CN105907881A - Application of gene marker to preparation of product for diagnosing ischemic cerebral stroke - Google Patents
Application of gene marker to preparation of product for diagnosing ischemic cerebral stroke Download PDFInfo
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Abstract
The invention discloses application of a gene marker to preparation of a product for diagnosing ischemic cerebral stroke. The marker is an SPAG9 gene; the expression of the SPAG9 gene in the ischemic cerebral stroke is up-regulated; and early diagnosis can be provided for a patient with the ischemic cerebral stroke through detecting the expression level of the SPAG9 gene, so that early treatment is realized and the death rate and the disability rate of the patient with the ischemic cerebral stroke is reduced.
Description
Technical field
The invention belongs to biological technical field, relate to gene marker at the product preparing diagnosing ischemia apoplexy
In purposes, specifically this gene marker is SPAG9.
Background technology
((stroke), is also called apoplexy or cerebrovas-cularaccident to apoplexy, is one group of unexpected onset, due to brain office
Acute cerebrovascular disease that portion's disturbance of blood circulation causes, with focal neurologic impairment as common trait.
Its because of high incidence, high mortality, high disability rate, high relapse rate become in world wide three big dead diseases it
One, rise to the first, urban residents' cause of the death second into urbanite's cause of the death in China, be also that adult is the first
Disabling disease.Over nearly 20 years, along with natural population increases and the impact of aging, the stroke onset of China
Rate, mortality rate are the most in rising trend, and according to statistics, new carbuncle in the occipital region apoplexy is more than 2,000,000 every year, the most extremely in China
In apoplexy person more than 1,000,000, and in survivor, patient 50-70% leaves over the handicaps such as paralysis, aphasia,
Give the burden that individual and composition of society are huge, become serious harm human health and the most public of social development is defended
One of raw problem.
Cerebral infarction is also called cerebral infarction, and it is to be supplied by the local brain tissue area blood caused by a variety of causes
Answer obstacle, cause brain tissue ischemia Hypoxic pathological changes downright bad, and then produce neurological deficit corresponding clinically
Performance.Ischemic Stroke is the main Types of apoplexy, account for that apoplexy is always fallen ill 60%～80%.Many popular
Sick research shows from different aspect, and cerebral infarction is a kind of coefficient by E&H factor
Complex Diseases.It is known that advanced age, smoking, drink, hypertension, hyperlipidemia, high blood are warded off, heart disease,
Fibrinogen level exception and disturbances of blood coagulation etc. are the conventional risk factors of cardiovascular and cerebrovascular disease, but this is only
The generation of about 50-60% apoplexy can be explained.Even if many people possess above-mentioned risk factor and apoplexy do not occur,
And some people not possessing above-mentioned risk factor there occurs apoplexy, the morbidity of prompting apoplexy also with other because of
Have pass.Majority all finds have apoplexy or the heart about the research of first degree relative Cerebral Vascular Disease situation in family
Disease of ZANG-organs Positive family history person is more of the same age, the other negative family history person of the same sex is more susceptible to suffer from apoplexy, and prompting family history is
One independent hazard factor of cerebrovascular；Long term follow-up twin to Denmark also indicates that, homozygotic twin is equal
The danger suffering from cerebral infarction is 2 times of non-identical twins, illustrates that cerebral infarction morbidity is close with inherited genetic factors
Relevant.In recent years, due to completing of the Human Genome Project, to the research of cerebral infarction from molecular genetic
Learn angle and have new breakthrough.
At present, the diagnosis of apoplexy relies primarily on clinical examination and various neuroimaging techniques, although the most than ever
For, Medical Technology is the most highly developed, but there is no reliable biomarker and can enter cerebral infarction
Row risk profile and early diagnosis, and prevent and treat cerebral infarction at present it is crucial that early discovery lesion vessels,
And be estimated intervening to it, instruct selection of clinical remedy measures timely and effectively.
Summary of the invention
In order to make up the deficiencies in the prior art, an object of the present invention, it is provided that examining of a kind of cerebral infarction
Disconnected mark；
The two of the purpose of the present invention, it is provided that the product of a kind of diagnosing ischemia apoplexy, it is achieved cerebral infarction
Early diagnosis.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides reagent the answering in preparing diagnosing ischemia apoplexy product of detection SPAG9 gene
With.
Further, diagnostic products described above includes: by RT-PCR, real-time quantitative PCR, immune detection,
The expression of in situ hybridization or chip detection SPAG9 gene is with the product of diagnosing ischemia apoplexy.
Further, the product of described RT-PCR diagnosing ischemia apoplexy at least includes a pair specific amplified
The primer of SPAG9 gene；The product of described real-time quantitative PCR diagnosing ischemia apoplexy at least includes a pair
The primer of specific amplified SPAG9 gene；The product of described immune detection diagnosing ischemia apoplexy includes: with
The antibody that SPAG9 protein-specific combines；The product of described in situ hybridization diagnosing ischemia apoplexy includes:
Probe with the nucleic acid array hybridizing of SPAG9 gene；The product of described micro-array chip diagnosing ischemia apoplexy
Product include: protein chip and gene chip；Wherein, protein chip includes being combined with SPAG9 protein-specific
Antibody, gene chip includes the probe of the nucleic acid array hybridizing with SPAG9 gene.
Further, described product includes chip and/or test kit；Wherein said chip includes gene chip, egg
White matter chip；Described test kit includes gene detecting kit and protein immunization detection kit.
Further, the primer pair of described gene detecting kit specific amplification SPAG9 gene.
In the detailed description of the invention of the present invention, the primer of described specific amplification SPAG9 gene is to sequence such as
Shown in SEQ ID NO.3 and SEQ ID NO.4.
The invention provides the product of a kind of diagnosing ischemia apoplexy, described product can be by detection sample
The expression of SPAG9 gene carrys out diagnosing ischemia apoplexy.
Further, product recited above includes chip or test kit.Wherein, described chip includes gene core
Sheet, protein chip；Described gene chip includes solid phase carrier and the oligonucleotide being fixed on solid phase carrier
Probe, described oligonucleotide probe include for detect SPAG9 gene transcription level for SPAG9 gene
Oligonucleotide probe；Described protein chip includes solid phase carrier and the SPAG9 being fixed on solid phase carrier
The specific antibody of albumen；Described test kit includes gene detecting kit and protein immunization detection kit；Institute
State gene detecting kit and include the reagent for detecting SPAG9 gene transcription level；Described protein immunization detects
Test kit includes the specific antibody of SPAG9 albumen.Wherein, described gene chip can be used for detection and includes
SPAG9 gene is at the expression water of interior multiple genes (such as, relevant to cerebral infarction multiple genes)
Flat.Multiple protein that described protein chip can be used for detecting including SPAG9 albumen are (such as with ischemia
Property the relevant multiple protein of apoplexy) expression.By by multiple and cerebral infarction mark
Detect simultaneously, be greatly improved the accuracy rate of cerebral infarction diagnosis.
Further, the reagent of described detection SPAG9 gene transcription level include the primer for SPAG9 gene and
/ or probe.
Further, described for the primer sequence such as SEQ ID NO.3 and SEQ ID NO.4 to SPAG9 gene
Shown in.
In the present invention, " antibody " covers monoclonal antibody, polyclonal antibody, multi-specificity antibody (example
Such as bi-specific antibody) and antibody fragment, combinatorial antibody etc., live as long as they show desired biology
Property." monoclonal antibody " refers to the antibody obtained from the antibody of a group substantially homogeneity, i.e. constitutes colony
Each antibody is identical and/or combines identical epi-position, except produce issuable during monoclonal antibody can
Outside energy variant, this type of variant is typically with indivisible existence.This type of monoclonal antibody is typically include to comprise and combines target
The antibody of the peptide sequence of thing, wherein target Binding peptide sequence is by selecting in many peptide sequences that includes comforming
Single target Binding peptide sequence obtains in interior process.
Monoclonal antibody also includes " being fitted together to " antibody, wherein a part for heavy chain and/or light chain with derived from spy
Earnest species or genus corresponding sequence in the antibody of specific antibodies classification or subclass is identical or homology, and the residue of chain
Part with derived from another species or belong to another antibody isotype or subclass antibody in corresponding sequence identical or
Homology, and the fragment of this antibody-like, as long as they show desired biologic activity.
" antibody or antibody fragment " refers to no matter derive from any species naturally occurring antibody, or by restructuring
DNA technique creates；No matter anti-from serum, B cell, hybridoma, transfectoma, yeast or bacteria distribution
Body (such as IgG, IgM, IgA, IgD or IgE) or fragment (such as Fab, F (ab ')2, Fv, two
The Fv of sulfide connection, scFv, Guan Bi conformation multispecific antibody, the scFv of disulphide connection, double antibody).
In the present invention, refer to can be with the particular sequence of another molecule or subsequence or other parts for term " probe "
In conjunction with molecule.Unless otherwise noted, term " probe " is often referred to pass through complementary base pairing and another are many
The polynucleotide probes that nucleotide (often referred to as " target polynucleotide ") combines.Tight according to hybridization conditions
Careful property, probe energy and the target polynucleotide lacking sufficient sequence complementary with this probe are combined.Probe can be made directly
Or indirectly labelling, its scope includes primer.Crossing system, includes, but are not limited to: solution phase, solid phase,
Mixed phase or in situ hybridization algoscopy.
In the present invention, term " microarray " is that hybridised arrays original paper is ordered in substrate, described miscellaneous
Hand over array original paper such as polynucleotide probe (such as oligonucleotide) or bonding agent (such as antibody).Described base
Matter can be solid matrix, such as, and glass or silicon dioxide slide, pearl, fibre optics binding agent or semisolid
Substrate, such as nitrocellulose filter.Nucleotide sequence can be any arrangement of DNA, RNA or therein.
Various probe arrays have been described above in the literature and may be used in the context of the invention detection may be with
The mark of phenotypic correlation described herein.Such as, the DNA probe array of DNA probe array chip or bigger
Wafer (otherwise, can obtain each individual chip by interrupting wafer) is for one embodiment of the invention.
DNA probe array chip generally comprises chip glass, and it placed high-density DNA probe (short dna sheet
Section) array.These wafers each can keep e.g., from about 60,000,000 for identifying longer sample DNA sequence
The DNA probe of (such as, from individual or colony, such as, comprising paid close attention to mark).Use glass
DNA probe group identification sample DNA on wafer is carried out by DNA hybridization.When DNA sample and DNA probe
During hybridization array, sample is incorporated into those probes of sample DNA complementary.By evaluating individual sample DNA
More securely hybridize with those probes, it is possible to determine whether known nucleotide sequence is present in sample, thus
Determine whether the mark found in nucleic acid exists.These means can also be used by controlling hybridization conditions to permit
Permitted to distinguish single nucleotide, such as, identify for SNP and the sample gene typing of one or more SNP enters
Row ASH.Array provides a kind of (or series winding) simultaneously and detects the convenience embodiment party of multiple polymorphic marker
Case.
Above-mentioned probe has the base sequence of the specific base sequence complementary with target gene.Here, so-called " mutual
Mend ", as long as hybridize, can not be complete complementary.It is specific that these polynucleotide are commonly angled relative to this
Base sequence has more than 80%, preferably more than 90%, more preferably more than 95%, particularly preferred 100% same
Source property.These probes can be DNA, it is also possible to is RNA, furthermore it is possible to be at one part or whole
The polynucleotide that nucleotide is obtained by the artificial replacement nucleic acid such as PNA, LNA, ENA, GNA, TNA.
In the context of the present invention, " SPAG9 gene " includes people's SPAG9 gene and people's SPAG9 gene
The polynucleotide of any function equivalent.SPAG9 gene includes and the most international common core sequence databank
In GeneBank, SPAG9 gene (NC_000017.11) DNA sequence has more than 70% homology, and compiles
Code-phase congenerous protein DNA sequence；In specific embodiments of the present invention, described SPAG9 gene
Coded sequence be the DNA sequence shown in SEQ ID NO.1.
The SPAG9 gene of the present invention can be natural or synthetic, or uses and can express
The vector-transfected cell of the DNA fragmentation of SPAG9 obtains.Carrier described in described carrier includes viral vector, true
Nuclear expression carrier.
Viral vector can be any suitable carrier, includes but not limited to that retroviral vector, adenovirus carry
Body, adeno-associated virus (AAV) carrier, herpesvirus (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus)
Carrier, alphavirus vectors.Carrier for expression of eukaryon can be any suitable expression vector, includes but not limited to
PCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP table
Reach carrier, pEF Bos expression vector, pTet expression vector, pTRE expression vector or in known expression
Carrier engineered on the basis of carrier, such as pBin438, pCAMBIA1301 etc..
In the context of the present invention, SPAG9 gene expression product includes people's SPAG9 albumen and people
The partial peptide of SPAG9 albumen.The partial peptide of described SPAG9 albumen contains the merit relevant to cerebral infarction
Can territory.
" SPAG9 albumen " includes any function equivalent of SPAG9 albumen and SPAG9 albumen.Institute
State function equivalent and include SPAG9 albumen conservative variation's protein or its active fragment, or its activity is spread out
Biology, allelic variant, natural mutation, induced mutants, energy and people under high or low stringent condition
Protein coded by the DNA of the DNA hybridization of SPAG9.In the detailed description of the invention of the present invention, institute
State the protein that SPAG9 albumen is made up of the aminoacid sequence shown in SEQ ID NO.2 in sequence table.
In the present invention, term " sample " refers to the compositions obtained from target patient, its comprise cell and/
Or other molecule bodies are carried out characterization and/or identification, such as, according to physics, biochemistry, chemistry and/or
Physiological feature.Such as, phrase " clinical sample " or " disease sample " and variant thereof, refer to from target patient
Any sample obtained, will be obtained in that cell and/or molecule body, such as will in expection or known described sample
The biomarker being characterized.
In the detailed description of the invention of the present invention, described " sample " is the blood of experimenter.
In the context of the present invention, " diagnosing ischemia apoplexy " both includes judging that experimenter has suffered from
There is cerebral infarction, also include judging whether experimenter exists the risk suffering from cerebral infarction.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that SPAG9 gene expression is relevant to cerebral infarction, by detection experimenter
The expression of SPAG9 in blood, it can be determined that whether experimenter suffers from cerebral infarction or judge tested
Whether person exists the risk suffering from cerebral infarction, thus instructs clinicist to provide prevention scheme to experimenter
Or therapeutic scheme.
Present invention finds the new molecular marked compound-SPAG9 gene of a kind of cerebral infarction, compare tradition
Detection means, gene diagnosis more in time, more special, sensitiveer, it is possible to realize the early stage of cerebral infarction
Diagnosis, thus reduce the mortality rate of cerebral infarction.
Accompanying drawing explanation
Fig. 1 show utilize QPCR detect SPAG9 gene expression in ischemic cerebral stroke patients.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are only used for
The bright present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment is logical
Often according to normal condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring HarborLaboratory Press, 1989) condition described in, or according to the bar proposed by manufacturer
Part.
The gene marker that embodiment 1 screening is relevant to cerebral infarction
1, sample collection
Each collection 10 example normal human bloods and ischemic cerebral stroke patients blood sample, taking of above-mentioned all specimen
Obtain all by the agreement of Ethics Committee, and obtain the informed consent of experimenter.
2, the preparation of RNA sample and quality analysis
The preparation of 2.1 RNA sample
(1) homogenized
Directly taking blood, add 3 times of volume erythrocyte cracked liquids, after mixing, room temperature places 10min,
10,000rpm is centrifuged 1min.Thoroughly inhale and abandon supernatant, collect leukocyte cell pellet.Every 100-200 μ l blood collecting
Leukocyte cell pellet add 1ml Trizol.
(2) layering
A., after sample adds Trizol, room temperature places 5min, makes sample fully crack.4 DEG C of 12,000rpm from
Heart 10min, takes supernatant；
B. often 1ml Trizol adds 200 μ l chloroforms, and acutely after vibration mixing, room temperature placement 3-5min makes it certainly
So split-phase.
(3) RNA precipitate
A.4 a DEG C 12,000rpm is centrifuged 10-15min.Sample can be divided into three layers: the organic facies of yellow, intermediate layer
With colourless aqueous phase, RNA, mainly in aqueous phase, transfers to careful for aqueous phase in new pipe.
B. adding the isopropanol that equal-volume is ice-cold in supernatant, room temperature places 10-20min.4℃12,000rpm
Centrifugal 10min, abandons supernatant, and RNA precipitate is at the bottom of pipe.
(4) RNA rinsing
Adding 1ml 75% ethanol (preparing with RNase-free water) in a.RNA precipitation, gentle vibration is centrifugal
Pipe, suspend precipitation.Every 1ml TRIzol adds 1ml 75% ethanol.
B.4 DEG C 5,000-8,000rpm is centrifuged 1-2min, abandons supernatant；Of short duration the most centrifugal, little with pipettor
The heart is inhaled and is abandoned supernatant, is careful not to suction and abandons precipitation.Room temperature is placed 1-2min and is dried precipitation.
(5) RNA is dissolved
Precipitation adds 50-100 μ l RNase-free water, flicks tube wall, fully to dissolve RNA ,-70 DEG C of guarantors
Deposit.
The quality analysis (NanoDrop1000 spectrophotometer) of 2.2 RNA sample
NanoDrop1000 spectrophotometer detection RNA sample, the sample requirement of RNA-seq order-checking:
OD260/OD280 is 1.8-2.2.
The quality analysis (Agilent Technologies 2100Bioanalyzer) of 2.3 RNA sample
The RNA of said extracted is carried out agarose gel electrophoresis, Agilent Technologies 2100
Bioanalyzer detects RNA sample quality, observes 28S rRNA and 18S rRNA master tape is obvious, without fall
Solve, RNA Perfection Index is qualified, concentration reaches the RNA-seq order-checking cDNA library that meets of requirement and builds
Requirement, may be used for library construction and order-checking.
3, high flux transcript profile order-checking
3.1 RNA-seq read section location
First low-quality reading section is removed and obtain cleaning reading section, then utilize TopHat v1.3.1 by cleansing tablet
Section is mated with UCSC H.sapiens reference genome (hg19), H.sapiens UCSC hg19 version pre-
The index first built is downloaded from TopHat homepage, and as with reference to genome, utilizes TopHat and genome
Timing, it is allowed to each reading section (defaulting to 20) has multiple coupling site, most 2 mispairing.TopHat root
Possible shearing site storehouse is set up, according to these shearing site storehouses according to exon region and GT-AG shear signal
The reading section not navigating to genome is navigated on genome.We use the system default of TopHat method
Parameter.
3.2 transcript abundance assessments
The reading segment file matched is by Cufflinks v1.0.3 process, and Cufflinks v1.0.3 is by RNA-seq sheet
Hop count mesh is standardized calculating the relative abundance of transcript.FPKM value refers in each million order-checking fragments
Match the segment number of the exon region of specific gene 1kb length.Calculated by Bayesian inference method
The confidence interval of FPKM estimated value.The GTF comment file of the reference that Cufflinks uses is from Ensembl number
(Homo_sapiens.GRCh37.63.gtf) is downloaded according to storehouse.
The detection of 3.3 difference expression genes
Ensembl GTF file and the original document mated by TopHat of download are transferred to Cuffdiff,
Cuffdiff uses original matching files to re-evaluate the gene expression abundance of the transcript listed in GTF file, inspection
Survey differential expression.Q value ＜ 0.01, test display is only had successfully more just to be considered in Cuffidff exports
It it is differential expression.
4, result
RNA-seq result shows, SPAG9 gene expression in ischemic cerebral stroke patients blood shows
Write the expression higher than normal person.
The differential expression of embodiment 2 QPCR sequence verification SPAG9 gene
1, SPAG9 gene is selected to carry out large sample QPCR checking according to the testing result of high-flux sequence.According to
Sample collection mode in embodiment 1 selects ischemic cerebral stroke patients blood and each 80 examples of normal human blood.
2, RNA extraction step is with embodiment 1.
3, reverse transcription: use the Reverse Transcription box of TAKARA company to operate.Specifically comprise the following steps that
(1) take total serum IgE 2 μ g and carry out reverse transcription, add Oligo (dT) 2 μ l, fully mix；70 DEG C of water-baths；
Ice bath 2-3min immediately after 5min；
(2) 25 μ l reaction systems are built, including 5 × RT Buffer 5 μ l, dNTP (2.5mM) 5 μ l,
RNasin 40U/ μ l, M-MLV 200U/ μ l, benefit nuclease free water to 25 μ l；
After (3) 42 DEG C of water-bath 60min, 95 DEG C of water-bath 5min are to inactivate M-MLV, and-20 DEG C store for future use.
4, QPCR amplification
(1) design of primers
Coded sequence design according to SPAG9 gene in Genebank and house-keeping gene GAPDH gene
QPCR amplimer, the synthesis of Beijing match promise genome research centered finite company.Wherein SPAG9 gene
Primer is as follows to sequence: forward primer sequence is 5 '-AGAGAATTGGAGGAAGAG-3 ' (SEQ ID
NO.3)；Reverse primer sequences is 5 '-ATCACTATCATCGTCATCT-3 ' (SEQ ID NO.4).
The primer sequence of GAPDH gene is to as follows: forward primer sequence is 5 '-
CTCTGGTAAAGTGGATATTGT-3’(SEQ ID NO.5)；Reverse primer sequences is 5 '-
GGTGGAATCATATTGGAACA-3’(SEQ ID NO.6)。
(2) PCR reaction system: forward primer and each 1 μ l of reverse primer, SYBR Green polymerase chain reaction
Answer system 12.5 μ l, template 2 μ l, add deionized water and complement to 25 μ l.
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 30 circulations.With
SYBR Green, as fluorescent marker, reacts at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument,
Determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
5, statistical method
Experiment uses and repeats experiment for 3 times, and result data is all to represent in the way of mean+SD, makes
Carrying out statistical analysis with SPSS13.0 statistical software, difference between the two uses t inspection, it is believed that when
P < has statistical significance when 0.05.
6, result
Result is as it is shown in figure 1, compared with normal human blood, SPAG9 gene is at ischemic cerebral stroke patients blood
Up-regulated in liquid, difference has statistical significance (P < 0.05), consistent with RNA-sep result.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It is right to it should be pointed out that,
For those of ordinary skill in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention
Carrying out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the reagent of detection SPAG9 gene application in preparing diagnosing ischemia apoplexy product.
Application the most according to claim 1, it is characterised in that described product includes: by RT-PCR,
Real-time quantitative PCR, immune detection, in situ hybridization or the expression of micro-array chip detection SPAG9 gene
Product with diagnosing ischemia apoplexy.
Application the most according to claim 2, it is characterised in that described RT-PCR diagnosing ischemia brain
The product of apoplexy at least includes the primer of a pair specific amplified SPAG9 gene；Described real-time quantitative PCR diagnoses
The product of cerebral infarction at least includes the primer of a pair specific amplified SPAG9 gene；Described immune detection
The product of diagnosing ischemia apoplexy includes: the antibody being combined with SPAG9 protein-specific；Described the most miscellaneous
The product handing over diagnosing ischemia apoplexy includes: with the probe of the nucleic acid array hybridizing of SPAG9 gene；Described use
The product of micro-array chip diagnosing ischemia apoplexy includes: protein chip and gene chip；Wherein, albumen core
Sheet includes the antibody being combined with SPAG9 protein-specific, and gene chip includes and the nucleotide sequence of SPAG9 gene
The probe of hybridization.
4. according to the application described in any one of claim 1-3, it is characterised in that described product include chip and
/ or test kit；Wherein said chip includes gene chip, protein chip；Described test kit includes that gene is examined
Test agent box and protein immunization detection kit.
Application the most according to claim 4, it is characterised in that described gene detecting kit specificity expands
Increase the primer pair of SPAG9 gene.
Application the most according to claim 5, it is characterised in that described specific amplification SPAG9 gene
Primer to sequence as shown in SEQ ID NO.3 and SEQ ID NO.4.
7. the product of a diagnosing ischemia apoplexy, it is characterised in that described product can be by detection
In sample, the expression of SPAG9 gene carrys out diagnosing ischemia apoplexy.
Product the most according to claim 7, it is characterised in that described product includes chip or test kit；
Wherein, described chip includes gene chip, protein chip；Described gene chip includes solid phase carrier and consolidates
Being scheduled on the oligonucleotide probe of solid phase carrier, described oligonucleotide probe includes for detecting SPAG9 genetic transcription
The oligonucleotide probe for SPAG9 gene of level；Described protein chip includes solid phase carrier and fixes
Specific antibody at the SPAG9 albumen of solid phase carrier；Described test kit includes gene detecting kit and albumen
Immunity detection reagent；Described gene detecting kit includes the reagent for detecting SPAG9 gene transcription level；
Described protein immunization detection kit includes the specific antibody of SPAG9 albumen.
Product the most according to claim 8, it is characterised in that described detection SPAG9 gene transcription level
Reagent include the primer for SPAG9 gene and/or probe.
Product the most according to claim 9, it is characterised in that described draw for SPAG9 gene
Thing sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4.
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| CN201610509639.1A CN105907881A (en) | 2016-07-01 | 2016-07-01 | Application of gene marker to preparation of product for diagnosing ischemic cerebral stroke |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106337089A (en) * | 2016-11-24 | 2017-01-18 | 汕头大学医学院第附属医院 | LncRNA for diagnosing cerebral arterial thrombosis |
| CN106434982A (en) * | 2016-11-24 | 2017-02-22 | 汕头大学医学院第附属医院 | Relevant molecular markers for cerebral ischemic stroke and application of molecular markers |
| CN110964802A (en) * | 2019-11-11 | 2020-04-07 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Application of ApoE gene in hematoma re-expansion and prognosis evaluation in acute stage of cerebral hemorrhage |
| CN112553358A (en) * | 2020-09-19 | 2021-03-26 | 河北医科大学第二医院 | Application of microbial flora in cerebral apoplexy |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590510A (en) * | 2012-02-02 | 2012-07-18 | 任碧琼 | Application of anti-SPAG9 (Sperm Associated Antigen 9) antibody in early auxiliary diagnosis of tumors |
| WO2013033169A1 (en) * | 2011-08-31 | 2013-03-07 | Sanofi | Methods of identifying genomic translocations associated with cancer |
| CN105296658A (en) * | 2015-11-30 | 2016-02-03 | 北京泱深生物信息技术有限公司 | Application of ALKBH2 gene to diagnosis of cerebral ischemic stroke |
| CN105296659A (en) * | 2015-11-30 | 2016-02-03 | 北京泱深生物信息技术有限公司 | Gene marker related to ischemic stroke |
-
2016
- 2016-07-01 CN CN201610509639.1A patent/CN105907881A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013033169A1 (en) * | 2011-08-31 | 2013-03-07 | Sanofi | Methods of identifying genomic translocations associated with cancer |
| CN102590510A (en) * | 2012-02-02 | 2012-07-18 | 任碧琼 | Application of anti-SPAG9 (Sperm Associated Antigen 9) antibody in early auxiliary diagnosis of tumors |
| CN105296658A (en) * | 2015-11-30 | 2016-02-03 | 北京泱深生物信息技术有限公司 | Application of ALKBH2 gene to diagnosis of cerebral ischemic stroke |
| CN105296659A (en) * | 2015-11-30 | 2016-02-03 | 北京泱深生物信息技术有限公司 | Gene marker related to ischemic stroke |
Non-Patent Citations (1)
| Title |
|---|
| DEEPIKA KANOJIA ET AL: "Sperm-Associated Antigen 9,a novel Biomarker for Early Detection of Breast cancer", 《CANCER EPIDEMIOLOGY, BIOMARKERS&PREVENTION》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106337089A (en) * | 2016-11-24 | 2017-01-18 | 汕头大学医学院第附属医院 | LncRNA for diagnosing cerebral arterial thrombosis |
| CN106434982A (en) * | 2016-11-24 | 2017-02-22 | 汕头大学医学院第附属医院 | Relevant molecular markers for cerebral ischemic stroke and application of molecular markers |
| CN110964802A (en) * | 2019-11-11 | 2020-04-07 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Application of ApoE gene in hematoma re-expansion and prognosis evaluation in acute stage of cerebral hemorrhage |
| CN110964802B (en) * | 2019-11-11 | 2023-05-02 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Application of ApoE gene in cerebral hemorrhage acute stage hematoma re-expansion and prognosis evaluation |
| CN112553358A (en) * | 2020-09-19 | 2021-03-26 | 河北医科大学第二医院 | Application of microbial flora in cerebral apoplexy |
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