CN104293885A - A kit for detecting a risk gene related to a rare genetic disease Gaucher disease - Google Patents
A kit for detecting a risk gene related to a rare genetic disease Gaucher disease Download PDFInfo
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- CN104293885A CN104293885A CN201310297184.8A CN201310297184A CN104293885A CN 104293885 A CN104293885 A CN 104293885A CN 201310297184 A CN201310297184 A CN 201310297184A CN 104293885 A CN104293885 A CN 104293885A
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- 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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Abstract
A kit for detecting a risk gene related to a rare genetic disease Gaucher disease is disclosed. The kit comprises a specific primer pair, a specific fluorescence probe pair, conventional components used for fluorogenic quantitative PCR detection, and the like, wherein the specific primer pair and the specific fluorescence probe pair are used for detecting the number rs131678 SNP site on a GBA gene. The kit evaluates the risk of suffering from the Gaucher disease of an individual by detecting the genotype of the single nucleotide polymorphic site of the GBA closely related to the Gaucher disease.
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 detecting rare inherited disease Gaucher disease relative risk gene, the risk that individuality suffers from Gaucher disease is assessed by the mononucleotide polymorphism site genotype detecting beta-glucoside lipase (β-the glucosidase)-glucocerebrosidase gene (glucerebrosidase gene, GBA) associated with Gaucher disease simultaneously.
Background technology
Gaucher disease (Gaucher disease, GD) is modal one in lysosomal storage disease, is autosomal recessive inheritance.Brady etc. find that in 1964 storing up of glucocerebroside is caused by beta-glucoside lipase (β-glucosidase)-glucocerebrosidase (glucerebrosidase, GBA) lacks.Glucocerebroside can be degraded into glucose and ceramide by β-glucocerebrosidase.This enzymatic defect can cause glucocerebroside to be assembled in mononuclear phygocyte system in vivo, is mainly gathered in spleen, lymphoglandula and myeloid tissue cell, hepatic stellate cell, central nervous system microglia, osteoclastic bone, pulmonary alveolar macrophage etc.Gaucher disease main clinical manifestation is: bone sticking is dead, skeleton deformity, hepatosplenomegaly, anaemia, thrombocytopenia.Minority case shows as the sings and symptoms of neurological involvement.Clinically, Gaucher disease is divided into three types according to neurological involvement degree: I type: non-neuropathy modification, modal type, accounts for 95%.Adult-onset, is slowly in progress, complicated clinical manifestation, and the organ from asymptomatic to serious is got involved and all can be showed, and minority case can show as parkinsonism; II type: acute forms pathotype, infancy falls ill, and has serious neurological system involvement, all with enclose the raw phase or death in 1 year of be born; III type: subacute or chronic forms pathotype, childhood onset, has a kind of Manifestations of nervous system at least, such as cerebellar ataxia, myoclonus, epileptic seizures or dementia, prognosis mala, usually in children or death in pubescence.
Gaucher disease is autosomal recessive inheritance, caused by glucocerebrosidase gene (glucerebrosidase gene, GBA) suddenlys change.GBA gene is positioned on karyomit(e) 1q21, has 11 exons.The GBA transgenation reported at present has kind more than 200, wherein common are N370S(c.1226 A > G), 84GG(c.84-85insG), L444P(c.1448T > C) and IVS2+1G → A.Research finds that N370S sudden change accounts for 78% in Jewry Gaucher disease people, and in non-Jewish national Gaucher patient, modal sudden change is then L444P(36%), be secondly N370S(29%).China's Gaucher patient GBA gene mutation analysis shows modal sudden change successively: L444P, F213I, R353W, and N370S sudden change is very rare in Chinese Gaucher patient.
Summary of the invention
Based on glucocerebrosidase gene (glucerebrosidase gene, GBA) 1 SNP site polymorphism on can be used to assessment individuality and suffers from the basis of Gaucher disease risk, the invention provides a kind of test kit detecting rare inherited disease Gaucher disease relative risk gene
Test kit comprises:
The Auele Specific Primer detecting rs131678 SNP Genetic polymorphism type on GBA 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 rs131678 SNP site on GBA gene, and energy specific amplification goes out to comprise the primer pair of the DNA fragmentation of this 1 SNP site.Designing this kind of primer pair is that those skilled in the art can be unlabored.Preferably, the primer pair with sequence shown in SEQ ID NO:1 and 2 is comprised in test kit.The synthetic technology of Auele Specific Primer to available routine is synthesized.Those skilled in the art will appreciate that primer of the present invention is not limited to this pair of primers.
Specificity fluorescent probe described in this test kit designs referring to for rs131678 SNP site on GBA gene, goes out the genotypic Taqman probe pair of this SNP site by fluorescent quantitative PCR technique specific detection.Designing this kind of probe is that those skilled in the art can be unlabored.Preferably, the Taqman probe pair with sequence shown in SEQ ID NO:3 and 4 is comprised in test kit.The probe synthetic technology of specificity fluorescent probe to available routine is synthesized.Those skilled in the art can understand, specificity fluorescent Taqman probe of the present invention is to being not limited to this pair probe, and all fluorescence quantitative PCR methods that can be used for detect and to be used for described in the present invention that assessment is individual suffers from the probe of a SNP site of Gaucher disease risk 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 μMs of Auele Specific Primers to each 0.225 μ l of every bar primer,
10 μMs of specificity fluorescent probes to each 0.25 μ l of every bar 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 DEG C.
Embodiment:
Below in conjunction with specific embodiment, set forth the present invention further.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.
The use of embodiment 1. detection kit
The extraction of step 1:DNA template
Gather human peripheral blood with vacuum test tube, extract the genomic dna in blood.
Step 2: quantitative fluorescent PCR reacts
Use can detect the fluorescent quantificationally PCR detecting kit of Gaucher disease relative risk gene, wherein, comprises following primer pair and fluorescent probe pair:
Sense primer 1:5 '-GTCTACTAAAAATACA – 3 ' (SEQ ID NO:1)
Antisense primer 1:5 '-GATTACAGGTGCCCAC – 3 ' (SEQ ID NO:2)
Band VIC fluorophor probe 1:5 '-TTAGCCaGGCTAG-3 ' (SEQ ID NO:3)
Band FAM fluorophor probe 1:5 '-TTAGCCgGGCTAG-3 ' (SEQ ID NO:4)
Sense primer 1, antisense primer 1, band VIC fluorophor probe 1, band FAM fluorophor probe 1 are specifically for the rs131678 SNP site polymorphism detected on GBA gene;
1 SNP site carries out 2 independently quantitative fluorescent PCR reactions respectively, the system of each reaction is cumulative volume 10 μ l, comprises DNA profiling 2 μ l, 1 μ l 10 X-fluorescence quantitative PCR reaction buffer, 0.1 μ l 25mM dNTP mixed solution, 0.6 μ l 25mM MgCl that concentration is 20ng/ μ l
2solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, the sense primer of 20 μMs and each 0.225 μ l of antisense primer, the band VIC fluorescent probe of 10 μMs and each 0.25 μ l of band FAM fluorescent probe, deionized water 5.325 μ l.
ABI9700 type PCR amplification instrument is reacted, and reaction conditions is 50 DEG C, 2 minutes, 95 DEG C, 10 minutes, 95 DEG C, 30 seconds that carry out 60 circulations, 60 DEG C, 1 minute.On ABI7900 type quantitative real time PCR Instrument, fluorescent amount is read after reaction terminates.
Step 3:SNP gene type assay
The those skilled in the art being familiar with fluorescent quantitative PCR technique can by the final sample fluorescence volume that identification quantitative real time PCR Instrument shows, and can determine the genotype of detected SNP site according to the power of different sequence probes VIC and FAM fluorescent signal.
Embodiment 2. couples of patients carry out the service of Gaucher disease relative risk gene detection
Step 1:DNA extracts
By hospital laboratory doctor, examinee is carried out to 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, respectively fluorescence quantitative PCR detection is carried out to the rs131678 SNP site on the GBA gene of examinee's genomic dna, determines the genotype of this SNP site.
Step 3: individuality suffers from the analysis of Gaucher disease risk
By to the genotypic analysis of detected person SNP, provide the analysis report list suffering from Gaucher disease risk.Describe the height that detected person suffers from Gaucher disease risk in report in detail, and suffer from the analysis report list of Gaucher disease risk to detected person's detailed description and deciphering by doctor.
Claims (6)
1. detect the test kit of rare inherited disease Gaucher disease relative risk gene, it is characterized in that: comprise the Auele Specific Primer that detects rs131678 SNP site on GBA gene 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, is characterized in that: described Auele Specific Primer designs referring to for rs131678 SNP site on GBA gene, and energy specific amplification goes out to comprise the primer pair of the DNA fragmentation of this 1 SNP site.
3. test kit according to claim 1, it is characterized in that: described specificity fluorescent probe designs referring to for rs131678 SNP site on GBA gene, goes out the genotypic Taqman probe pair of this 1 SNP site by fluorescent quantitative PCR technique specific detection.
4. test kit according to claim 1, is characterized in that: contained Auele Specific Primer has the primer pair of sequence shown in SEQ ID NO:1 and 2 to being selected from.
5. test kit according to claim 1, is characterized in that: contained specificity fluorescent probe is selected from the Taqman probe pair with sequence shown in SEQ ID NO:3 and 4.
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 μMs of Auele Specific Primers to each 0.225 μ l of every bar primer, 10 μMs of specificity fluorescent probes to each 0.25 μ l of every bar 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 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310297184.8A CN104293885A (en) | 2013-07-16 | 2013-07-16 | A kit for detecting a risk gene related to a rare genetic disease Gaucher disease |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310297184.8A CN104293885A (en) | 2013-07-16 | 2013-07-16 | A kit for detecting a risk gene related to a rare genetic disease Gaucher disease |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463085A (en) * | 2015-12-11 | 2016-04-06 | 上海新培晶医学检验所有限公司 | Kit for detecting Gaucher disease GBA gene mutation and detection method of kit |
| CN105755166A (en) * | 2016-05-19 | 2016-07-13 | 厦门人瑞生物医药科技有限公司 | GBA gene L444P mutation detection kit |
| CN106906280A (en) * | 2017-03-27 | 2017-06-30 | 广东华美众源生物科技有限公司 | A kind of glucocerebrosidase gene detecting kit and its detection method |
| CN107312834A (en) * | 2017-06-07 | 2017-11-03 | 广东华美众源生物科技有限公司 | Glucocerebrosidase gene detecting kit and its detection method |
Citations (1)
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| CN101787391A (en) * | 2009-01-23 | 2010-07-28 | 深圳出入境检验检疫局动植物检验检疫技术中心 | North American soybean sudden death pathogen detection probe, primer, kit and detection method |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101787391A (en) * | 2009-01-23 | 2010-07-28 | 深圳出入境检验检疫局动植物检验检疫技术中心 | North American soybean sudden death pathogen detection probe, primer, kit and detection method |
Non-Patent Citations (2)
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| GENBANK: "ss139369", 《GENBANK》 * |
| 田红和周淑芸: "Bcr基因断裂丛集区多态性分析", 《中国实验血液学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463085A (en) * | 2015-12-11 | 2016-04-06 | 上海新培晶医学检验所有限公司 | Kit for detecting Gaucher disease GBA gene mutation and detection method of kit |
| CN105755166A (en) * | 2016-05-19 | 2016-07-13 | 厦门人瑞生物医药科技有限公司 | GBA gene L444P mutation detection kit |
| CN106906280A (en) * | 2017-03-27 | 2017-06-30 | 广东华美众源生物科技有限公司 | A kind of glucocerebrosidase gene detecting kit and its detection method |
| CN106906280B (en) * | 2017-03-27 | 2020-06-19 | 广东华美众源生物科技有限公司 | Glucocerebrosidase gene detection kit and detection method thereof |
| CN107312834A (en) * | 2017-06-07 | 2017-11-03 | 广东华美众源生物科技有限公司 | Glucocerebrosidase gene detecting kit and its detection method |
| CN107312834B (en) * | 2017-06-07 | 2021-02-02 | 广东华美众源生物科技有限公司 | Glucocerebrosidase gene detection kit and detection method thereof |
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Application publication date: 20150121 |