CN105506172A - Osteoporosis blood diagnostic reagent with detection GFOD2 and application of osteoporosis blood diagnostic reagent - Google Patents
Osteoporosis blood diagnostic reagent with detection GFOD2 and application of osteoporosis blood diagnostic reagent Download PDFInfo
- Publication number
- CN105506172A CN105506172A CN201610116151.2A CN201610116151A CN105506172A CN 105506172 A CN105506172 A CN 105506172A CN 201610116151 A CN201610116151 A CN 201610116151A CN 105506172 A CN105506172 A CN 105506172A
- Authority
- CN
- China
- Prior art keywords
- gfod2
- gene
- expression
- osteoporosis
- application according
- 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
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/108—Osteoporosis
Abstract
The invention relates to an osteoporosis blood diagnostic reagent with detection GFOD2 and application of the osteoporosis blood diagnostic reagent. The osteoporosis blood diagnostic reagent and the application have the advantages that high-throughput sequencing is carried out on peripheral blood samples of nine osteoporosis patients and comparison peripheral blood samples of six healthy persons, genes are screened by the aid of bioinformatics methods, candidate genes GFOD2 are selected, and genes GFOD2 are in low-expression in peripheral blood of the osteoporosis patients as proved by molecular verification results; pathogenic target genes in the detected peripheral blood can be used for assisting clinical osteoporosis diagnosis and treatment, and accordingly novel solution can be provided for diagnosing, treating and predicting osteoporosis.
Description
Technical field
The present invention relates to biomedicine field, being specifically related to osteoporosis Blood diagnosis reagent and application thereof containing detecting GFOD2.
Background technology
Osteoporosis (Osteoporosis) is that the bone amount caused by a variety of causes reduces, osseous tissue Microstructure Fracture, and skeletal fragility increases, and is easy to a kind of general osteopathy that fragility fractures occurs.NIH is defined as osteoporosis and " declines with bone strength and be easy to the disease of skeletal system that fracture is feature.Its main pathology shows as Grafting Cancellous Bone Bolt girder and attenuates, ruptures and quantity minimizing, and cortex bone porous, bony structures are disorderly.Reason according to morbidity is different, and osteoporosis can be divided into: former osteoporosis and secondary osteoporosis two class.Primary osteoporosis, more common clinically, be mainly divided into 3 kinds: women's osteosporosis after menopause disease, senile osteoporosis and idiopathic osteoporosis.Secondary osteoporosis causes mainly due to following factor: endocrine factors, excessive use hormone medicine, trophic factor, organism disease factor and other factors.Osteoporosis is a kind of disease of bone remoulding obstacle in essence, and the bone amount caused reduces (bone density reduction), bony structures changes and the unbalance pathologic basis constituting osteoporosis of bone conversion.The molecular mechanism of research osteoporosis pathology, probes into the relation between the specific gene of unconventionality expression and disease, contributes to the pathogenesis of understanding disease in depth, be conducive to osteoporotic early diagnosis and prognosis.
For solving the rare problem of current osteoporosis molecule marker, contriver carries out high-flux sequence to 9 routine sufferers of osteoporosis face peripheral blood samples and 6 routine Healthy People contrast peripheral blood samples, carry out genescreen in conjunction with bioinformatics method, pick out candidate gene GFOD2.Further, invention has been the relation that molecular biology method confirms GFOD2 and osteoporosis: GFOD2 is low expression in sufferers of osteoporosis face peripheral blood, with osteoporosis, there is good dependency.Virus monitory has the advantages such as wound is little, method is easy, reliable results, and detecting GFOD2 in peripheral blood is that diagnosing osteoporosis, treatment and prediction provide new solution.Candidate gene GFOD2 provided by the invention can be used for preparation osteoporosis assisting in diagnosis and treatment preparation, is expected to the latent gene therapy target becoming osteoporosis clinical treatment, has a good application prospect.
Summary of the invention
GFOD2 gene and/its expression product is the object of the present invention is to provide to prepare the application in diagnosing osteoporosis and/or treatment preparation.
Detection GFOD2 gene and/its expression product is the object of the present invention is to provide to prepare the application in diagnosing osteoporosis preparation.
Further, diagnosing osteoporosis preparation detects the expression of GFOD2 gene and/or its expressing protein in peripheral blood.
For achieving the above object, first the present invention screens candidate gene GFOD2 by high-flux sequence in conjunction with bioinformatics method, GFOD2 and osteoporotic relation: GFOD2 low expression in sufferers of osteoporosis face peripheral blood is demonstrated further by molecular biology method, with osteoporosis, there is good dependency, can be used for preparation treatment osteoporosis preparations and/or diagnosing osteoporosis preparation, there is important clinical value.
GFOD2 gene is the object of the present invention is to provide to prepare the application in diagnosing osteoporosis preparation.
Further, described osteoporotic diagnostic preparation comprises the expression detecting GFOD2 gene in osteoporosis peripheral blood with fluorescence quantifying PCR method, method for gene chip.
Fluorescence quantitative PCR method is by fluorescence dye or fluorescently-labeled specific probe, and carry out mark to PCR primer and follow the tracks of, real time and on line monitoring reaction process, can analyze product in conjunction with corresponding software, calculates the starting point concentration of testing sample template.The appearance of quantitative fluorescent PCR, greatly simplifies the process of detection by quantitative, and really achieves absolute quantitation.The appearance of multiple detection system, makes the selectivity of experiment stronger.Automated operation improves working efficiency, reacts quick, reproducible, highly sensitive, high specificity, result be clear.
Gene chip is also called DNA microarray (DNAmicroarray), three kinds of main Types can be divided into: 1) be fixed on polymer matrix film (nylon membrane, nitrocellulose membrane etc.) nucleic acid probe on surface or cDNA fragment, usually be hybrid with it with isotope-labeled target gene, detected by radiography technology.2) fixing DNA probe array on a glass by point sample method, detecting by hybridizing with fluorescently-labeled target gene.3) oligonucleotide probe array of directly synthesis on the hard surfaces such as glass, hybridizes with fluorescently-labeled target gene and detects.Gene chip is as a kind of advanced person, extensive, high throughput testing technology, and be applied to the diagnosis of disease, its advantage has the following aspects: one is susceptibility highly and accuracy; Two is fast and convenient; Three is can detect various diseases simultaneously.
Described contains the primer of a pair specific amplification GFOD2 gene for the product of GFOD2 gene in fluorescence quantifying PCR method detection osteoporosis; Described gene chip comprises the probe with the nucleic acid array hybridizing of GFOD2 gene.
GFOD2 gene expression product is the object of the present invention is to provide to prepare the application in diagnosing osteoporosis preparation.Further, described osteoporotic diagnostic preparation comprises the expression detecting GFOD2 albumen with immunization method.In preferred described immunologic detection method detection osteoporosis, GFOD2 protein expression is westernblot and/or ELISA and/Radioactive colloidal gold detection method.
Enzyme-linked immunosorbent assay (ELISA) is adsorbed on surface of solid phase carriers by known antigen or antibody, makes the technology that the antigen antibody reaction of enzyme labelling is carried out at solid phase surface.This technology can be used for detecting macromole antigen and specific antibody etc., has quick, sensitive, easy, carrier and is easy to the advantages such as stdn.ELISA detection kit can be divided into indirect method, double-antibody method, competition law, dibit point single stage method, prize law to survey the ELISA of IgM antibody, application avidin and vitamin H according to testing goal and operation steps.In ELISA detection kit, chromogenic substrate can select horseradish peroxidase (HRP) or alkaline phosphatase (AP).
Conventional immune colloid gold detection technique: (1) immune colloid gold light microscopic staining cell suspension smear or peripheral blood section, the antibody of available colloid gold label dyes, also can on the basis of colloid gold label, mark is strengthened with silver-colored developing solution, the gold grain surface silver atoms be reduced being deposited on marked, obviously can strengthen the susceptibility of colloid gold label.(2) immune colloid gold staining method for electron microscopy can be combined with negative staining Virus Sample or peripheral blood ultrathin section(ing) with the antibody of colloid gold label or anti-antibody, then carries out negative staining.Can be used for observation and the Viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application millipore filtration makes carrier, first by antigen or antibody point on film, add sample to be checked after closing, wash the corresponding antigen of antibody test or the antibody of rear colloid gold label.(4) specific antigen or antibody are fixed on film with ribbon by colloidal gold immunity chromatography, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on pad, after in the sample pad that sample to be checked is added to test strip one end, moved forward by wicking action, react to each other after dissolving the colloid gold label reagent on pad, when moving to the region of fixing antigen or antibody, there is specific binding with it again and be trapped in the binding substances of thing to be checked and gold marked reagent, be gathered in detection zone, by being observed visually colour developing result.This method now develops into diagnosis test paper, uses very convenient.
Further, the ELISA method of described detection GFOD2 albumen is for using ELISA detection kit.Antibody in described test kit can adopt commercially available GFOD2 monoclonal antibody, preferred abcam antibody.Further, described test kit comprises: wrap by the solid phase carrier of GFOD2 monoclonal antibody, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, washings, enzyme reaction stop buffer etc.
Further, the colloidal gold method of described detection GFOD2 albumen is for using detection kit, and described antibody can adopt commercially available GFOD2 monoclonal antibody.Further, described gold-immunochromatographyreagent reagent for assay box adopts colloidal gold immunochromatographimethod technology or Radioactive colloidal gold percolation process.Further, detection zone (T) specking on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-GFOD2 monoclonal antibody, quality control region (C) specking has immunoglobulin IgG.
The object of the present invention is to provide the osteoporotic PCR kit for fluorescence quantitative of a kind of detection, it is characterized in that, described test kit detects gene GFOD2, adopts special upstream primer and downstream primer, upstream primer sequence is SEQIDNO.1, and downstream primer sequence is SEQIDNO.2.
Further, this PCR kit is suitable for all types fluorescence quantitative gene extender deposited at present commercially, highly sensitive, quantitatively quick and precisely, good stability, has a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component comprises: Auele Specific Primer, internal reference primer, fluorescence quantitative PCR reaction solution.Wherein said Auele Specific Primer comprises upstream primer and downstream primer, and upstream primer sequence is SEQIDNO.1, and downstream primer sequence is SEQIDNO.2.Described internal reference primer is β-actin internal reference primer, and upstream primer sequence is SEQIDNO.3, and downstream primer sequence is SEQIDNO.4.
Described test kit also comprises RNA extraction agent.Preferred health is that century blood rna extraction test kit carries out sample rna extraction.
The present invention also have detected this test kit susceptibility, and minimum concentrations is 100copies/ μ l.
The object of the invention there is provided a kind of osteoporosis detection kit, and described detection kit detects GFOD2 albumen.Further, described test kit also comprises other detection reagent.
The object of the invention there is provided the osteoporotic gene chip of a kind of detection, and described gene chip comprises the probe with the nucleic acid array hybridizing of GFOD2 gene.
The object of the present invention is to provide one to treat osteoporosis reagent, described treatment osteoporosis reagent promotes the expression of GFOD2 gene.Further, containing the carrier promoting GFOD2 genetic expression in described treatment osteoporosis reagent.
Further, described treatment osteoporosis reagent refers to the reagent of the expression that can promote GFOD2 gene.Those skilled in the art know and promote that the expression of gene can adopt the one in following method and/or several usually: regulate and control goal gene by DNA level: include but not limited to increase the copy number of GFOD2 gene, transfection containing the over-express vector of GFOD2 gene; By transcriptional level control GFOD2 gene: include but not limited to the expression of activation GFOD2 gene, activate the promotor of regulation and control GFOD2 genetic expression, suppress the transcription factor of negative regulation GFOD2 genetic expression, adopt suppression of RNA perturbation technique to suppression GFOD2 genetic expression to disturb; By post-transcriptional level regulation and control GFOD2 gene: include but not limited to suppress the microRNA transcriptional expression of promotion GFOD2 gene mRNA degraded, import the microRNA promoting GFOD2 genetic expression; By translating rear level modulation GFOD2 gene: include but not limited to import the molecule promoting goal gene proteins encoded, the albumen suppressing negative regulation GFOD2 genetic expression, promote the factor of GFOD2 genetic expression and the expression of albumen.
Above-mentioned treatment osteoporosis reagent is the object of the present invention is to provide to prepare the application in osteoporosis therapy preparation.
Accompanying drawing explanation
Fig. 1 is GFOD2 gene relative expression's spirogram in osteoporosis peripheral blood and healthy human peripheral blood
Embodiment
Below in conjunction with specific embodiment, setting forth the present invention further, only for explaining the present invention, and can not limitation of the present invention be interpreted as.Those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition examinations of advising according to manufacturer.
Embodiment 1 high-flux sequence and analysis
Samples sources, in BJ Union Hospital, all obtains subjects informed consent.Collect 9 routine osteoporosis Patients with Peripheral blood samples and 6 routine Healthy People contrast peripheral blood samples respectively, carry out RNA extraction, RNA extract after agarose gel electrophoresis, from electrophoresis result can preliminary judgement extract RNA sample whether up-to-standard, whether may be used for further transcriptome analysis.And then detected the extraction situation of RNA sample by NanoDrop1000 spectrophotometer, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.
Order-checking platform is the HiSeq2500 high-flux sequence platform of Illumina company, carry out the order-checking of the high-throughput transcript profile degree of depth, after order-checking, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) software to carry out total evaluation to the quality of sequencing data, comprise the mass value distribution of base, the position distribution of mass value, GC content, PCRduplication content, the frequency etc. of kmer.When differential genes expression analysis, according to the FPKM value obtained, internationally recognized algorithm EBSeq is adopted to carry out differential screening.Wherein, during screening, LOG2FC>1 or <-1, FDR<0.05.In order to better understand the function of difference expression gene, we have carried out GeneOnlogy and signal path analysis to difference expression gene, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of the result of above data analysis, in conjunction with document, we have screened difference expression gene GFOD2.
Embodiment 2 sufferers of osteoporosis face peripheral blood and healthy human peripheral blood GFOD2 expression conditions
One materials and methods
1, material
Collect 74 routine sufferers of osteoporosis face peripheral bloods and 46 routine healthy human peripheral bloods, it is divided into groups and numbers.
2, method
The extraction of 2.1 sufferers of osteoporosis face peripheral bloods and healthy human peripheral blood total serum IgE
Adopt health to be century blood rna extraction test kit (article No. CW0538) carry out sample rna extraction, experimental implementation is undertaken by product description, and specification sheets is shown in concrete operations.
RNA quality judging standard: the OD260/OD280 value of RNA sample is between 1.7-2.2; Total serum IgE electrophoretogram has 28S, 18S band clearly; The electrophoretogram of 70 DEG C of water bath heat preservations after 1 hour and the collection of illustrative plates no significant difference before water bath heat preservation.
2.2GFOD2 detects design of primers and synthesis
According to PCR primer principle of design, the OligoArchitect of application Premier5.0 and enhanced edition
tMsoftware carries out design of primers.
The upstream and downstream primer sequence of GFOD2 (NM_030819) is respectively:
Upstream primer: 5'-CTTGCTTTATTAAGTGAG-3'; SEQIDNO.1
Downstream primer: 5'-TTTCTGAATTATTTATTAACAT-3'; SEQIDNO.2
Product length is 119bp.
Internal reference β-actin (NM_001101) upstream and downstream primer sequence is respectively:
Upstream primer: 5'-TAATCTTCGCCTTAATACT-3'; SEQIDNO.3
Downstream primer: 5'-CCTTCATACATCTCAAGT-3'; SEQIDNO.4
Product length is 103bp.
The foundation of 2.3 quantitation curves
The preparation of standard DNA template
To specifications, from peripheral blood, utilize health to extract total serum IgE for century blood rna extracts test kit (article No. CW0538), then be that century SuperRTcDNA first chain synthetic agent box (article No. CW0741) carries out reverse transcription reaction by health, concrete steps are as follows:
1. RNA template, PrimerMix, dNTPMix, RTBuffer, SuperRT and RNase-FreeWater are dissolved and be placed in for subsequent use on ice.
2. configure reaction system, cumulative volume is 20 μ l: final concentration is RNA template, 2 μ lPrimerMix, 4 μ ldNTPMix, 4 μ lRTBuffer, the 1 μ lSuperRT of 50pg-5 μ g, adds RNase-FreeWater and fills 20 μ l.
3. vortex concussion mixing, of short duration centrifugal, makes solution collection on tube wall at the bottom of pipe.
Hatch 30-50 minute for 4.42 DEG C, hatch 5 minutes for 85 DEG C.After reaction terminates, of short duration centrifugal, be placed in cooled on ice.
The cDNA health obtained by reverse transcription reaction is that century 2 × TaqMasterMix (article No. CW0682) carries out Standard PCR, reaction system and condition as follows: each 2 μ l of 2 × TaqMasterMix25 μ l, upstream and downstream primer, cDNA0.5 μ g, to fill to 50 μ l.Reaction conditions is 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 30s, 30cycles; Last 72 DEG C extend 2min.
Sample 5 μ L, agarose gel electrophoresis detection is carried out to the product of pcr amplification, carry out cutting glue and reclaim and purifying, purified product is connected to pGM-T cloning vector, is transformed into subsequently in DH5 α competent cell.By the Auele Specific Primer screening positive clone that sequence is SEQIDNO.1 and SEQIDNO.2.Plasmid DNA is extracted after positive colony amplification, plasmid DNA adopts NanoDropND-1000 nucleic acid quantification instrument quantitatively (NanoDropTechnologies, Wilmington, Delaware) and for the preparation of typical curve, (standard DNA template concentration range is 10 as standard substance to do 10 times of serial dilutions
8-10
2copies/ μ l).
2.3 sensitivity experiments
Getting that recombinant plasmid dilutes in proportion is 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2individual copy/μ L, carries out quantitative fluorescent PCR, the detection sensitivity being the method with the minimum concentration of test positive.The method sensing range that this institute sets up is 10
8-10
2copies/ μ L, minimum concentrations is 100copies/ μ L.
2.4qRT-PCR detects GFOD2 gene expression amount
Get above-mentioned 74 routine sufferers of osteoporosis face peripheral bloods and 46 routine healthy human peripheral blood health be century blood rna extract test kit (article No. CW0538) and extract total serum IgE, and then carry out RT-PCR with UltraSYBR single stage method PCR kit for fluorescence quantitative (article No. CW0660).Concrete steps:
1. RNA template, primer, 2 × UltraSYBROneStepRT-qPCRBuffer (WithROX), SuperEnzymeMix and RNase-FreeWater are dissolved and be placed in for subsequent use on ice.
2.RT-PCR reaction system (25 μ l): 2 × UltraSYBROneStepRT-qPCRBuffer (WithROX) 12.5 μ l, upstream primer (10 μMs) 0.5 μ l, downstream primer (10 μMs) 0.5 μ l, SuperEnzymeMix0.5 μ l, add RNA template (final concentration is 10pg – 100ng), RNase-FreeWater fills to 25 μ l.
3. vortex concussion mixing, of short duration centrifugal, at the bottom of solution collection to pipe.
4. thermal cycler is preheating to 45 DEG C, PCR pipe is placed in thermal cycler, react by following reaction conditions: reverse transcription: 45 DEG C of 10min; 95 DEG C of 10min denaturations, connect 45 circulations: 95 DEG C of 15s, 60 DEG C of 60s.
Use SPSSForWindows11.5 software to qRT-PCR reaction result, related data adopts χ2-test,chi-square test and Fisher exact method method to process, and P < 0.05 has statistical significance; QRT-PCR reaction utilizes MedCalc statistical analysis software to calculate.
Two results
Real-time quantitative PCR amplification curve flex point is clear, and the overall collimation of amplification curve is good, shows that the amplification efficiency of each reaction tubes is close, and the limit is flat and without raising up now, exponent phase slope is comparatively large, illustrates that amplification efficiency is higher; Sample amplified production solubility curve is all unimodal, illustrates that amplified production only has one, is specific amplification; According to the relative quantification formula of qRT-PCR, compare the expression level of GFOD2 gene in osteoporosis peripheral blood and healthy human peripheral blood.Result display (specifically seeing Fig. 1): qRT-PCR stable amplification result, wherein the expression level of GFOD2 in sufferers of osteoporosis face peripheral blood is lower than healthy human peripheral blood, the former is about the latter's 3/10ths, the result of confluence analysis GFOD2 low expression in osteoporosis peripheral blood of above result verification high-throughput transcript profile expression data.
Embodiment 3 one kinds detects osteoporotic detection kit and using method
RNA extracts reagent: ultrapure RNA extracts test kit (article No. CW0597)
Fluorescent quantitation reagent: UltraSYBR single stage method PCR kit for fluorescence quantitative (article No. CW0660)
Quantitative fluorescent PCR reaction system and method:
RT-PCR reaction system (25 μ l): 2 × UltraSYBROneStepRT-qPCRBuffer (WithROX) 12.5 μ l, upstream primer (10 μMs) 0.5 μ l, downstream primer (10 μMs) 0.5 μ l, SuperEnzymeMix0.5 μ l, add RNA template (final concentration is 10pg – 100ng), RNase-FreeWater fills to 25 μ l.
Reaction regulates: reverse transcription: 45 DEG C of 10min; 95 DEG C of 10min denaturations, connect 30-40 circulation: 95 DEG C of 15s, 60 DEG C of 60s.
The present invention adopts high-flux sequence to filter out osteoporosis pathogenic related gene GFOD2, and binding molecule biological experiment is verified, confirms that GFOD2 has important effect in bone loss disorders.The present invention is that osteoporosis clinic diagnosis provides new target, has good potential applicability in clinical practice.
Claims (10)
1. the application in diagnosing osteoporosis preparation prepared by the reagent detecting GFOD2 gene and/or its expression product.
2. application according to claim 1, is characterized in that, diagnosing osteoporosis preparation detects the expression of GFOD2 gene and/or its expressing protein in peripheral blood.
3. application according to claim 1, is characterized in that, described diagnosing osteoporosis preparation comprises the expression detecting GFOD2 gene with fluorescence quantifying PCR method, method for gene chip.
4. application according to claim 3, is characterized in that, contains the primer of a pair specific amplification GFOD2 gene with the product of fluorescence quantifying PCR method detection GFOD2 gene; Gene chip comprises the probe with the nucleic acid array hybridizing of GFOD2 gene.
5. application according to claim 4, is characterized in that, the primer of described a pair specific amplification GFOD2 gene, and upstream primer sequence is SEQIDNO.1, and downstream primer sequence is SEQIDNO.2.
6. application according to claim 1, it is characterized in that, diagnosing osteoporosis preparation adopts immunization method to detect the expression of GFOD2 albumen, and preferably, what described immunization method detected GFOD2 protein expression is ELISA detection kit and/gold-immunochromatographyreagent reagent for assay box.
7.GFOD2 gene and/or its expression product are preparing the application in osteoporosis therapy preparation.
8. application according to claim 7, is characterized in that, treatment osteoporosis preparations promotes the expression of GFOD2 gene.
9. application according to claim 7, is characterized in that, containing the carrier promoting GFOD2 genetic expression in treatment osteoporosis preparations.
10. application according to claim 7, it is characterized in that, osteoporosis therapy preparation can adopt the expression of a kind of and/or several promotion GFOD2 gene in following method: comprise increase GFOD2 gene copy number, transfection is containing the over-express vector of GFOD2 gene; Activating the expression of GFOD2 gene, activate the promotor of regulation and control GFOD2 genetic expression, suppress the transcription factor of negative regulation GFOD2 genetic expression, adopting RNA perturbation technique to disturb suppressing suppression of GFOD2 genetic expression; Suppress to promote the microRNA transcriptional expression of GFOD2 gene mRNA degraded, import the microRNA promoting GFOD2 genetic expression; Import the molecule promoting goal gene proteins encoded, the albumen suppressing negative regulation GFOD2 genetic expression, promote the factor of GFOD2 genetic expression and the expression of albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610116151.2A CN105506172B (en) | 2016-03-01 | 2016-03-01 | The osteoporosis Blood diagnosis reagent of the GFOD2 containing detection and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610116151.2A CN105506172B (en) | 2016-03-01 | 2016-03-01 | The osteoporosis Blood diagnosis reagent of the GFOD2 containing detection and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105506172A true CN105506172A (en) | 2016-04-20 |
| CN105506172B CN105506172B (en) | 2019-04-09 |
Family
ID=55714462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610116151.2A Active CN105506172B (en) | 2016-03-01 | 2016-03-01 | The osteoporosis Blood diagnosis reagent of the GFOD2 containing detection and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105506172B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105981026A (en) * | 2014-02-06 | 2016-09-28 | 因姆内克斯普雷斯私人有限公司 | Biomarker signature method, and apparatus and kits therefor |
-
2016
- 2016-03-01 CN CN201610116151.2A patent/CN105506172B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105981026A (en) * | 2014-02-06 | 2016-09-28 | 因姆内克斯普雷斯私人有限公司 | Biomarker signature method, and apparatus and kits therefor |
Non-Patent Citations (5)
| Title |
|---|
| AURORA E. SERRALDE-ZUNIGA，ET AL: "Omental adipose tissue gene expression, gene variants,branched-chain amino acids, and their relationship with metabolic syndrome and insulin resistance in humans", 《GENES NUTR》 * |
| J. J. LI,ET AL: "Identification of candidate genes in osteoporosis by integrated microarray analysis", 《BONE JOINT RES》 * |
| MARTHA GUEVARA-CRUZ，ET AL: "Effect of a GFOD2 variant on responses in total and LDL cholesterol in Mexican subjects with hypercholesterolemia after soy protein and soluble fiber supplementation", 《GENE》 * |
| ZHAN-CHUN LI，ET AL: "Functional Annotation of Rheumatoid Arthritis and Osteoarthritis Associated Genes by Integrative Genome-Wide Gene Expression Profiling Analysis", 《PLOS ONE》 * |
| 叶青等: "类风湿关节炎患者骨质疏松情况分析", 《中西医结合研究》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105506172B (en) | 2019-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3469551B2 (en) | Novel method for detection and monitoring of endometrial and uterine cancer | |
| CN105063194A (en) | Parkinson diagnostic marker and application thereof | |
| CN110257499A (en) | A kind of diagnostic system or product and its application for predicting susceptibility of stroke or prognostic risk | |
| CN107937515B (en) | A kind of diagnosis and treatment gene target of Alzheimer and its application | |
| CN108034707B (en) | SPAG7 gene is preparing the application in diagnosis of dementia preparation | |
| CN105288659A (en) | Application of TENM1 gene and its expression product on diagnosis and treatment of papillary adenocarcinoma | |
| CN115948544A (en) | Use of CITED4 and/or METRN for differential diagnosis of degree of disc degeneration | |
| CN114164273B (en) | Squamous carcinoma prognosis marker, establishment method of prognosis risk assessment model and application of prognosis risk assessment model | |
| CN104984363B (en) | Applications of the ZMYM1 in Parkinson's diagnosis and treatment reagent is prepared | |
| CN110656169B (en) | Diagnostic markers for atrial fibrillation | |
| CN107779503A (en) | The related difference expression gene of Alzheimer and its application | |
| CN111690744B (en) | Biomarker for evaluating breast tumor progression and application thereof | |
| JP2019007759A (en) | Method for evaluating aging degree and kit for evaluating aging degree | |
| CN105506172A (en) | Osteoporosis blood diagnostic reagent with detection GFOD2 and application of osteoporosis blood diagnostic reagent | |
| EP4036233A1 (en) | Method for detecting brain tumor | |
| CN105695617A (en) | Tumor marker of papillary carcinoma and application of tumor marker | |
| CN110988346A (en) | Marker for auxiliary diagnosis of lung cancer and detection method | |
| US11497817B2 (en) | Senile dementia treatment formulation and application thereof | |
| CN109576361A (en) | A kind of biomarker relevant to ischemic cardiomyopathy occurrence and development | |
| KR102665392B1 (en) | Composition for diagnosing subclinical hypothyrodism and kit comprising the same | |
| CN105648079B (en) | A kind of osteoporosis blood testing target spot and its application | |
| CN108179181B (en) | Application of RDX gene in clinical medication | |
| CN105506171A (en) | LYRM2 (LYR motif-containing 2) gene and new use of expression product thereof | |
| CN109735617A (en) | Leucoderma genetic test marker and its application | |
| WO2014182726A2 (en) | Genetic markers for macular degeneration disorder treatment |
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 |
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 Yang Shen biology information technology company limited Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Patentee before: Beijing Yang Shen biology information technology company limited |
|
| CP02 | Change in the address of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211117 Address after: 266000 room 2503, Qianshan building, D2, Qingdao International Innovation Park Phase II, No.1 Keyuan Weiyi Road, Laoshan District, Qingdao City, Shandong Province Patentee after: Qingdao Yangshen biomedical 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. |
|
| TR01 | Transfer of patent right |