CN105675863B - One group of multi-drug resistance tuberculosis diagnosis marker and application thereof - Google Patents
One group of multi-drug resistance tuberculosis diagnosis marker and application thereof Download PDFInfo
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- CN105675863B CN105675863B CN201610089177.2A CN201610089177A CN105675863B CN 105675863 B CN105675863 B CN 105675863B CN 201610089177 A CN201610089177 A CN 201610089177A CN 105675863 B CN105675863 B CN 105675863B
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- tuberculosis
- drug resistance
- diagnosis
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- resistance tuberculosis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
Abstract
The invention discloses one group of multi-drug resistance tuberculosis diagnosis marker and application thereof, the sequence pair of this group of multi-drug resistance tuberculosis diagnosis marker answers SEQ：ID：NO：1‑SEQ：ID：NO：8.8 antigens of this group of diagnosis of tuberculosis mark from mycobacterium tuberculosis, whether judge tested person is multi-drug resistance tuberculosis, testing result shows, the specificity of the best operating point for the protein combination diagnosis multi-drug resistance tuberculosis that the present invention is provided is 80.4%, sensitiveness is 80%, it is above the index of multi-drug resistance tuberculosis diagnosis in the prior art, and this technology is Serologic detection, sample is easily obtained, suitable for various suspicious multi-drug resistance tuberculosis, include the diagnosis of Drug resistant pulmonary tubeculosis and extrapulmonary tuberculosis.
Description
Technical field
The invention belongs to technical field of bioengineering, it is related to one group of multi-drug resistance tuberculosis diagnosis marker and application thereof.
Background technology
Mycobacterium tuberculosis (Mycobacterium Tuberculosis, Mtb) is to cause pathogen lungy, to invade
Violating lung causes pulmonary tuberculosis to be most common, can also invade its outer hetero-organization of lung and cause whole body multiple organ multiple location tuberculosis.Due to controlling
The reason such as improper is treated, increasing mycobacterium tuberculosis generates drug resistance to main flow antituberculotic, especially multi-drug resistant
It is to work as and the appearance and propagation of Drug-Resistant Mycobacterium tuberculosis have turned into the one of the main reasons that global tuberculosis epidemic situation is gone up extensively
Problem in former world tuberculosis diagnosis and treatment, severe challenge is brought to the work of global tuberculosis prevention and treatment.Multi-drug resistance tuberculosis
(multidrug-resistant tuberculosis, MDR-TB, i.e. mycobacterium tuberculosis are at least same to rifampin and isoniazid
When resistance) appearance and propagation, add the difficulty of tuberculotherapy, two wires antituberculotic used not only toxic side effect
Greatly, somewhat expensive, and curative effect can not show a candle to the pulmonary tuberculosis of medicaments insensitive, as the disaster in our times tuberculotherapy
Topic.It is more severe, extensive resistant tuberculosis (extensively drug resistant tuberculosis, XDR-
TB, i.e. mycobacterium tuberculosis are on the basis of to rifampin and isoniazid simultaneously resistance, also in fluorine quinolone druge any one
Drug-resistant is planted, and 3 kinds of two wires treating tuberculosis are injected at least 1 in medicine capreomycin sulfate, kanamycins and amikacins
Plant the tuberculosis with drug resistance) quietly occur and persistently spreading, the current disease almost pasts medical help.Therefore, in knot
Core disease epidemic situation control faces today of resistant tuberculosis, especially multi-drug resistance tuberculosis severe challenge, is clinically badly in need of efficient, valency
A kind of method of new diagnosis resistant tuberculosis honest and clean, easy to spread is extremely urgent.At present on being diagnosed with serological technique
There is not been reported for multi-drug resistance tuberculosis.
The conventional bacteriology and molecular biology method detection of relying primarily on of multi-drug resistant/extensive resistant tuberculosis of current diagnosis has
Mutation without drug resistance related gene：1st, culture-based method：It is cumbersome and time-consuming, isolate tuberculosis branch bar from clinical samples
Time of bacterium generally >=6 weeks, be unfavorable for the early of disease and find and diagnosis；2nd, PCR-single-stranded conception polymorphism point
Analysis method (PCR-SSCP methods)：Its influence factor is more (DNA fragmentation length, gel strength etc.), and repeatability is poor, in clinical work
It is not used widely in work；3rd, RT-PCR methods：High are required to testing conditions, its popularization is limited；4th, oligonucleotide probe hybridization
Method：It is a kind of fast method for detecting mycobacterium tuberculosis, but there is certain false negative rate and false positive rate, and reagent price phase
To costliness, clinical expansion is not obtained；5th, gene chips：With the time limit is short, few required sample, thermal cycle and cooling effectiveness height etc.
Advantage, but because analysis software requires high, the shortcomings of cost of manufacture of chip is high limits its application；6、GeneXpert MTB/RIF
The detection that pulmonary tuberculosis phlegm applies yin disease people and resistance to rifampin tulase can be completed in 2 hours, be that pulmonary tuberculosis quick diagnosis is carried
For facility, but in countries and regions underdeveloped, using being still restricted.
In a word, prior art do not reach it is quick, effective, inexpensive, easily diagnose multi-drug resistance tuberculosis.
The content of the invention
It is an object of the invention to the defect for overcoming above-mentioned technology presence, there is provided one group of multi-drug resistance tuberculosis diagnosis marker
And application thereof, 8 antigens of this group of diagnosis of tuberculosis mark from mycobacterium tuberculosis, whether judge tested person is multi-drug resistant
Tuberculosis, testing result shows that the protein combination that the present invention is provided diagnoses the special of the best operating point of multi-drug resistance tuberculosis
Property be 80.4%, sensitiveness is 80%, is above the index of the diagnosis of resistant tuberculosis in the prior art, and this technology is serology
Detection, sample easily obtained, it is adaptable to various suspicious multi-drug resistance tuberculosis, including Drug resistant pulmonary tubeculosis and extrapulmonary tuberculosis are examined
It is disconnected.
Its concrete technical scheme is：
One group of multi-drug resistance tuberculosis diagnosis marker, including it is anti-Rv0363cIgM antibody, anti-Rv2396IgM antibody, anti-
Rv1100IgM antibody, anti-Rv3509cIgM antibody, anti-Rv3653IgM antibody, anti-Rv0350IgG antibody, anti-Rv0865IgG resist
Body and anti-Rv2031cIgG antibody, its sequence pair answer SEQ：ID：NO：1-SEQ：ID：NO：8.
Purposes of the multi-drug resistance tuberculosis diagnosis marker of the present invention in screening multi-drug resistance tuberculosis diagnostic products.
Compared with prior art, beneficial effects of the present invention are：
The present invention is diagnosed by commercialization MtbProtTM mycobacterium tuberculosis protein matter group cDNA microarrays multi-drug resistance tuberculosis
Molecular marker, the chip covers ＞ 95% encoding gene, includes 4262 recombinant proteins of Mycobacterium tuberculosis.Clinical serum
Sample includes Normal group, active tuberculosis group and multi-drug resistance tuberculosis group, is separately reacted with chip, to examine
Survey for a certain or several antigen proteins in experimental group, the specific TB antibody (IgG or IgM) with generality.
Based on primary dcreening operation result, the more obvious 94 mycobacterium tuberculosis protein matter of selection differences and this research team are both by two-way solidifying
6 mycobacterium tuberculosis differential expression proteins that the separation of gel electrophoresis technology is obtained, self-control is small containing 100 differential expression proteins
Chip, send company's spot film, and each albumen repeats two point point systems, carries out the clinical verification of more than 200 part serum samples, finally obtains 8
It is individual diagnosis multi-drug resistance tuberculosis candidate markers (Rv0363cIgM, Rv2396IgM, Rv1100IgM, Rv3509cIgM,
Rv3653IgM, Rv0350IgG, Rv0865IgG and Rv2031cIgG).The detection of this 8 protein corresponding antibodies of Conjoint Analysis
As a result, whether be multi-drug resistance tuberculosis, as a result show if judging tested person, the present invention provides the small multi-drug resistant tuberculosis of chip auxiliary diagnosis
The specificity of the best operating point of disease is specificity 80.4%, and sensitivity 80% is above multi-drug resistance tuberculosis in the prior art
The index of diagnosis.
Brief description of the drawings
Discoveries and screening process schematic diagram of the Fig. 1 for the diagnosis of tuberculosis mark based on MtbProtTM protein-chips.
Embodiment
Technical scheme is described in more detail with specific embodiment below in conjunction with the accompanying drawings.
First, the discovery and checking of serodiagnosis mark
(1) discovery and screening of the diagnosis of tuberculosis mark based on MtbProtTM protein-chips：
1. Cleaning Principle：Buy BoChong biotech firm MtbProtTM mycobacterium tuberculosis protein matter group chips, the chip bag
Containing 4262 recombinant proteins of Mycobacterium tuberculosis, cover ＞ 95% encoding gene.Clinical serum sample include Normal group,
Active tuberculosis medicaments insensitive group, and multi-drug resistant group of active tuberculosis, separately react with chip, to detect reality
Test for a certain or several antigen proteins in group, the specific TB antibody (IgG or IgM) with generality, these
Antigen protein can characterize morbid state as diagnosis marker.
2. experimental implementation people：By chip, service group producers perform operation.
3. flow chart is as shown in Figure 1.
4. laboratory operating procedures：
1) pre- closing：Box (self-control of Guangzhou ChongBo companies) is incubated using chip, 5ml confining liquids is added, Quality Control chip will be treated
From -80 DEG C of taking-ups, face-up it is placed in incubation box；Laterally it is placed on again on side-sway shaking table (domestic), 50-60rpm, room temperature,
10min。
2) close：Confining liquid is abandoned, the new confining liquids of 5ml, side-sway shaking table 20-30rpm, room temperature 1h is rapidly added.
3) serum sample is incubated：Confining liquid is abandoned, preprepared albumen sample incubation liquid 3ml, side-sway is rapidly added
Shaking table 20-30rpm, room temperature 3h.Albumen sample incubation liquid：3ml Incubating Solutions add 30 μ l serum, and (fresh preparation is frozen in -80
Degree, is provided by patent applicant).
4) clean：Chip is taken out, the chip cleaning box for adding and there are 40ml cleaning fluids is placed in, acutely rocks 10-15 times, and
Liquid is replaced and cleaned, is acutely rocked 10-15 times again, free primary antibody is fully removed；Liquid is replaced and cleaned again, as horizontal shaker
On, 60-70rpm, 5min every time, is cleaned 3 times.
5) secondary antibody is incubated：Cleaning fluid is abandoned, preprepared secondary antibody Incubating Solution 3ml, side-sway shaking table 20- is rapidly added
30rpm, lucifuge, room temperature 45min.Secondary antibody Incubating Solution：Using anti-human IgG fluorescence secondary antibody, (549anti- human IgGs, are excited
Light 532nm, or 635anti- people IgM, exciting light 635nm, Jackson ImmunoResearch), Incubating Solution dilution antibody is extremely
Its working solution concentration, cumulative volume is 3ml.Lucifuge when secondary antibody is incubated.
6) clean：Same step " 4 ".Notice that the process answers lucifuge to operate.
7) 5min x are cleaned with ddH20 after the completion of 2 times, and rinses 10s.Notice that the process answers lucifuge to operate.
8) dry.Chip is placed in chip drier (rich difficult to understand biological, Slidewasher), centrifugal drying.
9) scan.According to scanner (rich difficult to understand biological, Luxscan 10K) working specification and operation instruction operation, ginseng is set
Number is：532nm (or correspondence wavelength of fluorescence), Power 100%, PMT value 500
10) data are extracted：By correspondence GAL File Opens, chip image and each array of GAL files are integrally alignd, pressed
Lower automatic aligning button, extracts data and preserves LPR files.
(2) checking of the multi-drug resistance tuberculosis diagnosis marker based on the small chip of designed, designed (self-control) protein
Primary dcreening operation result based on the first step, the more obvious 94 mycobacterium tuberculosis protein matter of selection differences and this research
Team both separated 6 differential expression proteins obtained by two dimensional gel electrophoresis, and second step designed, designed (self-control) contains
100 small chips of differential expression protein, serve and state company's spot film, and each albumen repeats two point point systems, carry out more than 200 part serum
The clinical verification of sample.
1. Cleaning Principle：Specific antibody (including IgG, IgM or other type antibodies) and the egg being fixed on chip
Be combined in vain, cleaning removes uncombined antibody and other oroteins, then with anti-human IgM fluorescence labelings secondary antibody (cy5 is marked,
It is presented red) and anti-human igg fluorescence secondary antibody (cy3 is marked, and green is presented) detection, signal, signal are read by Fluorescence Scanner
Power and the affinity and quantity of antibody be proportionate.
2. sample requirement：Patients serum, freezes and spends (being provided by patent applicant) in -80.
3. prepare following solution：
1) confining liquid：1ml 10%BSAm, add 9ml 1x PBSml solution, mix.
2) Incubating Solution：1x PBST 1x PBST1 solution.
3) cleaning fluid：1x PBST.
4. operating procedure：
1) pre- closing：Box is incubated using chip, 5ml confining liquids are added, by chip from -80 DEG C of taking-ups, is face-up placed in
It is incubated in box；Laterally it is placed on again on side-sway shaking table, 50-60rpm, room temperature, 10min.
2) close：Confining liquid is abandoned, the new confining liquids of 5ml, side-sway shaking table 20-30rpm, room temperature 2h is rapidly added.
3) sample incubation：Confining liquid is abandoned, respectively using 1XPBS, 0.2XPBS ddH 20q are cleaned 1 time, 5min/ times；So
Centrifugal drying afterwards.Fence special is installed and adds sample：Incubating Solution dilution is then centrifuged for drying.Fence special is installed and adds sample
This：Incubating Solution dilution is then centrifuged for drying.Fence special is installed and adds sample：Incubating Solution dilution 1: 200, and add chip
200ul volumes, side-sway shaking table 20-30rpm, 4 spend night.
4) clean：Chip is taken out and (notes the upper surface that can not be touched or scratch) and extracts fence, being placed in addition has 40ml
The chip cartridges of cleaning fluid, are acutely rocked 10-15 times, and are replaced and cleaned liquid, then are acutely rocked 10-15 times, fully remove free one
It is anti-；Liquid is replaced and cleaned again, every time, is cleaned 3 times as 60-70,5min is fully removed on horizontal shaker.
5) fluorescence labeling IgG/IgM secondary antibodies are incubated：Cleaning fluid is abandoned, preprepared secondary antibody Incubating Solution is rapidly added
3ml (1: 1000 dilution), side-sway shaking table 20-30rpm, lucifuge, room temperature 45min.Note needing lucifuge.
6) clean：Same step " 4 ".Notice that the process answers lucifuge to operate.
7) 5min x are cleaned 2 times with ddH 20 after the completion of, and rinses 10s.Notice that the process answers lucifuge to operate.
8) dry.Chip is placed in chip machine, centrifugal drying.
9) scan.According to the working specification and operation instruction of scanner, arrange parameter is：635nm, Power 100%
Power, PMT value 400；532nm, Power 100%, PMT value 400.
10) data are extracted：By correspondence GALGAL File Opens, each array of chip image GAL files is integrally alignd, pressed
Lower automatic aligning button, extracts data and preserves gpr file.
2nd, data analysis and documents and materials are consulted
1. in order to eliminate error that the systematic divergence between different samples, different chip brings, it is necessary to chip data
It is normalized.During normalization, corresponding two points of each albumen are averaged the signal responded as the albumen
Value.
2. data analysis：It it is 3 groups by sample components by diagnosis of tuberculosis meaning：Normal group, active tuberculosis medicine
Thing sensitivity group, and multi-drug resistant group of active tuberculosis, carry out statistical analysis to find multi-drug resistant group of diagnosis of active tuberculosis
Property serum molecules mark.
3. candidate markers are relatively independently filtered out first：According to response signal value of each albumen to each sample, it is assumed that
The albumen there is significant difference in the two groups of samples contrasted and in TB group samples be in stronger response (immune response compared with
By force), and its credibility is calculated, is characterized with T test P values, as P value ＜ 0.05, represent that there is significant difference；
The signal average diversity ratio of two groups of samples is calculated simultaneously, and table is come with fold change (fc, fc=log2 (TB groups/control group))
Show, fc is bigger, represent that responsiveness is higher；In addition, for the combination of candidate markers, according to the response of known packet samples
Value, based on SPSS statistical analysis softwares, builds discriminant function, and finally draws ROC curve, provides clinical diagnosis evaluation index：
Specificity and sensitivity.
4. further by SPSS statistical softwares, optimization optimum combination, and generate discriminant function (Discrimination
Function), parameter is specially：Using step-forward methods, using average as characterising parameter, the statistics based on fisher function coefficients
Analysis, serum molecules mark is preferably gone out from mark.
5. the practical problem diagnosed for multi-drug resistance tuberculosis, from great amount of samples, preferentially carries out Normal group, activity
Property the sensitive group of tubercular drugs, and multi-drug resistant group of active tuberculosis diagnosed, final to determine 8 marks combinations, point
Not Wei Rv0363cIgM, Rv2396IgM, Rv1100IgM, Rv3509cIgM, Rv3653IgM, Rv0350IgG, Rv0865IgG and
Rv2031cIgG, under the integration of discriminant function, realizes specificity 80.4%, sensitivity 80%.
The foregoing is only a preferred embodiment of the present invention, protection scope of the present invention not limited to this, any ripe
Those skilled in the art are known in the technical scope of present disclosure, the letter for the technical scheme that can be become apparent to
Altered or equivalence replacement are each fallen within protection scope of the present invention.
Claims (1)
1. one group of multi-drug resistance tuberculosis diagnosis marker, it is characterised in that including Rv0363cIgM, Rv2396IgM,
Rv1100IgM, Rv3509cIgM, Rv3653IgM, Rv0350IgG, Rv0865IgG and Rv2031cIgG, wherein, Rv0363c,
Rv2396, Rv1100, Rv3509c, Rv3653, Rv0350, Rv0865 SEQ corresponding with Rv2031c nucleotide sequence：ID：NO：
1-SEQ：ID：NO：8.
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| CN106226520B (en) * | 2016-08-31 | 2018-08-28 | 中国疾病预防控制中心传染病预防控制所 | The application of antigen of mycobacterium tuberculosis albumen Rv0865 and its B cell epitope peptide |
| CN107653313B (en) * | 2017-09-12 | 2021-07-09 | 首都医科大学附属北京胸科医院 | Application of RETN and KLK1 as tuberculosis detection markers |
| CN108918890B (en) * | 2018-07-23 | 2021-11-16 | 北京市结核病胸部肿瘤研究所 | Method for screening human ligand protein specifically targeting mycobacterium tuberculosis |
| CN108948175B (en) * | 2018-07-23 | 2020-11-20 | 首都医科大学附属北京胸科医院 | Tuberculosis protein interacting with human protein SMAD2 and application thereof |
| CN108918891B (en) * | 2018-07-23 | 2021-08-20 | 首都医科大学附属北京胸科医院 | Tuberculosis protein interacting with human protein NRF1 and application thereof |
| CN110596397B (en) * | 2019-08-09 | 2023-05-09 | 佛山市第四人民医院(佛山市结核病防治所) | Serum protein marker for tuberculosis drug resistance diagnosis and application thereof |
| CN114778656B (en) * | 2022-03-29 | 2023-02-14 | 浙江苏可安药业有限公司 | Serum metabolic marker for detecting drug-resistant tuberculosis and kit thereof |
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| WO2006060484A2 (en) * | 2004-12-01 | 2006-06-08 | Gene Therapy Systems, Inc. | Tuberculosis nucleic acids, polypeptides and immunogenic compositions |
| WO2007108829A2 (en) * | 2005-10-26 | 2007-09-27 | Gene Therapy Systems, Inc. | Tuberculosis nucleic acids, polypeptides and immunogenic compositions |
| EP2429575A1 (en) * | 2009-05-13 | 2012-03-21 | Immport Therapeutics, INC. | Compositions and methods for immunodominant antigens of mycobacterium tuberculosis |
| KR20120129927A (en) * | 2010-01-27 | 2012-11-28 | 글락소 그룹 리미티드 | Modified tuberculosis antigens |
| WO2012027200A2 (en) * | 2010-08-23 | 2012-03-01 | New York University | Epitope therapy for infectious diseases |
| CN102154527A (en) * | 2011-05-12 | 2011-08-17 | 遵义医学院附属医院 | Method for rapidly detecting multi-drug resistant tuberculosis |
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