CN106435009A - Application of TUBB2A used as blood diagnostic marker - Google Patents
Application of TUBB2A used as blood diagnostic marker Download PDFInfo
- Publication number
- CN106435009A CN106435009A CN201611222765.5A CN201611222765A CN106435009A CN 106435009 A CN106435009 A CN 106435009A CN 201611222765 A CN201611222765 A CN 201611222765A CN 106435009 A CN106435009 A CN 106435009A
- Authority
- CN
- China
- Prior art keywords
- tubb2a
- sample
- gene
- instrument
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
Abstract
The invention discloses application of TUBB2A used as a blood diagnostic marker, and particularly discloses application of TUBB2A used as a marker for diagnosing Budd-Chiari syndromes. The application has the advantages that as proved by high-throughput sequencing and PCR (polymerase chain reaction) experiments, TUBB2A genes are differentially expressed in blood of patients with the Budd-Chiari syndromes and blood of normal persons, and accordingly the TUBB2A genes can be used as molecular markers for diagnosing the Budd-Chiari syndromes; a novel method for diagnosing the Budd-Chiari syndromes is discovered and is non-invasive, diseases can be judged at early stages, and accordingly the application is favorable for increasing the survival rates of the patients.
Description
Technical field
The present invention relates to medical diagnosis on disease field, more particularly it relates to TUBB2A is in Bgudd-Chiari Syndrome diagnosis
Application.
Background technology
Budd-Chiari syndrome (Budd-Chiari syndrome, BCS) be by hepatic vein and (or) its opening with epimere cavity of resorption
Property portal hypertension after a kind of liver with the characteristics of what vein occlusion pathological changes caused be often accompanied by inferior vena cava syndrome.From global model
From the point of view of enclosing, BCS sickness rate is relatively low, and its cause of disease has obvious regional difference, and western countries are common with hepatic venous obstruction type BCS, greatly
There are the clear and definite cause of disease, such as oral contraceptive, gestation, blood disease etc. more, have document report, hepatic vein type BCS and gene
The high blood coagulation state that mutation causes, Hepatic venous thrombosis are relevant；And in Asia and Southern African region then with IVC obstructive type BCS
Common, pathogenic factor is mostly unclear.
BCS can cause liver injury, portal hypertension, even result in liver cirrhosis, liver failure, upper gastrointestinal hemorrhage etc. serious
Complication, Nature prognosis extreme difference, survival rate is very low within 5 years, non-operative treatment mortality rate height.The clinical manifestation of BCS patient's early stage is hidden
Hide, no specificity, and lack early stage diagnosis and treatment effective ways, patient when medical more belong to late period, complicated clinical manifestation is various, hold
Easily cause mistaken diagnosis, wrong treatment.The domestic research to BCS is focused mostly in terms of clinical treatment, by contrast epidemiology and etiology
Research delayed, lack the whole nation generaI investigation and Study of Etiology.Therefore, carry out BCS Study of Etiology in a deep going way, improve the early stage of BCS
Diagnosis are the key issues for improving BCS survival of patients and prognosis.
Content of the invention
The present invention relates to a kind of diagnosis marker of Bgudd-Chiari Syndrome, the present invention is experimentally confirmed and suffers from Bgudd-Chiari Syndrome
In the blood of person, TUBB2A gene mRNA content is significantly higher than normal population, and therefore the property of the differential expression of TUBB2A gene makes
Which becomes the molecular marker that can be used to diagnose whether experimenter suffers from Bgudd-Chiari Syndrome.
Specifically, the invention provides application of the product of detection TUBB2A in Bgudd-Chiari Syndrome diagnostic tool is prepared.
Further, the product of the detection TUBB2A includes the product of TUBB2A gene expression amount in detection sample.
Further, the product of the detection TUBB2A include can in quantitative sample TUBB2A gene mRNA product,
And/or can in quantitative sample TUBB2A albumen product.
In the quantitative sample of the present invention product of TUBB2A gene mRNA can based on the known method using nucleic acid molecules come
Play its function：As PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA are micro-
Array, ASO method, high-flux sequence platform etc..Can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
The nucleic acid being included in the said goods can be obtained by chemosynthesis, or passes through to contain from biomaterial preparation
Expecting the gene of nucleic acid, then it is expanded using the primer for being designed for amplification expectation nucleic acid to obtain.
Further, the PCR method is known method, for example ARMS (Amplification Refractory
Mutation System, abruptly-changing system is not answered in amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR method and PCR-SSCP (single strand conformation polymorphism) method are detecting.
Described can include the specific amplified used in real-time quantitative PCR by the product of TUBB2A gene mRNA in quantitative sample
The primer of TUBB2A gene, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
The primer that product includes can be prepared by chemosynthesis, by using those skilled in the art will know that side
Method is suitably designed with reference to Given information, and is prepared by chemosynthesis.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemosynthesis, by using this
The method that skilled person knows appropriately is designed with reference to Given information, and is prepared by chemosynthesis, or can be led to
Cross and prepare containing the gene for expecting nucleotide sequence from biomaterial, and expanded using the primer for being designed for expanding expectation nucleotide sequence
Increase it to prepare.
In the quantitative sample of the present invention product of TUBB2A albumen can based on using antibody known method playing its work(
Energy：For example, it is possible to including ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
In the quantitative sample of the present invention product of TUBB2A albumen include to specifically bind the antibody of TUBB2A albumen or its
Fragment.Antibody or its fragment of any structure, size, immunoglobulin class, origin etc. can be used, as long as it combines target egg
White matter.The antibody that the detection product of the present invention includes or its fragment can be monoclonal or polyclones.Antibody piece
Section refers to retain peptide of the antibody to an antibody part (Partial Fragment) of the binding activity of antigen or containing an antibody part.Antibody piece
Section can include F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization
Change V area (double antibody) or the peptide containing CDR.In the quantitative sample of the present invention, the product of TUBB2A albumen can include that coding is anti-
The detached nucleic acid of the aminoacid sequence of body or Encoding Antibody Fragment, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be by well known to a person skilled in the art method be obtaining.For example, prepare and retain target all or in part
The polypeptide of protein or integration encode the mammalian cell expression vector of their polynucleotide as antigen.Exempted from using antigen
After epidemic disease animal, from through immunity animal adaptive immune cell and merge myeloma cell to obtain hybridoma.Then from hybridization
Antibody collected by tumor culture.Finally can by using be used as TUBB2A albumen of antigen or part thereof to obtain antibody reality
Apply antigenic specificity purification to obtain the monoclonal antibody for TUBB2A albumen.Polyclonal antibody is prepared as follows can：With with
Identical antigen-immunized animal, from the animal collect blood sample through immunity, isolates serum from blood, then makes above
Implement antigenic specificity with above-mentioned antigen to serum to purify.Can pass through with the antibody of ferment treatment acquisition or by using acquisition
The sequence information of antibody is obtaining antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.For example, may be used
With following fluorescent marker protein or peptide：Clean protein or peptide with phosphate buffer, add with DMSO, buffer agent, etc. standard
Standby dyestuff, then mixed solution, places 10 minutes then at room temperature.In addition, labelling can commodity in use labelling kit, all
As biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark test kit such as alkali phosphatase enzyme mark test kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling test kit such as peroxidase mark
Note test kit-NH2, peroxidase labelling test kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2,
B- phycoerythrin labelling kit-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- label protein mark
Note test kit (Funakoshi Corporation).For correct labeling, it is possible to use suitable instrument is detecting through labelling
Antibody or its fragment.
Sample as the detection product according to the present invention, it is possible to use the tissue sample for for example obtaining from biopsy experimenter
Or fluid.Sample is not particularly limited, as long as it is suitable to the measure of the present invention；For example, it can include tissue, blood, blood plasma,
Serum, lymph fluid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, the sample source is in blood.
Further, the product of the quantitative TUBB2A gene or TUBB2A albumen can be detection TUBB2A gene or
The reagent of TUBB2A albumen, can also be the test kit comprising the reagent, chip, reagent paper etc., or using the examination
The high-flux sequence platform of agent.
Present invention also offers a kind of instrument of diagnosis Bgudd-Chiari Syndrome, the instrument can detect TUBB2A.
Further, the instrument being capable of TUBB2A gene expression amount in detection sample.
Further, the instrument include can in quantitative sample TUBB2A gene mRNA reagent, and/or being capable of quantitative sample
The reagent of TUBB2A albumen in product.
Further, described can be used in the reagent of TUBB2A gene mRNA be real-time quantitative PCR in quantitative sample
The primer of specific amplified TUBB2A gene, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.Described can
In quantitative sample, the reagent of TUBB2A albumen includes the antibody for being combined with TUBB2A protein-specific.
Further, the instrument of the diagnosis Bgudd-Chiari Syndrome includes but is not limited to chip, test kit, reagent paper or high flux
Microarray dataset；High-flux sequence platform is a kind of instrument of special diagnosis Bgudd-Chiari Syndrome, with high throughput sequencing technologies
Development, will become to the structure of the gene expression profile of a people and very easily work.By contrasting Disease and normal person
The gene expression profile of group, the exception for easily analyzing which gene is related to disease.Therefore, know in high-flux sequence
The exception of the TUBB2A gene purposes that fall within TUBB2A related to Bgudd-Chiari Syndrome, equally protection scope of the present invention it
Interior.
The aminoacid recognized by anti-TUBB2A antibody or its fragment used in the detection product of the present invention, diagnostic tool
Number is not particularly limited, as long as antibody can be in conjunction with TUBB2A.
Present invention also offers a kind of method of diagnosis Bgudd-Chiari Syndrome, methods described comprises the steps：
(1) sample of experimenter is obtained；
(2) expression of TUBB2A gene or albumen in Samples subjects is detected；
(3) whether the expression of the TUBB2A gene for measuring or albumen is associated with the illness of experimenter.
(4) compared with normal control, the expression of TUBB2A gene or albumen is raised, then the experimenter is judged and has
Tendency with Bgudd-Chiari Syndrome or suffered from Bgudd-Chiari Syndrome, or Bgudd-Chiari Syndrome patient be judged as recurrence or
Person Bgudd-Chiari Syndrome patient is judged as prognosis malas.
In the context of the present invention, " diagnosis Bgudd-Chiari Syndrome " includes to judge whether experimenter adds synthesis with cloth
Levy, judge that experimenter whether there is risk with Bgudd-Chiari Syndrome, judge whether Bgudd-Chiari Syndrome patient has been recurred, judged
The prognosis of Bgudd-Chiari Syndrome patient.
The advantages of the present invention：
The present invention is found that a kind of molecular marker of diagnosis Bgudd-Chiari Syndrome, can be in cloth using the molecular marker
Plus the early stage that syndrome occurs can judge, there is provided the survival rate of patient.
Description of the drawings
Fig. 1 shows the cartogram for detecting TUBB2A gene differential expression situation using QPCR in mRNA level in-site.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1 screens difference expression gene
1st, object of study and sample collection
(1) object of study：3 Bgudd-Chiari Syndrome patients and 5 Healthy Volunteers.
(2) sample collection：Object of study at least 12h on an empty stomach is required, in m seq 7：00～8：Under 00 room temperature, extract
10ml venous blood adds 3 times of volume erythrocyte cracked liquids in ethylenediaminetetraacetic acid (EDTA) anticoagulant tube, and after mixing, room temperature places 10
Minute, 10,000rpm centrifugations 1 minute.Thoroughly inhale and supernatant is abandoned, collect leukocyte cell pellet.White thin per 100-200 μ l blood collecting
Born of the same parents' precipitation adds 1ml TRIzol, and -80 DEG C frozen standby.
2nd, the acquisition of total serum IgE
Conventionally using TRIzol, the total serum IgE in blood leucocyte is extracted.
3rd, RNA concentration and purity testing
NanoDrop1000 spectrophotometer detects RNA sample, the sample requirement of RNA-seq sequencing：OD260/OD280 is
1.8-2.2.
4th, the quality analysiss (Agilent Technologies 2100Bioanalyzer) of RNA sample
Agilent Technologies 2100Bioanalyzer detects RNA sample quality, observes 28S rRNA and 18S
RRNA master tape substantially, is no degraded, RNA Perfection Index is qualified, concentration reaches and meeting for requirement wanting for cDNA library structure is sequenced
Ask, can be used for library construction and sequencing.
5th, high flux transcript profile sequencing
(1) RNA-seq read positioning
First by low-quality read remove obtain clean read, then using TopHat v1.3.1 will clean fragment and
UCSC H.sapiens reference gene group (hg19) is mated, the index of the advance structure of H.sapiens UCSC hg19 version
Download from TopHat homepage, and as reference gene group, when being mated with genome using TopHat, it is allowed to each read (acquiescence
To 20) there are multiple coupling sites, most 2 mispairing.TopHat sets up possible according to exon region and GT-AG shear signal
Shearing site storehouse, navigates on genome according to the read that these shearing site storehouses are not navigated to genome.We use
The system default parameter of TopHat method.
(2) transcript abundance assessment
The read file for matching is by Cufflinks v1.0.3 process, and Cufflinks v1.0.3 is by RNA-seq piece
Hop count mesh is standardized calculating the relative abundance of transcript.FPKM value refers to specific per matching in 1,000,000 sequencing fragments
The segment number of the exon region of gene 1kb length.The confidence interval of FPKM estimated value is calculated by Bayesian inference method.
The GTF comment file of the reference that Cufflinks is used downloads (Homo_ from Ensembl data base
sapiens.GRCh37.63.gtf).
(3) detection of difference expression gene
The original document for mating by the Ensembl GTF file of download and by TopHat is transferred to Cuffdiff,
Cuffdiff re-evaluates the gene expression abundance of the transcript that lists in GTF file using original matching files, detects difference table
Reach.Only has q value ＜ 0.01 in Cuffidff output, test shows to be considered as successfully more just differential expression.
6th, result
Gene expression difference relatively in Bgudd-Chiari Syndrome patient and normal human blood, has 405 difference expression genes,
317 rises, 88 downwards.Wherein, compared with normal person, on TUBB2A gene is expressed in Bgudd-Chiari Syndrome blood samples of patients
Adjust, mRNA relative expression quantity is that 4.71 ± 0.93, difference has statistical significance (P<0.05).
The difference expression gene that the checking of 2 large sample of embodiment is filtered out
TUBB2A gene is selected to be verified.
1st, sample collection
The peripheral blood sample that method according to embodiment 1 collects Bgudd-Chiari Syndrome patient and normal population is each 50.
2nd, verified in mRNA level in-site
2.1 extract blood total serum IgE
Step is with embodiment 1.
2.2 reverse transcription
Reverse transcription uses Primescript 1stStrand cDNA synthesis kit test kit, operating procedure is as follows
Carry out：
(1) following reaction liquid is added in microcentrifugal tube, as shown in table 1：
1 reaction liquid of table
| Reagent | Dosage |
| RNA | 2.0μg |
| dNTP | 1.0μl |
| Oligo(dT) | 2.0μl |
| Rnase free dH2O | Add to 10.0 μ l |
(2) 70 DEG C of incubation 5min, are rapidly cooled to 4 DEG C；
Following reaction reagent is added in microcentrifugal tube, makes reaction system：
The preparation of 2 reaction system of table
| Reagent | Dosage |
| 5x1st Strand Synthesis Buffer | 4.0μl |
| PrimeScript RTase | 1.0μl |
| RNase Inhibitor | 1.0μl |
| Rnase free dH2O | 4.0μl |
Gently shake, after quick centrifugation, 42 DEG C of reaction 1h, 70 DEG C of 10min terminating reactions, 4 DEG C of coolings, -20 DEG C of preservations.
Using SYBP Premix Ex TapTMII test kit, is carried out in Eppendorf Real-time PCR analyser,
Concrete operations are as follows：
(1) following PCR reactant liquor is prepared on ice：
The preparation of table 3PCR reactant liquor
| Reagent | Dosage |
| SYBR | 10.0μl |
| Forward primer | 1.0μl |
| Reverse primer | 1.0μl |
| cDNA | 2.0μl |
| ddH2O | 6.0μl |
| Total amount | 20.0μl |
Primer sequence design is as follows：
TUBB2A gene：
5’-GCATCCTTAGTGAACTTCT-3’(SEQ ID NO.1)；
5’-CCACATCATTACATCAACAG-3’(SEQ ID NO.2)
β-actin：
5’-GTGGGGCGCCCCAGGCACCA-3’(SEQ ID NO.3)；
5’-CTCCTTAATGTCACGCACGATTT-3’(SEQ ID NO.4)
(2) upper machine, executes following programs：95 DEG C of denaturations 3min；95 DEG C of degeneration 15s.60 DEG C of annealing 15s, 72 DEG C of extensions
20s, totally 40 circulations.
As a result relative quantification method, using formula 2 are used-△△ctCalculate.Experiment is repeated 3 times.
△ ct=ct (A)-ct (β-actin)
△ △ ct=△ ct (experimental group)-△ ct (matched group)
As a result as shown in figure 1, compared with normal control population, the mRNA of TUBB2A gene in Bgudd-Chiari Syndrome blood samples of patients
Level is substantially raised, and difference has statistical significance (P<0.05).
The preparation of 2 Bgudd-Chiari Syndrome diagnostic kit of embodiment
According to the dependency of TUBB2A gene and Bgudd-Chiari Syndrome, can by detect the expression of TUBB2A gene come
Diagnosis Bgudd-Chiari Syndrome, accordingly the invention provides a kind of diagnose the examination of Bgudd-Chiari Syndrome based on detection TUBB2A gene expression
Agent box, the component in the diagnostic kit is as follows：SYBR Green polymerase chain reaction system；Amplification TUBB2A gene and β-
The primer pair of actin gene.The forward primer sequence of amplification TUBB2A gene is 5 '-GCATCCTTAGTGAACTTCT-3 ', instead
It is 5 '-CCACATCATTACATCAACAG-3 ' to primer sequence；Amplification β-actin forward primer sequence be 5 '-
GTGGGGCGCCCCAGGCACCA-3 ', reverse primer sequences are 5 '-CTCCTTAATGTCACGCACGATTT-3 '.SYBR
Green polymerase chain reaction system includes PCR buffer, dNTPs, SYBR Green fluorescent dye.PCR buffer components
For：25mM KCL, 2.5mM MgCL2、200mM(NH4)2SO4.
The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these improve and modification will be also fallen in the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Beijing Yang Shen biology information technology company limited
<120>TUBB2A is used as the purposes of Blood diagnosis mark
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
gcatccttag tgaacttct 19
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ccacatcatt acatcaacag 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
gtggggcgcc ccaggcacca 20
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
ctccttaatg tcacgcacga ttt 23
Claims (10)
1. application of the product of detection TUBB2A in diagnosis Bgudd-Chiari Syndrome instrument is prepared.
2. application according to claim 1, it is characterised in that the product of the detection TUBB2A is included in detection sample
The product of TUBB2A gene expression amount.
3. application according to claim 2, it is characterised in that the product of the detection TUBB2A includes being capable of quantitative sample
The product of middle TUBB2A gene mRNA, and/or can in quantitative sample TUBB2A albumen product.
4. application according to claim 3, it is characterised in that described can in quantitative sample TUBB2A gene mRNA examination
Agent includes the primer of the specific amplified TUBB2A gene used in real-time quantitative PCR, the primer sequence such as SEQ ID NO.1 and
Shown in SEQ ID NO.2；Described the reagent of TUBB2A albumen can include to specifically bind TUBB2A albumen in quantitative sample
Antibody.
5. the application according to any one of claim 2-4, it is characterised in that the sample source is in blood.
6. a kind of instrument of diagnosis Bgudd-Chiari Syndrome, it is characterised in that the instrument includes being capable of TUBB2A in detection sample
Instrument.
7. instrument according to claim 6, it is characterised in that the instrument includes being capable of TUBB2A gene in detection sample
The instrument of expression.
8. instrument according to claim 7, it is characterised in that the instrument includes being capable of TUBB2A in quantitative sample
The reagent of gene mRNA, and/or can in quantitative sample TUBB2A albumen reagent.
9. instrument according to claim 8, it is characterised in that described can in quantitative sample TUBB2A gene mRNA examination
Agent is the primer of the specific amplified TUBB2A gene used in real-time quantitative PCR, the primer sequence such as SEQ ID NO.1 and
Shown in SEQ ID NO.2；Described the reagent of TUBB2A albumen can include to specifically bind TUBB2A albumen in quantitative sample
Antibody.
10. the application according to any one of claim 6-9, it is characterised in that the sample source is in blood.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611222765.5A CN106435009B (en) | 2016-12-27 | 2016-12-27 | Purposes of the TUBB2A as Blood diagnosis marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611222765.5A CN106435009B (en) | 2016-12-27 | 2016-12-27 | Purposes of the TUBB2A as Blood diagnosis marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106435009A true CN106435009A (en) | 2017-02-22 |
| CN106435009B CN106435009B (en) | 2019-08-23 |
Family
ID=58215950
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611222765.5A Active CN106435009B (en) | 2016-12-27 | 2016-12-27 | Purposes of the TUBB2A as Blood diagnosis marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106435009B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101778637A (en) * | 2007-03-05 | 2010-07-14 | 新南创新私人有限公司 | methods for detecting and modulating the sensitivity of tumour cells to anti-mitotic agents |
-
2016
- 2016-12-27 CN CN201611222765.5A patent/CN106435009B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101778637A (en) * | 2007-03-05 | 2010-07-14 | 新南创新私人有限公司 | methods for detecting and modulating the sensitivity of tumour cells to anti-mitotic agents |
Non-Patent Citations (3)
| Title |
|---|
| V. PARADIS等: "DIFFERENTIAL GENE EXPRESSION PROFILE IN BUDD-CHIARI SYNDROME AND CIRRHOSIS", 《JOURNAL OF HEPATOLOGY》 * |
| V. PARADIS等: "Quantitative gene expression in Budd-Chiari syndrome: a molecular approach to the pathogenesis of the disease", 《GUT》 * |
| 都晓英: "应用基因芯片表达谱筛选MOVC型布加综合征患者外周血白细胞相关基因的研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106435009B (en) | 2019-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105296659B (en) | A kind of gene marker relevant to cerebral arterial thrombosis | |
| CN106636413B (en) | It is a kind of for diagnosing the molecular marker of asthma | |
| CN105925714A (en) | Molecular marker for diagnosing cerebral ischemic thrombosis | |
| CN106555004A (en) | The lncRNA marks of cerebral infarction | |
| CN110656169B (en) | Diagnostic markers for atrial fibrillation | |
| CN107227358A (en) | Purposes of the NUMB in postmenopausal women's diagnosis of primary osteoporosis or prognosis | |
| CN107043811B (en) | Application of the CFAP20 genes in diagnosis of osteoporosis | |
| CN105567862B (en) | CDK18 is preparing the purposes in diagnosis of coronary heart disease product | |
| KR101745297B1 (en) | Composition for diagnosis of obesity and uses thereof | |
| CN105543400B (en) | The molecular marker of type-1 diabetes mellitus | |
| CN105821152B (en) | Coronary heart disease biomarker | |
| CN108624690A (en) | A kind of half adenocarcinoma of colon early screening detection kit of left and right and detection method | |
| CN106435009B (en) | Purposes of the TUBB2A as Blood diagnosis marker | |
| CN106367484A (en) | Application of molecular marker to sepsis diagnosis | |
| CN106435011B (en) | The diagnostic uses of MYOM2 | |
| CN106435010B (en) | The diagnosis marker of Bgudd-Chiari Syndrome | |
| KR102505617B1 (en) | Urinary exosome-derived miRNA gene biomarkers for diagnosis of T cell-mediated rejection in kidney allografts and use thereof | |
| CN110184346A (en) | The diagnosis marker of Male Osteoporosis | |
| KR20210024986A (en) | Urinary exosome-derived miRNA gene biomarkers for diagnosis of antibody-mediated rejection in kidney allografts and use thereof | |
| CN106755427A (en) | Applications of the OTOF in Bgudd-Chiari Syndrome diagnosis | |
| CN106636408A (en) | Budd-Chiari syndrome diagnosis tool | |
| CN106755426A (en) | Purposes of the IFI27 in diagnosis Bgudd-Chiari Syndrome product is prepared | |
| CN110229903A (en) | Molecular marker of the PODN as Diagnosis of Thyroid Carcinoma | |
| CN106048005B (en) | A kind of molecular marker of diagnosis of coronary heart disease | |
| CN110184358A (en) | The OIT3 gene of thyroid cancer early diagnosis and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Patentee after: BEIJING MEDINTELL BIOMED Co.,Ltd. Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Patentee before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221108 Address after: 102208 Room 704, Unit 2, Building 1, Yard 1, Longyu Middle Street, Huilongguan Town, Changping District, Beijing Patentee after: Beijing Micro Future Technology Co.,Ltd. Address before: Room 1210, building 3, Ronghua Xintai building, 10 ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Patentee before: BEIJING MEDINTELL BIOMED Co.,Ltd. |