CN110156886A - The marker and its detection method of mycobacterium tuberculosis infection and application - Google Patents
The marker and its detection method of mycobacterium tuberculosis infection and application Download PDFInfo
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Abstract
The invention discloses a kind of marker of mycobacterium tuberculosis infection and its detection method and applications, are related to diagnosis technical field.The marker of mycobacterium tuberculosis infection disclosed by the invention, which contains peptide fragment shown in SEQ ID NO.1 or 2, or contains missing, addition or the homologous peptide fragment of mutation relative to SEQ ID NO.1 or 2 with one or more amino acid.Using the polypeptide fragment as the marker of mycobacterium tuberculosis infection, with preferable specificity, sensitivity and accuracy, illustrate that the polypeptide fragment can be used for the fields such as clinical detection, diagnosis or curative effect evaluation, to combine detection, diagnosis and the curative effect evaluation etc. of bacillus infection to provide a kind of new marker.
Description
Technical field
The present invention relates to diagnosis technical fields, a kind of marker in particular to mycobacterium tuberculosis infection and
Its detection method and application.
Background technique
About tuberculosis:
Tuberculosis is that disease is infected as caused by mycobacterium tuberculosis, in addition to pulmonary tuberculosis, the other organs and tissue of body
It is likely to be involved.Pulmonary tuberculosis is respiratory infectious disease, and main droplet by patient's cough or during sneezing carries out
It propagates.It is considered as leading to dead most disease by single pathogenic bacteria at present.
According to the estimation of the World Health Organization (WHO), the infection rate of global tubercle bacillus is about one third, small part meeting
Switch to tuberculosis, it is even more serious in Asia and Africa.And active tuberculosis period from falling ill to making a definite diagnosis can about infect 10-15
People.If patient's uncomplaisance is effectively treated, the death rate is about 66%.
About resistant tuberculosis:
Using the method for microculture, if the mycobacterium tuberculosis isolated from patient is in one or more anti-tubercular drugs
It remains to grow under object existence condition, resistant tuberculosis can be diagnosed as.Four seed types can be divided into, including single drug resistance, more drug resistances, resistance to
Multiple medicine (MDR-TB) and extensive drug resistance (XDR-TB).
There are mainly two types of resistance mechanisms: chromosomal variation causes M tuberculosis complex (MTBC) to generate drug resistance,
And the mutation of atp synthase generates drug tolerance (such as shellfish reaches quinoline resistance).
Epidemiology:
2006, first appeared the report in relation to XDR-TB, in addition to isoniazid and rifampin-resistance, while to appoint
1 kind of fluoroquinolones of anticipating and (kanamycins and amikacin, curling are mould to 3 kinds of two wires anti-tubercular drug composition injections
Element) at least one kind of drug resistance.2009, there is report to claim, 15 Iranian patients all have the antituberculotic of all detections
Drug resistance.
China is one of 27 MDR-TB high burden areas in the whole world.The whole nation 2007-2008 drug resistant tuberculosis baseline tune
It looks into the results show that multi-drug resistant rate is 8.32% in lunger, extensive resistant rate is 0.68%.
Clinical diagnosis:
Existing diagnostic method includes: Chest X-rays iconography, sputum smear microscopy, microculture, tuberculin skin test, γ dry
Disturb plain release test and genetic test (Gene X-pert MTB/RIF).Wherein microculture is diagnosis goldstandard, but is detected
Period is long.Gene X-pert is the phthisical method of the resistance to rifampin of diagnosis that WHO recommends, and technical foundation is that real-time fluorescence is poly-
Synthase chain reaction (qPCR).This sensitivity is high, specificity is good, time-consuming is short, but with high costs, and to place and operator
It is required that harsh.
Existing diagnostic method includes: Chest X-rays iconography, sputum smear microscopy, microculture, tuberculin skin test, γ dry
Disturb plain release test and genetic test (Gene X-pert MTB/RIF).Wherein microculture is diagnosis goldstandard, but is detected
Period is long.Gene X-pert is the phthisical method of the resistance to rifampin of diagnosis that WHO recommends, and technical foundation is that real-time fluorescence is poly-
Synthase chain reaction (qPCR).This sensitivity is high, specificity is good, time-consuming is short, but with high costs, and to place and operator
It is required that harsh.
Countermeasure:
The measure of prevention and control MDR and XDR-TB that U.S. CDC proposes includes: to explore better therapeutic scheme, is established
Fast diagnosis method carries out specialized intervention to all types of cases of tuberculosis, and reinforce global cooperation so as to improve
Diagnosing and treating.Wherein, fast and accurately Examination and diagnosis is very important.
Summary of the invention
The purpose of the present invention is to provide a kind of markers of mycobacterium tuberculosis infection.
Another object of the present invention is to provide the applications for the substance for detecting above-mentioned marker.
Another object of the present invention is to provide a kind of kits.
Another object of the present invention is to provide a kind of methods for detecting tubercle bacillus.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of marker of mycobacterium tuberculosis infection, the marker contains SEQ ID NO.1
Or peptide fragment shown in 2, or containing there are one or more amino acid relative to SEQ ID NO.1 or 2 missing, addition or prominent
The homologous peptide fragment become.
It is of the invention the study found that there are a species specific polypeptide fragment, the polypeptide pieces in the blood of tuberculosis patient
Section is not present in healthy normal human blood, and is obtained by further analysis, using the polypeptide fragment as mycobacterium tuberculosis infection
Marker, there is preferable specificity, sensitivity and accuracy, illustrate that the polypeptide fragment can be used for clinical detection, examine
The fields such as disconnected or curative effect evaluation, to combine detection, diagnosis and the curative effect evaluation etc. of bacillus infection to provide a kind of new marker.
It is commented in addition, the substance for detecting above-mentioned marker can be used for preparing for mycobacterium tuberculosis infection detection, diagnosis of tuberculosis or curative effect
Estimate kit, provides a kind of new, kit easy to use for the detection of mycobacterium tuberculosis infection.
And by analysing in depth discovery, the amino acid sequence of the polypeptide fragment is as shown in SEQ ID NO.1 or 2.
Based on of the invention the study found that there is SEQ ID by whether there is in detection sample to be tested such as blood
The marker of peptide fragment shown in NO.1 or 2, if there is any one in above-mentioned marker as the result is shown, illustrate provide to
There are the oldness TB focus re-ignition of mycobacterium tuberculosis infection or the main body for the main body (such as people) of test sample sheet.
Wherein, the amino acid sequence of the peptide fragment of SEQ ID NO.1 is as follows:
FAQKVSEWYNYNETLDMVNSTESGQVYVNGDAL；
The amino acid sequence of the peptide fragment of SEQ ID NO.2 is as follows:
KAQGVSEWYMPNETPDMHNSTEWGPAESNIDAL。
On the other hand, the present invention provides the substances for detecting as above described in any item markers to be used for tuberculosis bar in preparation
Application in the kit of bacterium infection detection, diagnosis of tuberculosis or curative effect evaluation.
Optionally, in some embodiments of the present invention, the kit is based on any in following method or technique
One or more of combinations are to detect the marker: method, two-way gel based on antigen and antibody specificity reaction principle
Electrophoresis, chromatography and polymerase chain reaction technology, wherein the polymerase chain reaction technology is by detecting the mark
The corresponding encoding gene expression quantity of will object is to realize the detection to the marker；
Preferably, the method based on antigen and antibody specificity reaction principle is selected from following method or technique: immune colloid
Technology for gold, Western blot, enzyme-linked immunosorbent assay, immunofluorescence technique, immunodiffusion, unidirectional quantitative immunoelectrophoretic
Method and immunoturbidimetry；
Preferably, the immunodiffusion is simple immunodiffusion method or double immunodiffusion；
Preferably, the chromatography is gas chromatography or high performance liquid chromatography；
Preferably, the polymerase chain reaction technology includes: reverse transcription PCR, quantitative fluorescent PCR and droplet type number
One or more of PCR.
These methods are all based on detection of the antigen and antibody specific reaction principle realization to target substance.For the present invention
Marker pass through method or skill based on antigen Yu antibody specificity reaction principle using the specific antibody of the marker
Art (such as immune colloidal gold technique, Western blot, enzyme-linked immunosorbent assay, immunofluorescence technique, immunodiffusion,
Unidirectional quantitative immunoelectriophoresis, immunoturbidimetry etc.) detection to marker may be implemented.
It is detected according to chromatography, it can be by comparing the chromatographic peak of sample to be tested and the color of the standard items containing the marker
Spectral peak is to determine in sample to be tested whether there is the marker.Optionally, in some embodiments of the present invention, the object
Matter is the antibody for specifically binding the marker or the substance is the corresponding encoding gene expression quantity of the detection marker
Primer.
Optionally, in some embodiments of the present invention, the antibody by deposit number CGMCC NO.17483 and
The cell of the hybridoma of CGMCC NO.17484.
The hybridoma of deposit number CGMCC NO.17483 was stored in positioned at Beijing's southern exposure on March 28th, 2019
The China Committee for Culture Collection of Microorganisms's common micro-organisms center of the institute 3 of area North Star West Road 1.
The hybridoma of deposit number CGMCC NO.17484 was stored in positioned at Beijing's southern exposure on March 28th, 2019
The China Committee for Culture Collection of Microorganisms's common micro-organisms center of the institute 3 of area North Star West Road 1.
On the other hand, the present invention provides a kind of kit, the kit includes detecting as above described in any item marks
The substance of will object, the kit have below on the way any one:
(1) it is used for the detection of mycobacterium tuberculosis infection；
(2) it is used for diagnosis lungy；
(3) curative effect evaluation after patients received medication's treatment for infecting tubercle bacillus.
Optionally, in some embodiments of the present invention, the kit is based on any in following method or technique
One or more of combinations are to detect the marker:
Method or technique, two dimensional gel electrophore- sis method, chromatography based on antigen and antibody specificity reaction principle and poly-
Polymerase chain reaction technology, wherein the polymerase chain reaction technology is by detecting the corresponding encoding gene of the marker
Expression quantity is to realize the detection to the marker；
Preferably, the method or technique based on antigen and antibody specificity reaction principle is selected from following method or technique: exempting from
Epidemic disease colloidal gold technique, enzyme-linked immunosorbent assay, immunofluorescence technique, immunodiffusion, is unidirectionally quantitatively exempted from Western blot
Epidemic disease electrophoresis and immunoturbidimetry；
Preferably, the immunodiffusion is simple immunodiffusion method or double immunodiffusion；
Preferably, the chromatography is gas chromatography or high performance liquid chromatography；
The polymerase chain reaction technology includes: one in reverse transcription PCR, quantitative fluorescent PCR and droplet type digital pcr
Kind is several.
Optionally, in some embodiments of the present invention, the substance is the antibody for specifically binding the marker,
Or the substance is the primer of the corresponding encoding gene expression quantity of the detection marker.
On the other hand, the present invention provides it is a kind of detection tubercle bacillus and by non-disease diagnose for the purpose of method, it is described
Method includes:
Detection comes in the sample to be tested of autonomous agent with the presence or absence of described in any item markers as above；
If sample to be tested there are the marker, indicates the main body infection mycobacterium tuberculosis or the main body
Oldness TB focus re-ignition.
Optionally, in some embodiments of the present invention, the sample to be tested is blood.
Optionally, in some embodiments of the present invention, with the following method or technology detects the marker:
Method or technique, two dimensional gel electrophore- sis method, chromatography based on antigen and antibody specificity reaction principle and poly-
Polymerase chain reaction technology, wherein the polymerase chain reaction technology is by detecting the corresponding encoding gene of the marker
Expression quantity is to realize the detection to the marker；
Preferably, the method or technique based on antigen and antibody specificity reaction principle is selected from following method or technique: exempting from
Epidemic disease colloidal gold technique, enzyme-linked immunosorbent assay, immunofluorescence technique, immunodiffusion, is unidirectionally quantitatively exempted from Western blot
Epidemic disease electrophoresis and immunoturbidimetry；
Preferably, the immunodiffusion is simple immunodiffusion method or double immunodiffusion；
Preferably, the chromatography is gas chromatography or high performance liquid chromatography；
The polymerase chain reaction technology includes: one in reverse transcription PCR, quantitative fluorescent PCR and droplet type digital pcr
Kind is several.
Optionally, in some embodiments of the present invention, the method is for the purpose of the diagnosis of non-disease or treatment.
Optionally, in some embodiments of the present invention, the main body behaviour or inhuman mammal.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the tubercle bacillus testing result of active stage tuberculosis sample in embodiment.
Fig. 2 is the tubercle bacillus testing result of nonmobile phase tuberculosis sample in embodiment.
Fig. 3 is the ROC curve of activity tuberculosis.
Fig. 4 is the ROC curve of inactive tuberculosis.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
(1) hybridoma is prepared
Use antibody protein (using peptide fragment shown in SEQ ID NO.1 or 2 as antigen) to mix respectively with adjuvant CFA, makes
Standby immunogene, injects mouse peritoneal；
14th day extraction mouse tail blood (Balb/C mouse: the Balb/C of SPF (Specific Pathogen Free) rank
Mouse)；
Using albumen coated elisa plate (1 μ g/mL), 100 μ L, 4 DEG C of reaction overnights are added in every hole；
Using PBS solution board-washing 3 times, 1hr is closed in room temperature using 5%milk-PBS；
Then it using after PBS solution board-washing 1 time, is added and uses the diluted mouse tail blood of 5%milk-PBS solution gradient, room
Temperature reaction 1hr；Then it uses PBS solution board-washing 3 times, and after being patted dry, the goat-anti that the diluted HRP label of 1:2000 is added is small
Mouse IgG (Fc) secondary antibody reacts at room temperature 1hr；
It after PBS solution board-washing 5 times, pats dry, isometric A liquid and B liquid is added, is protected from light, reacts 20min under room temperature；
Then 50 μ L terminate liquids are added, read OD450 value after mixing in microplate reader；
Choose immune success rate mouse, be used as cell fusion agent using PEG (polyethylene glycol), make immune mouse boosting cell with
Murine myeloma cell fusion；
Under the action of HAT selective medium, only allows and merge successful Growth of Hybridoma Cell.Selection secretion is positive anti-
The hybridoma of body carries out preservation.
It is stored on March 28th, 2019 positioned at north for the positive hybridoma cell of peptide fragment shown in SEQ ID NO.1
The China Committee for Culture Collection of Microorganisms's common micro-organisms center of the institute 3 of the Chaoyang District Jing Shi North Star West Road 1, preservation are compiled
Number CGMCC NO.17483；
It is stored on March 28th, 2019 positioned at north for the positive hybridoma cell of peptide fragment shown in SEQ ID NO.2
The China Committee for Culture Collection of Microorganisms's common micro-organisms center of the institute 3 of the Chaoyang District Jing Shi North Star West Road 1, preservation are compiled
Number CGMCC NO.17484.
Cell substep is freezed, 30 DEG C → -70 DEG C → liquid nitrogen.It is spare.
Wherein, it antigen is done with peptide fragment shown in SEQ ID NO.1 carries out immune obtained hybridoma deposit number and be
CGMCC NO.17483.Antigen, which is done, with peptide fragment shown in SEQ ID NO.2 carries out immune hybridoma deposit number obtained
For CGMCC NO.17484.
(2) antibody purification
The coupling of 1mL is taken to have the column material of G-protein to be added in void column, after cleaning using PBS solution, by 2mL ascites
With upper prop after the PBS dilution of 8mL, then flowing through liquid, upper prop is primary again；
Then it is eluted using the glycine elution liquid of pH 2.7, every 1mL eluent collects a pipe and (is previously added 100 μ
L neutralizer), 5 pipes are collected altogether；
Then it is eluted using the glycine elution liquid of pH 1.9, every 1mL eluent collects a pipe and (is previously added 300 μ
L neutralizer), 3 pipes are collected altogether；
Then OD280 reading is carried out to each pipe eluent respectively, the eluent by OD280 greater than 0.5 mixes, and mixes
The OD280 for redeterminating mixed liquor after conjunction again, according to 1.4 coefficient calculating antibody concentration；Antibody concentration=OD280/1.4.
Conclusion: two antibody cross match overall trends are preferable, can be used for later period kit developing.
(3) antibody is prepared
Recovery: the hybridoma (CGMCC NO.17483 or CGMCC NO.17484) frozen is taken out, immerses 37 immediately
In DEG C -40 DEG C of water-baths, it is made to melt rapidly, recover.
1. taking out 25cm2Culture bottle, 75% alcohol disinfecting, removes sealed membrane, is put into 37 DEG C, 5%CO2In cell incubator.
It stands 6-8 hours or stays overnight, to stablize cell state.
2. when cell reach 80% converge when be ready for secondary culture.
3. cell passes on
1) 25cm is sucked out2It is primary to clean cell with PBS for culture medium in culture bottle；
2) addition 0.125% tryptic digestive juice about 1mL is into culture bottle, 37 DEG C of warm bath 3min or so；It is inverted micro-
Under mirror；
Observation inhales after cell retraction is rounded and abandons digestive juice, adds complete culture solution and terminate digestion；
3) mixing is gently blown and beaten with suction pipe, is carried out inoculation passage in the ratio appropriate such as 1:2 or 1:3, is then supplemented fresh
It is complete；
Culture medium is put into 37 DEG C, 5%CO to 5mL2It is cultivated in cell incubator；
4) after cell is completely adherent, culture observation.Complete medium fresh every replacement in 2-3 days later.
5) suspension cell passes on: cell suspension is transferred in sterile centrifugation tube, 1000rpm is centrifuged 5min, and it discards supernatant,
Fresh culture medium is added, precipitating is carefully dispelled with suction pipe, cell suspension is made, cell suspension is inoculated into other 2 respectively
In~3 cell bottles, fresh culture is added, sets 37 DEG C of incubator cultures.
6) culture supernatant, centrifugation removal cell and its fragment are collected, can be obtained required monoclonal antibody.
Embodiment 2
1 has the marker of any one peptide fragment in SEQ ID NO.1 or 2 in tuberculosis using immune colloidal gold technique detection
Expression in bacillus infection or tuberculosis patient in blood
Take the blood of the patient or normal healthy controls that are confirmed as mycobacterium tuberculosis infection by hospital, total sample size 1048,
In, the TB clinical samples amount 375 of active stage, nonmobile phase TB sample size 513, nonmobile phase TB sample size 513, non-TB
Clinical samples amount 148, non-disease healthy person sample size 112.It is utilized respectively by the miscellaneous of deposit number CGMCC NO.17483
The peptide fragment marker of antibody test SEQ ID NO.1 secreted by oncocyte is handed over, the hybridoma using CGMCC NO.17484 is thin
The peptide fragment marker of antibody test SEQ ID NO.2 secreted by born of the same parents.The antibody of specificity is fixed on film with ribbon, glue
Body gold labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be examined is added to the sample pad of test strips one end
It after upper, moves forward, reacts to each other after dissolving the colloid gold label reagent on bonding pad, then be moved to fixation through capillary action
Antigen or antibody region when, the conjugate of object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and gathers
Collection takes in detection, can observe by the naked eye colour developing result.
As a result as (figure Chinese and English abbreviation is respectively STP: strong positive by Fig. 1 and Fig. 2；WTP: weakly positive；FP: false positive；FN:
False negative；TN: true negative) shown in, tubercle bacillus detection, sample size 1048, the TB clinical samples amount 375 of active stage: strong
It is positive: 89.5%, weakly positive: 4.7%, it is negative: 5.8%.Nonmobile phase TB sample size 513: strong positive: 14.3%, weak sun
Property: 8.2%, it is negative: 77.5%.Non- TB clinical samples amount 148: strong positive: 3.0%, weakly positive: 0.38%, negative:
93.2%.Non-disease healthy person sample size 112: strong positive: 1.04%, weakly positive: 0%, it is negative: 98.96%.
According to above-mentioned testing result, Receiver operating curve's analysis is carried out, as a result sees Fig. 3 and Fig. 4.
Fig. 3 is active tuberculosis ROC curve (testee's indicatrix), and Fig. 4 is inactive tuberculosis ROC curve, knot
Fruit shows the sensitivity and specificity to active tuberculosis and inactive tuberculosis.Sensitivity is 94.13%, and specificity is
84.24%.This is the result shows that can be used for distinguishing tuberculosis bar using the marker of any one peptide fragment in SEQ ID NO.1 or 2
Bacterium the infected and healthy person.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Chengdu Ren Qin Biotechnology Co., Ltd
<120>marker of mycobacterium tuberculosis infection and its detection method and application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 33
<212> PRT
<213>artificial sequence
<400> 1
Phe Ala Gln Lys Val Ser Glu Trp Tyr Asn Tyr Asn Glu Thr Leu Asp
1 5 10 15
Met Val Asn Ser Thr Glu Ser Gly Gln Val Tyr Val Asn Gly Asp Ala
20 25 30
Leu
<210> 2
<211> 33
<212> PRT
<213>artificial sequence
<400> 2
Lys Ala Gln Gly Val Ser Glu Trp Tyr Met Pro Asn Glu Thr Pro Asp
1 5 10 15
Met His Asn Ser Thr Glu Trp Gly Pro Ala Glu Ser Asn Ile Asp Ala
20 25 30
Leu
Claims (10)
1. a kind of marker of mycobacterium tuberculosis infection, which is characterized in that the marker contains shown in SEQ ID NO.1 or 2
Peptide fragment, or contain the homologous of missing, addition or the mutation relative to SEQ ID NO.1 or 2 with one or more amino acid
Peptide fragment.
2. detect the substance of marker described in claim 1 preparation for mycobacterium tuberculosis infection detection, diagnosis of tuberculosis or
Application in the kit of curative effect evaluation.
3. application according to claim 2, which is characterized in that the kit is based on any in following method or technique
One or more of combinations are to detect the marker:
Method, two dimensional gel electrophore- sis method, chromatography and polymerase chain reaction based on antigen Yu antibody specificity reaction principle
Answer technology, wherein the polymerase chain reaction technology is by detecting the corresponding encoding gene expression quantity of the marker with reality
Now to the detection of the marker；
Preferably, the method based on antigen and antibody specificity reaction principle is selected from following method or technique: immune colloid gold skill
Art, Western blot, enzyme-linked immunosorbent assay, immunofluorescence technique, immunodiffusion, unidirectional quantitative immunoelectriophoresis with
And immunoturbidimetry；
Preferably, the immunodiffusion is simple immunodiffusion method or double immunodiffusion；
Preferably, the chromatography is gas chromatography or high performance liquid chromatography；
Preferably, the polymerase chain reaction technology includes: in reverse transcription PCR, quantitative fluorescent PCR and droplet type digital pcr
One or more.
4. application according to claim 3, which is characterized in that the substance is the anti-of the specific binding marker
Body or the substance are the primer of the corresponding encoding gene expression quantity of the detection marker.
5. application according to claim 4, which is characterized in that the antibody by deposit number be CGMCC NO.17483 or
The hybridoma of CGMCC NO.17484 is secreted to obtain.
6. a kind of kit, which is characterized in that the kit includes the substance for detecting marker as described in claim 1,
The kit have below on the way any one:
(1) it is used for the detection of mycobacterium tuberculosis infection；
(2) it is used for diagnosis lungy；
(3) curative effect evaluation after patients received medication's treatment for infecting tubercle bacillus.
7. kit according to claim 6, which is characterized in that the kit is based on appointing in following method or technique
One or more of combinations anticipate to detect the marker:
Method or technique, two dimensional gel electrophore- sis method, chromatography and polymerase based on antigen Yu antibody specificity reaction principle
Chain reaction technology, wherein the polymerase chain reaction technology is by detecting the corresponding encoding gene expression of the marker
Amount is to realize the detection to the marker；
Preferably, the method or technique based on antigen and antibody specificity reaction principle is selected from following method or technique: immune glue
Body technology for gold, Western blot, enzyme-linked immunosorbent assay, immunofluorescence technique, immunodiffusion, unidirectional quantitative immunological electricity
Swimming method and immunoturbidimetry；
Preferably, the immunodiffusion is simple immunodiffusion method or double immunodiffusion；
Preferably, the chromatography is gas chromatography or high performance liquid chromatography；
Preferably, the polymerase chain reaction technology includes: in reverse transcription PCR, quantitative fluorescent PCR and droplet type digital pcr
One or more.
8. kit according to claim 7, which is characterized in that the substance is the anti-of the specific binding marker
Body or the substance are the primer of the corresponding encoding gene expression quantity of the detection marker.
9. it is a kind of detection mycobacterium tuberculosis infection and by non-disease diagnose for the purpose of method, which is characterized in that the described method includes:
Detection to whether there is marker as described in claim 1 in the sample to be tested of autonomous agent；If sample to be tested exists
The marker then indicates the main body infection mycobacterium tuberculosis or the oldness TB focus re-ignition of the main body.
10. according to the method described in claim 9, it is characterized in that, the sample to be tested is blood.
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| CN101661044A (en) * | 2008-08-27 | 2010-03-03 | 复旦大学附属华山医院 | Diagnostic reagent of tuberculosis and kit |
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