CN103667433A - Kit for detecting susceptibility gene of glaucoma - Google Patents
Kit for detecting susceptibility gene of glaucoma Download PDFInfo
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- CN103667433A CN103667433A CN201210359914.8A CN201210359914A CN103667433A CN 103667433 A CN103667433 A CN 103667433A CN 201210359914 A CN201210359914 A CN 201210359914A CN 103667433 A CN103667433 A CN 103667433A
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- 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
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- 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
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- 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
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses a kit for detecting a susceptibility gene of glaucoma. The kit comprises specific primer pairs, specific fluorescent probe pairs, conventional components and the like, wherein the specific primer pairs and the specific fluorescent probe pairs are used for simultaneously detecting a No.rs3825942 SNP (single nucleotide polymorphism) site on an LOXL (lysyl oxidase-like)1 gene and a No.rs4236601 SNP site on a CAV (chicken anemia virus)1/CAV2 gene; the conventional components are used for fluorescent quantitative PCR (polymerase chain reaction) detection. The kit is used for evaluating the risk of suffering from glaucoma by individuals by simultaneously detecting SNP site genotypes of LOXL1 and CAV1/CAV2 closely related to glaucoma.
Description
Technical field
The present invention relates to molecular biology and medical field, more specifically, the present invention relates to a kind of test kit that detects glaucoma tumor susceptibility gene, by the mononucleotide polymorphism site genotype of the gene LOXL1 associated with glaucoma of detection simultaneously and CAV1/CAV2, assess individuality and suffer from glaucoma risk.
Background technology
Glaucoma is tertiary diseases causing blindness in the world, and typical clinical manifestation is that optic atrophy, carrying out property defect of visual field and intraocular pressure increase.Although definite pathogenesis is still not known, according to primary glaucoma, morbidity presents familial, and the frequently-occurring and gemellary research in glaucoma relatives, all shows that inherited genetic factors plays an important role in glaucomatous morbidity.Along with deepening continuously that molecular genetics and molecular biology are studied and applied in field of ophthalmology, glaucomatous research has had development at full speed and significant progress.
Glaucoma has heredity, represents that glaucoma has certain inherited pathogenic factor.In family, had the needs of patients of glaucoma medical history regularly toward examination in hospital, early discovery timely treatment are for curing glaucomatous key.
Glaucoma is that a kind of meeting affect optical neuron, and causes blind disease.Research office worker has checked 271 siblings and the discovery of glaucoma patients, and about 12% people suffers from glaucoma; Those initial screening results are not suffered from glaucoma again again by (159 people) in the people of screening, 7% people after developed glaucoma during the follow of 6 to 8 years, and 19% people is under a cloud, suffer from glaucoma.These data presentation glaucomas are heredity in the family, and risk is along with the age increases and increases.
Glaucoma has heredity, according to one, Britain, recently studies show that, if you have siblings to suffer from glaucoma, the risk that your eyes occur to damage so may be 4 times of common people's calculated risk.Based on these investigation results, there are the siblings of glaucoma patients preferably every two years will do one time eye examination.
At present, LOXL1 is confirmed relevant to the generation of glaucoma disease by scientist with CAV1/CAV2 gene.
Researchist has investigated the health data that a plurality of countries exceed 40,000 people, has found this genetic marker on the karyomit(e) of code name 7Q31.It is the variant of a mononucleotide, is the hereditary unit less than gene, and its position is near known two the gene C AV1s and CAV2 relevant with glaucoma.
Analyze and show, approximately 6% European carries a pair of this genetic marker, and they suffer from glaucomatous risk and exceed 60% than other Europeans.Compare with European, the ratio that Chinese carry this genetic marker is very low, exists the people of this mark to be less than 1% in genome.But, it is much bigger that the Chinese that carry this genetic marker suffer from glaucomatous risk--than other Chinese, exceed more than 5 times.
LOXL1 be scientific research personnel by large-scale genome is analyzed, finally find that LOXL1 gene and glaucoma on No. 15 karyomit(e) of human body are closely related.All exfoliative glaucoma cases are all attributable to the variation of this gene.Research group has altogether chosen 1.6 ten thousand glaucoma patients and has studied.Statistical study demonstration, the people who carries mutant gene suffers from the glaucomatous risk of exfoliative is much higher than normal people.
Research group further analyzes discovery, and the protein of LOXL1 genes encoding is relevant with the formation of spandex fiber, and if spandex fiber in eye abnormal accumulation, will bring out exfoliative glaucoma.
Summary of the invention
2 SNPs loci polymorphisms based on LOXL1 gene, CAV1/CAV2 gene can be used to assessment individuality and suffer from the basis of glaucoma risk, the invention provides a kind of test kit that detects glaucoma tumor susceptibility gene.
Test kit comprises:
The Auele Specific Primer that detects rs3825942 SNP Genetic polymorphism type on LOXL1 gene to and specificity fluorescent probe pair;
The Auele Specific Primer that detects rs4236601 SNP Genetic polymorphism type on CAV1/CAV2 gene to and specificity fluorescent probe pair;
Quantitative fluorescent PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl
2solution, reaction buffer, deionized water etc.).
Auele Specific Primer described in this test kit designs referring to for rs4236601 SNP site on rs3825942 SNP site on LOXL1 gene and CAV1/CAV2 gene, and energy specific amplification goes out to comprise the primer pair of the DNA fragmentation in these 2 SNPs sites.Designing this class primer pair is that those skilled in the art can be unlabored.Preferably, in test kit, comprise there is SEQ ID NO:1 and 2, the primer pair of sequence shown in 3 and 4.Auele Specific Primer synthesizes the synthetic technology of available routine.Those skilled in the art will appreciate that primer of the present invention is not limited to this two pairs of primers.
Specificity fluorescent probe described in this test kit designs referring to for rs4236601 SNP site on rs3825942 SNP site on LOXL1 gene and CAV1/CAV2 gene, can go out by fluorescent quantitative PCR technique specific detection the Taqman probe pair of these two SNPs loci gene types.Designing this class probe is that those skilled in the art can be unlabored.Preferably, in test kit, comprise there is SEQ ID NO:5 and 6, the Taqman probe pair of sequence shown in 7 and 8.Specificity fluorescent probe synthesizes the probe synthetic technology of available routine.Those skilled in the art can understand, specificity fluorescent Taqman probe of the present invention is to being not limited to this two pairs of probes, all can be used for fluorescence quantitative PCR method detect described in the present invention, be used for individual two the SNPs sites of suffering from glaucoma risk of assessment probe all within the scope of the present invention.
Component and the content of test kit of the present invention comprise:
5 μ l 10 X-fluorescence quantitative PCR reaction buffers,
0.5 μ l 25mM dNTP mixed solution,
3 μ l 25mM MgCl
2solution,
0.125 μ l (5units/ μ l) Taq archaeal dna polymerase,
20 μ M Auele Specific Primers are to each 0.225 μ l of every primer,
10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe,
Deionized water 26.625 μ l.
This test kit detects application for a person-portion, and the storage temperature of test kit is-20 ℃.
Embodiment:
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.
The use of embodiment 1. detection kit
The extraction of step 1:DNA template
With vacuum test tube, gather human peripheral blood, extract the genomic dna in blood.
Step 2: quantitative fluorescent PCR reaction
Use can detect the fluorescent quantificationally PCR detecting kit of glaucoma tumor susceptibility gene, wherein, comprises following primer pair and fluorescent probe pair:
Sense primer 1:5 '-TCCGTCTCCCAGCAA – 3 ' (SEQ ID NO:1)
Antisense primer 1:5 '-CGAAGCCGAGACCGA – 3 ' (SEQ ID NO:2)
Sense primer 2:5 '-TGACAGTCCTTTTCT – 3 ' (SEQ ID NO:3)
Antisense primer 2:5 '-AATTATATTAACAAA – 3 ' (SEQ ID NO:4)
Band VIC fluorophor probe 1:5 '-CACGGGGaCTCCGC-3 ' (SEQ ID NO:5)
Band FAM fluorophor probe 1:5 '-CACGGGGgCTCCGC-3 ' (SEQ ID NO:6)
Band VIC fluorophor probe 2:5 '-GTATTGTaTTTGTT-3 ' (SEQ ID NO:7)
Band FAM fluorophor probe 2:5 '-GTATTGTgTTTGTT-3 ' (SEQ ID NO:8)
Sense primer 1, antisense primer 1, with VIC fluorophor probe 1, with FAM fluorophor probe 1 specifically for the rs3825942 SNP loci polymorphism detecting on LOXL1 gene;
Sense primer 2, antisense primer 2, with VIC fluorophor probe 2, with FAM fluorophor probe 2 specifically for the rs4236601 SNP loci polymorphism detecting on CAV1/CAV2 gene;
2 independently quantitative fluorescent PCR reactions are carried out respectively in 2 SNPs sites, the system of each reaction is cumulative volume 10 μ l, and comprising concentration is DNA profiling 2 μ l, 1 μ l 10 X-fluorescence quantitative PCR reaction buffers, 0.1 μ l 25mM dNTP mixed solution, the 0.6 μ l 25mM MgCl of 20ng/ μ l
2the band VIC fluorescent probe of the sense primer of solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and antisense primer each 0.225 μ l, 10 μ M and each 0.25 μ l of band FAM fluorescent probe, deionized water 5.325 μ l.
On ABI9700 type pcr amplification instrument, react, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, and 95 ℃, 30 seconds of carrying out 60 circulations, 60 ℃, 1 minute.After finishing, reaction reads fluorescent amount on ABI7900 type quantitative real time PCR Instrument.
Step 3:SNP gene type assay
The those skilled in the art that are familiar with fluorescent quantitative PCR technique can, by the final sample fluorescence volume showing on identification quantitative real time PCR Instrument, can determine the genotype in detected SNP site according to the power of different sequence probe VIC and FAM fluorescent signal.
2. couples of patients of embodiment carry out the service of glaucoma tumor susceptibility gene detection
Step 1:DNA extracts
By hospital laboratory doctor, examinee is carried out the collection of peripheral blood, adopt vacuum test tube to gather peripheral blood, and extract genomic dna wherein.
Step 2: genotype tests
Use test kit provided by the invention, fluorescence quantitative PCR detection is carried out respectively in rs3825942 SNP site on the LOXL1 gene of examinee's genomic dna and the rs4236601 SNP site on CAV1/CAV2 gene, determine the genotype in these two SNPs sites.
Step 3: suffer from the analysis of glaucoma risk
By to the genotypic analysis of detected person SNPs, provide the analysis report list of suffering from glaucoma risk.In report, describe the height that detected person suffers from glaucomatous risk in detail, and to detected person, describe and understand the analysis report list of suffering from glaucoma risk by doctor in detail.
Claims (6)
1. a test kit that detects glaucoma tumor susceptibility gene, is characterized in that: comprise detect on LOXL1 gene on rs3825942 SNP site and CAV1/CAV2 gene simultaneously the Auele Specific Primer in rs4236601 SNP site to and specificity fluorescent probe to, Taq enzyme, dNTP mixed solution, MgCl
2solution, quantitative fluorescent PCR reaction buffer, deionized water.
2. test kit according to claim 1, it is characterized in that: described Auele Specific Primer designs referring to for rs4236601 SNP site on rs3825942 SNP site on LOXL1 gene and CAV1/CAV2 gene, energy specific amplification goes out to comprise the primer pair of the DNA fragmentation in these 2 SNPs sites.
3. test kit according to claim 1, it is characterized in that: described specificity fluorescent probe designs referring to for rs4236601 SNP site on rs3825942 SNP site on LOXL1 gene and CAV1/CAV2 gene, can go out by fluorescent quantitative PCR technique specific detection the Taqman probe pair of these 2 SNPs loci gene types.
4. test kit according to claim 1, is characterized in that: contained Auele Specific Primer has SEQ ID NO:1 and 2 to being selected from, the primer pair of sequence shown in 3 and 4.
5. test kit according to claim 1, is characterized in that: contained specificity fluorescent probe be selected from there is SEQ ID NO:5 and 6, the Taqman probe pair of sequence shown in 7 and 8.
6. test kit according to claim 1, is characterized in that: the component of test kit and content comprise 10 X-fluorescence quantitative PCR reaction buffers, 0.5 μ l 25mM dNTP mixed solution, 3 μ l 25mM MgCl
2solution, 0.125 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M Auele Specific Primers to each 0.225 μ l of every primer, 10 μ M specificity fluorescent probes to every probe each 0.25 μ l, deionized water 26.625 μ l, this test kit detects application for a person-portion, and the storage temperature of test kit is-20 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210359914.8A CN103667433A (en) | 2012-09-25 | 2012-09-25 | Kit for detecting susceptibility gene of glaucoma |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210359914.8A CN103667433A (en) | 2012-09-25 | 2012-09-25 | Kit for detecting susceptibility gene of glaucoma |
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| Publication Number | Publication Date |
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| CN103667433A true CN103667433A (en) | 2014-03-26 |
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|---|---|---|---|
| CN201210359914.8A Pending CN103667433A (en) | 2012-09-25 | 2012-09-25 | Kit for detecting susceptibility gene of glaucoma |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164498A (en) * | 2014-07-28 | 2014-11-26 | 电子科技大学附属医院·四川省人民医院 | Glaucoma screening kit |
| CN113430263A (en) * | 2021-08-27 | 2021-09-24 | 中国医学科学院北京协和医院 | Biomarker-based product for diagnosing glaucoma and application thereof |
-
2012
- 2012-09-25 CN CN201210359914.8A patent/CN103667433A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164498A (en) * | 2014-07-28 | 2014-11-26 | 电子科技大学附属医院·四川省人民医院 | Glaucoma screening kit |
| CN104164498B (en) * | 2014-07-28 | 2016-03-09 | 电子科技大学附属医院·四川省人民医院 | A kind of glaucomatous kit for screening |
| CN113430263A (en) * | 2021-08-27 | 2021-09-24 | 中国医学科学院北京协和医院 | Biomarker-based product for diagnosing glaucoma and application thereof |
| CN113430263B (en) * | 2021-08-27 | 2021-11-05 | 中国医学科学院北京协和医院 | Biomarker-based product for diagnosing glaucoma and application thereof |
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Application publication date: 20140326 |