection of antigen, but is limited because being prepared by high activity specific antibody difficulty so that up to this point apply good antigen detecting agent the most clinically both at home and abroad.
3, cytokines measurement
Cellular immunization is the important method of non-active tuberculosis examination, the memory T lymphocyte that body produces after being infected first, is proliferated into effect T cell, secrete cytokines after antigen stimulates again.The cytokine amount (ELISA) of cellular immunization main pairing effect T cell number (ELISPOT) of detection and secretion detects at present, but cellular immunization detectable thinks that gamma interferon release test is primarily adapted for use in the diagnosis of tuberculosis infection rather than active tuberculosis in cannot be distinguished by " suggestion that gamma interferon release test is applied " that active tuberculosis patient and latent infection person, phthisiology branches of Chinese Medical Association in 2014 and " China's tuberculosis and breathing magazine " tissue expert formulate in China at present.
(3) molecular biology method
Completing of mycobacterium tuberculosis genome sequencing work in 1998, brings brand-new idea to diagnosis of tuberculosis.The initial success of rapid gene diagnostic techniques, brings revolutionary variation to treatment lungy and control, shows wide prospect.
DNA detection
The 90's of last century Hance
Utilize PCR technology for detection mycobacterium tuberculosis, bring revolutionary breakthrough to clinical diagnosis lungy.On this basis, real-time fluorescence quantitative PCR compensate for the deficiency of Standard PCR detection to a certain extent, by utilizing specific primer and fluorescent probe, monitors whole PCR process in real time and carries out quantitative analysis with standard curve, making testing result relatively reliable.Loop-mediated isothermal amplification technique (LAMP) need not change temperature, is independent of specialized equipment equipment, the most with the naked eye result of determination, simple efficiently low cost, avoids the cross-contamination of laboratory to a certain extent.The most novel molecular detecting method Xpert
MTB/RIF(rifampicin) receive significant attention, in December, 2010 World Health Organization accreditation Xpert MTB/RIF is as a kind of brand-new tuberculosis New Method for Rapid.But said method is all with DNA as template, the stability of DNA make PCR testing result not distinguish viable bacteria and dead bacterium produce false positive results.
But, detect relative to Sputum smears, Molecular Detection or complexity height, and mainly originally detect with sputum sample equally, inquire into the molecular diagnosis method testing result for blood sample currently without correlational study.
In sum, these results all show that it is feasible for setting up bacteriological detection method based on blood simple and efficient, that positive rate is high, also in the urgent need to.And object of this investigation is to set up a kind of bacteriological detection method that brand-new based on blood pathogenic mycobacterium tuberculosis is special.First, the method utilizes Conventional blood sample to detect, difficult by this sampling of sputum sample, sample the problems such as lack of standardization or sample is defective and affected, with expectorant bacterium positive tubercular negative for expectorant bacterium has identical Detection results, and can detect the mycobacterium tuberculosis in same patients blood plasma and leukocyte respectively；Second, the method utilizes the monoclonal antibody for pathogenic mycobacterium tuberculosis distinctive RD1 district antigen ESAT-6 and CFP10, only detecting the existence of pathogenic mycobacterium tuberculosis, and do not affected by bacillus calmette-guerin vaccine immunity and other mycobacteria, specificity is better than conventional expectorant bacterium detection；3rd, the method uses Immunofluorescence test technology, the LED fluorescence microscope can recommended by WHO, it is easy to find positive bacteria, improves recall rate.
Summary of the invention
The invention provides the method for mycobacterium tuberculosis in detection leukocyte, including step:
A. from blood, hemocyte is collected by centrifugal or natural sinking method；
B. add erythrocyte cracked liquid and make erythrocyte splitting；
C. centrifugal collection leukocyte；
The most alternatively, washing leukocyte；
The most resuspended leukocyte；
F. leukocyte smear is made；
G. fixative is used to fix leukocyte；
H. penetrating liquid is used thoroughly to change leukocyte；
I. confining liquid is used to close；
G. Killing Mycobacterium Tuberculosis ESAT-6 or the polyclone of CFP-10 or monoclonal antibody is used to carry out directly or indirectly fluorescence staining；
K. descend and under fluorescence microscope, see whether whether leukocyte smear exists fluorescence, then make clear in cell if there is fluorescence and there is mycobacterium tuberculosis, then make clear if there is no fluorescence and cell does not exist mycobacterium tuberculosis.
Wherein:
Erythrocyte cracked liquid in step b contains NH4Cl, KHCO3 and Na2EDTA, and slowly mixes cracking 5-15 minute at shaking table；
Step c collects leukocyte in centrifugal 5-10 minute for 1500-1800 rev/min；
Step d uses normal saline or PBS, 1500-1800 rev/min, within centrifugal 5-10 minute, washs leukocyte；
Step e is added in PBS or normal saline the 0.1%BSA suspension leukocyte of preparation；
Step g use fixative methanol or 1%-8% paraformaldehyde room temperature fix 10 minutes；
Step h uses cell membrane penetration liquid 0.1%-3.0%TritonX-100 penetrating 5-30 minute；
Step i use the confining liquid 37 DEG C containing 0.1%-15% calf serum or blood plasma or 0.1%-15% donkey serum or blood plasma close 5-40 minute；
Direct fluorescence detection wherein uses the polyclone of fluorescent material anti-ESAT-6 or CFP-10 of such as marked by fluorescein isothiocyanate or monoclonal antibody, and wherein use fluorescent material such as marked by fluorescein isothiocyanate second antibody or staphylococcal protein A in indirect fluorescent method.
Correspondingly, present invention also offers the test kit carrying out said method, including:
A. erythrocyte splitting；
B. fixative；
The most penetrating liquid；
D. confining liquid；
E. Killing Mycobacterium Tuberculosis ESAT-6 or the polyclone of CFP-10 or monoclonal antibody；
F. directly or indirectly reagent needed for fluorescence staining is carried out.
(1) test kit Main Ingredients and Appearance
The fluorescent dyeing reagent box of mycobacterium tuberculosis in a kind of quick detection blood, its Main Ingredients and Appearance includes:
1) fixative: in blood, the fixative of mycobacterium tuberculosis and cell includes that methanol and paraformaldehyde, paraformaldehyde concentration are 1%-8%；
2) cell membrane penetration liquid: 0.1%-3.0%TritonX-100 solution；
3) confining liquid: include 0.1%-15% calf serum；0.1%-15% donkey serum (or blood plasma)；
4) monoclonal antibody: include unlabelled ESAT-6 and CFP-10 monoclonal antibody, fluorescence (fluorescent dye such as Fluorescein isothiocyanate) ESAT-6 and the CFP-10 monoclonal antibody of labelling, fluorescently-labeled second antibody and fluorescently-labeled staphylococcal protein A (SPA)；
5) washing liquid: 0.01mol/L PBS；
6) erythrocyte cracked liquid: containing NH4Cl, KHCO3 and Na2EDTA；
7) microfluid mycobacterium tuberculosis enriching apparatus based on film and microporous membrane.The material of microporous membrane can be any material, such as cellulose esters, polyamide-based, polysulfones, fluorine material class, polyesters, TPO, inorganic material etc., and optimization polycarbonate film, the size of microporous membrane can be 0.4-1.5 micron；
8) polyclonal antibody can be to be prepared by rabbit, horse, cattle, sheep, mule, Cavia porcellus or chicken；
9) monoclonal antibody is mouse monoclonal antibody, has cloned the gene of ESAT-6 and CFP10 antigen respectively, builds prokaryotic expression carrier and prepares purification ESAT-6 and CFP10 antigen, and immune mouse is prepared for specific monoclonal antibody；
10) the detection sample described in is blood but is not limited to blood (blood plasma and hemocyte), it is also possible to be cerebrospinal fluid.
(2) immunofluorescence dyeing basic skills
1, separated plasma and hemocyte
(1) examinee blood 5-10ml (anticoagulant, EDTA anticoagulant can do Molecular Detection and resistance test after beneficially finding mycobacterium tuberculosis) is gathered；
(2) 500-1500 turns/is centrifuged 5-10 minute (separated plasma and hemocyte after hemocyte also can be allowed naturally to sink)；
(3) separated plasma and hemocyte.
2, the filtration of mycobacterium tuberculosis and enrichment in blood plasma
(1) microfluid defecator based on film filters enrichment mycobacterium tuberculosis；
(2) blood plasma 2-10ml, illustration method and process as a example by 2ml；
(3) blood plasma 2ml adds normal saline or PBS2ml, mixing；
(4) 6% sodium hypochlorite 8ml is added, mixing, stand 5-20 minute；
(5) 95 ethanol (containing 1%TritonX-100) 48ml is added, mixing, stand 5-15 minute；
(6) in aforesaid liquid being added microfluidic device or syringe-driven filter based on film (microporous membrane aperture 0.4-1.5 micron) filter enrichment mycobacterium tuberculosis；
(7) the mycobacterium tuberculosis immunofluorescence dyeing to be done being enriched on microporous membrane is filtered.
3, the preparation of hemocyte (leukocyte) detection mycobacterium tuberculosis leukocyte sheet smear
(1) drawing the hemocyte that blood plasma stays, add erythrocyte cracked liquid (1:5-10), shaking table slowly mixes 5-15 minute；
(2) 1500-1800 rev/min, centrifugal 5-10 minute, inhale and abandon supernatant；
(3) normal saline or PBS are added
5ml, 1500-1800 rev/min, centrifugal 5-10 minute, inhale and abandon supernatant；
(4) 0.1%BSA(PBS or normal saline are added) 0.2ml suspension leukocyte；
(5) leukocyte smear, immunofluorescence dyeing to be done.
4, immunofluorescence dyeing
(1) plasmapheresis enrichment microporous membrane and leukocyte smear；
(2) fixing 10 minutes (also can use methanol) by 1-8% paraformaldehyde (pH7.4) room temperature, pure water rinses；
(3) 0.1%-3.0%
Penetrating 5-30 minute of TritonX-100, PBS or pure water rinsing；
(4) 0.5%-15%
BSA (bSA) or calf serum or donkey serum, 370C closes 5-40 minute, PBS or pure water rinsing；
(5) fluorescence direct staining: add pH7.2
Fluorescent labeling ESAT-6 that PBS suitably dilutes and the monoclonal antibody of CFP-10, dye 30 minutes-2 hours (also can 40C refrigerator stained over night).Rinse with PBS, be dried, get final product the mycobacterium tuberculosis of aobvious fluorescence on fluorescence microscope microporous membrane and leukocyte sheet.Fluorescently-labeled ESAT-6 and CFP-10 monoclonal antibody, can individually dye, also can both combined staining；
(6) dyeing indirectly: add pH7.2
The monoclonal antibody of unmarked ESAT-6 and CFP-10 that PBS suitably dilutes, dye 30 minutes-2 hours (also can 40C refrigerator stained over night).After rinsing with PBS, adding fluorescently-labeled second antibody (anti-mouse antibody) or fluorescently-labeled SPA dyes 20-40 minute, PBS rinses, and is dried, and gets final product the mycobacterium tuberculosis of aobvious fluorescence on fluorescence microscope microporous membrane and leukocyte sheet.Unlabelled ESAT-6 and CFP-10 monoclonal antibody, can individually dye, also can both combined staining.
Accompanying drawing explanation
Fig. 1 is individually added into anti-ESAT-6 for using test kit staining of the present invention
Mycobacterium tuberculosis in the blood of monoclonal antibody detection；
Fig. 2 is to use test kit staining of the present invention to be individually added into mycobacterium tuberculosis in the blood of anti-CFP-10 monoclonal antibody detection；
Fig. 3 successively adds anti-ESAT-6 for using test kit staining of the present invention
Mycobacterium tuberculosis in the blood of monoclonal antibody and the detection of anti-CFP-10 monoclonal antibody.
Detailed description of the invention
The detection of mycobacterium tuberculosis and qualification in embodiment 1 blood plasma
(1) examinee blood 5-10ml (anticoagulant, EDTA anticoagulant can do Molecular Detection and resistance test after beneficially finding mycobacterium tuberculosis) is gathered；
(2) 500-1500 turns/is centrifuged 5-10 minute (separated plasma and hemocyte after hemocyte also can be allowed naturally to sink)；
(3) microfluid defecator based on film filters enrichment mycobacterium tuberculosis；
(4) blood plasma 2-10ml, illustration method and process as a example by 2ml；
(5) blood plasma 2ml adds normal saline or PBS 2ml, mixing；
(6) 6% sodium hypochlorite 8ml is added, mixing, stand 5-20 minute；
(7) 95 ethanol (containing 1%TritonX-100) 48ml is added, mixing, stand 5-15 minute；
(8) in aforesaid liquid being added microfluidic device or syringe-driven filter based on film (microporous membrane aperture 0.4-1.5 micron) filter enrichment mycobacterium tuberculosis；
(9) fixing 10 minutes (also can use methanol) by 1-8% paraformaldehyde (pH7.4) room temperature, pure water rinses；
(10) 0.1%-3.0%
Penetrating 5-30 minute of TritonX-100, PBS or pure water rinsing；
(11) 0.5%-15%
BSA (bSA) or calf serum or donkey serum, close 5-40 minute for 37 DEG C, PBS or pure water rinsing；
(12) fluorescence direct staining: add pH7.2
Fluorescent labeling ESAT-6 that PBS suitably dilutes and the monoclonal antibody of CFP-10, dye 30 minutes-2 hours (also can 40C refrigerator stained over night).Rinse with PBS, be dried, get final product the mycobacterium tuberculosis of aobvious fluorescence on fluorescence microscope microporous membrane.Fluorescently-labeled ESAT-6 and CFP-10 monoclonal antibody, can individually dye, also can both combined staining；
(13) dyeing indirectly: add pH7.2
The monoclonal antibody of unmarked ESAT-6 and CFP-10 that PBS suitably dilutes, dye 30 minutes-2 hours (also can 40C refrigerator stained over night).After rinsing with PBS, adding fluorescently-labeled second antibody (anti-mouse antibody) or fluorescently-labeled SPA dyes 20-40 minute, PBS rinses, and is dried, and gets final product the mycobacterium tuberculosis of aobvious fluorescence on fluorescence microscope microporous membrane.Unlabelled ESAT-6 and CFP-10 monoclonal antibody, can individually dye, also can both combined staining.
The detection of the mycobacterium tuberculosis on embodiment 2 leukocyte sheet and qualification
(1) examinee blood 5-10ml (anticoagulant, EDTA anticoagulant can do Molecular Detection and resistance test after beneficially finding mycobacterium tuberculosis) is gathered；
(2) 500-1500 turns/is centrifuged 5-10 minute (separated plasma and hemocyte after hemocyte also can be allowed naturally to sink)；
(3) hemocyte stayed after drawing blood plasma, adds erythrocyte cracked liquid (1:5-10), and shaking table slowly mixes 5-15 minute；
(4) 1500-1800 rev/min, centrifugal 5-10 minute, inhale and abandon supernatant；
(5) normal saline or PBS are added
5ml, 1500-1800 rev/min, centrifugal 5-10 minute, inhale and abandon supernatant；
(6) 0.1%BSA(PBS or normal saline are added) 0.2ml suspension leukocyte；
(7) coat and on slide, make leukocyte smear；
(8) fixing 10 minutes (also can use methanol) by 1-8% paraformaldehyde (pH7.4) room temperature, pure water rinses；
(9) 0.1%-3.0%
Penetrating 5-30 minute of TritonX-100, PBS or pure water rinsing；
(10) 0.5%-15%
BSA (bSA) or calf serum or donkey serum, close 5-40 minute for 37 DEG C, PBS or pure water rinsing；
(11) fluorescence direct staining: add pH7.2
Fluorescent labeling ESAT-6 that PBS suitably dilutes and the monoclonal antibody of CFP-10, dye 30 minutes-2 hours (also can 40C refrigerator stained over night).Rinse with PBS, be dried, get final product the mycobacterium tuberculosis of aobvious fluorescence on fluorescence microscope microporous membrane and leukocyte sheet.Fluorescently-labeled ESAT-6 and CFP-10 monoclonal antibody, can individually dye, also can both combined staining；
(12) dyeing indirectly: add pH7.2
The monoclonal antibody of unmarked ESAT-6 and CFP-10 that PBS suitably dilutes, dye 30 minutes-2 hours (also can 40C refrigerator stained over night).After rinsing with PBS, adding fluorescently-labeled second antibody (anti-mouse antibody) or fluorescently-labeled SPA dyes 20-40 minute, PBS rinses, and is dried, and gets final product the mycobacterium tuberculosis of aobvious fluorescence on fluorescence microscope microporous membrane and leukocyte sheet.Unlabelled ESAT-6 and CFP-10 monoclonal antibody, can individually dye, also can both combined staining.
The detection of mycobacterium tuberculosis and qualification in embodiment 3 cerebrospinal fluid
(1) the cerebrospinal fluid 0.5-2ml of fresh collection,
Avoiding placing because of specimen causing the substance decomposition such as cytoclasis, glucose, bacterolysis etc. for a long time, specimen should be avoided solidification as far as possible and be mixed into blood；
(2) microfluid defecator based on film filters enrichment mycobacterium tuberculosis；
(3) cerebrospinal fluid 0.5-2ml,
Illustration method and process as a example by 0.5ml；
(4) cerebrospinal fluid 0.5ml adds normal saline or PBS 3.5ml, mixing；
(5) 6% sodium hypochlorite 8ml is added, mixing, stand 5-20 minute；
(6) 95 ethanol (containing 1%TritonX-100) 48ml is added, mixing, stand 5-15 minute；
(7) in aforesaid liquid being added microfluidic device or syringe-driven filter based on film (microporous membrane aperture 0.4-1.5 micron) filter enrichment mycobacterium tuberculosis；
(8) fixing 10 minutes (also can use methanol) by 1-8% paraformaldehyde (pH7.4) room temperature, pure water rinses；
(9) 0.1%-3.0%
Penetrating 5-30 minute of TritonX-100, PBS or pure water rinsing；
(10) 0.5%-15%
BSA (bSA) or calf serum or donkey serum, close 5-40 minute for 37 DEG C, PBS or pure water rinsing；
(11) fluorescence direct staining: add pH7.2
Fluorescent labeling ESAT-6 that PBS suitably dilutes and the monoclonal antibody of CFP-10, dye 30 minutes-2 hours (also can 40C refrigerator stained over night).Rinse with PBS, be dried, get final product the mycobacterium tuberculosis of aobvious fluorescence on fluorescence microscope microporous membrane.Fluorescently-labeled ESAT-6 and CFP-10 monoclonal antibody, can individually dye, also can both combined staining；
(12) dyeing indirectly: add pH7.2
The monoclonal antibody of unmarked ESAT-6 and CFP-10 that PBS suitably dilutes, dye 30 minutes-2 hours (also can 40C refrigerator stained over night).After rinsing with PBS, adding fluorescently-labeled second antibody (anti-mouse antibody) or fluorescently-labeled SPA dyes 20-40 minute, PBS rinses, and is dried, and gets final product the mycobacterium tuberculosis of aobvious fluorescence on fluorescence microscope microporous membrane.Unlabelled ESAT-6 and CFP-10 monoclonal antibody, can individually dye, also can both combined staining.
Claims (12)
1. detect a method for mycobacterium tuberculosis in leukocyte, including step: a. collects hemocyte by centrifugal or natural sinking method from blood；B. add erythrocyte cracked liquid and make erythrocyte splitting；C. centrifugal collection leukocyte；The most alternatively, washing leukocyte；The most resuspended leukocyte；F. leukocyte smear is made；G. fixative is used to fix leukocyte；H. penetrating liquid is used thoroughly to change leukocyte；I. confining liquid is used to close；G. Killing Mycobacterium Tuberculosis ESAT-6 or the polyclone of CFP-10 or monoclonal antibody is used to carry out directly or indirectly fluorescence staining；K. descend and under fluorescence microscope, see whether whether leukocyte smear exists fluorescence, then make clear in cell if there is fluorescence and there is mycobacterium tuberculosis, then make clear if there is no fluorescence and cell does not exist mycobacterium tuberculosis.
2. the method described in claim 1, wherein the erythrocyte cracked liquid in step b contains NH4Cl、KHCO3And Na2EDTA, and slowly mix cracking 5-15 minute at shaking table；Step c collects leukocyte in centrifugal 5-10 minute for 1500-1800 rev/min；Step d uses normal saline or PBS, 1500-1800 rev/min, within centrifugal 5-10 minute, washs leukocyte；Step e is added in PBS or normal saline the 0.1%BSA suspension leukocyte of preparation；Step g use fixative methanol or 1%-8% paraformaldehyde room temperature fix 10 minutes；Step h uses cell membrane penetration liquid 0.1%-3.0%TritonX-100 penetrating 5-30 minute；Step i use the confining liquid 37 DEG C containing 0.1%-15% calf serum or blood plasma or 0.1%-15% donkey serum or blood plasma close 5-40 minute.
3. the method described in claim 1 or 2, uses polyclone or the monoclonal antibody of anti-ESAT-6 or CFP-10 of fluorescent material labelling in wherein said direct fluorescence detection.
4. the method described in claim 3, wherein fluorescent material is Fluorescein isothiocyanate.
5. the method described in claim 1 or 2, uses fluorescent material labelling second antibody or staphylococcal protein A in wherein said indirect fluorescent method.
6. the method described in claim 5, wherein fluorescent material is Fluorescein isothiocyanate.
7. detect a test kit for mycobacterium tuberculosis in leukocyte, including: a. erythrocyte cracked liquid；B. fixative；The most penetrating liquid；D. confining liquid；E. Killing Mycobacterium Tuberculosis ESAT-6 or the polyclone of CFP-10 or monoclonal antibody；F. directly or indirectly reagent needed for fluorescence staining is carried out.
8. the test kit described in claim 7, wherein said erythrocyte cracked liquid contains NH4Cl、KHCO3And Na2EDTA；Described fixative is methanol or 1%-8% paraformaldehyde；Described cell membrane penetration liquid is 0.1%-3.0%TritonX-100；Described confining liquid is 0.1%-15% calf serum or blood plasma or 0.1%-15% donkey serum or blood plasma.
9. the test kit described in claim 7 or 8, wherein uses polyclone or the monoclonal antibody of anti-ESAT-6 or CFP-10 of fluorescent material labelling in described direct fluorescence detection.
10. the test kit described in claim 9, wherein fluorescent material is Fluorescein isothiocyanate.
Test kit described in 11. claim 7 or 8, wherein uses fluorescent material labelling second antibody or staphylococcal protein A in described indirect fluorescent method.
Test kit described in 12. claim 11, wherein fluorescent material is Fluorescein isothiocyanate.
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