CN109722475A - A kind of kit and its application method and application for the detection of senile dementia risk genes - Google Patents
A kind of kit and its application method and application for the detection of senile dementia risk genes Download PDFInfo
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- 206010039966 Senile dementia Diseases 0.000 title claims abstract description 41
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- 238000000034 method Methods 0.000 title claims abstract description 14
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- 101150115910 Kcnj15 gene Proteins 0.000 claims abstract description 14
- 101150003482 SORL1 gene Proteins 0.000 claims abstract description 14
- 101150004665 GCH1 gene Proteins 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 238000003753 real-time PCR Methods 0.000 claims abstract description 6
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Abstract
The invention discloses a kind of kit for the detection of senile dementia risk genes and its application method and application, the kit includes: the probe pair and primer pair for detecting the site rs72713460 of GCH1 gene；Detect the probe pair and primer pair in the site rs928771 of KCNJ15 gene；Detect the probe pair and primer pair in the site rs3824968 of SORL1 gene；2X PCR amplification liquid.The present invention is directed to Chinese group alzheimer's disease risk genes, with specific reference to KCNJ15 gene, GCH1 and SORL1 gene, design the kit containing specific primer and Taqman probe, fluorescent quantitative PCR is carried out using genomic DNA of the component in kit to the blood sample from people, the genotype in site to be checked is known that according to the type of the amount of the fluorescence of generation and fluorescence.Kit provided by the invention is ingenious in design, without accurate and reliable, high sensitivity of extracting genome, testing result, and senile dementia is vigilant in the reference that the result of genetic test is prevented as senile dementia, raising.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to it is a kind of for senile dementia risk genes detection kit and
Its application method and application.
Background technique
Senile dementia (Alzheimer, AD), also known as alzheimer's disease are a kind of to be mainly in the slow of the elderly group
Brain retrograde degeneration's disease of property.The disease in first discoveries in 1906 and is recorded by German doctor earliest, clinical manifestation early stage
For slight cognitive disorder, but with the development of the state of an illness, the loss of memory of patient is gradually aggravated, the decline of analysis and understanding ability,
There is mood change, behavioral disorder or even clouding of consciousness, final nervous system is seriously damaged, and leads to death.
According to statistics, there is patients with Alzheimer disease 1700-2500 ten thousand in the whole world.In China, estimation has about 6,450,000 patients.And
With the increase at age, the illness rate of AD will be also obviously improved, and have 20% the elderly to suffer from old age in 80-85 years old patient
It is dull-witted.Senile dementia also becomes the 4th cause of the death after heart disease, tumour and cerebral apoplexy, causes to patient home, society
Very big burden and pressure.However, being easy to be ignored by family numbers of patients, to lose due to the atypism of the disease early symptom
Remove optimal therapic opportunity.Many factors are related with the disease pathogenesis and the cause of disease.Most important of which is that inherent cause: AD tool
There is clustering of disease in family, 40% patient has Positive family history, is in autosomal dominant inheritance and multiple-factor inheritance.
Up to the present studies have shown that the variation of multiple genes and the onset risk of senile dementia are closely related, packet
Including is APOE1 gene, APOE2 gene, CYP46 gene, CTSD gene and IL1A gene, and late hair style AD patient is to a certain extent
With senile dementia genotype close relation.It is directed to the research of Chinese group Alzheimer's disease recently, it was found that in
The relevant risk genes of compatriots group Alzheimer's disease are such as: KCNJ15, GCH1 and SORL1 gene.
Therefore, the generation for preventing senile dementia earlier for Chinese group needs just to mention before senile dementia occurs
It is preceding that High risk group is gone out by the accurate screening of genetic test, and targeted health control is carried out to this crowd, it can just do
To real early prevention, in conjunction with other means periodic detections, early discovery, the early treatment of senile dementia are realized, really to reach
The purpose of personalized prevention is carried out in advance.
Summary of the invention
The present invention provides a kind of kit for the detection of senile dementia risk genes and its application method and applications.It should
The application method of kit is easy to operate, testing result is accurate and reliable, can be whether further to take targetedly health pipe
Reason and subsequent detection means provide reference frame.
The technical scheme adopted by the invention is as follows:
A kind of kit for the detection of senile dementia risk genes, the kit include: detection GCH1 gene
The probe pair and primer pair in the site rs72713460；Detect the probe pair and primer pair in the site rs928771 of KCNJ15 gene；
Detect the probe pair and primer pair in the site rs3824968 of SORL1 gene；2X PCR amplification liquid.
Further, the probe pair in the site rs72713460 of the detection GCH1 gene are as follows: will be as shown in SEQ NO:1
Probe A and the probe B equal proportion as shown in SEQ NO:2 be mixed to get；The rs72713460 of the detection GCH1 gene
The primer pair in site is P1 and P2；The sequence of described P1, P2 are respectively as shown in SEQ NO:3 and SEQ NO:4.
Further, the 5 ' ends of the probe A carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe B
VIC label is carried out, 3 ' ends carry out TAMRA label.
Further, the probe pair in the site rs928771 of the detection KCNJ15 gene are as follows: will be as shown in SEQ NO:5
Probe C and the probe D equal proportion as shown in SEQ NO:6 be mixed to get；The rs928771 of the detection KCNJ15 gene
The primer pair in site is P3 and P4；The sequence of described P3, P4 are respectively as shown in SEQ NO:7 and SEQ NO:8.
Further, the 5 ' ends of the probe C carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe D
VIC label is carried out, 3 ' ends carry out TAMRA label.
Further, the probe pair in the site rs3824968 of the detection SORL1 gene are as follows: will be as shown in SEQ NO:9
Probe E and the probe F equal proportion as shown in SEQ NO:10 be mixed to get；The detection SORL1 gene
The primer pair in the site rs3824968 is P5 and P6；The sequence of described P5, P6 are respectively as shown in SEQ NO:11 and SEQ NO:12.
Further, the 5 ' ends of the probe E carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe F
VIC label is carried out, 3 ' ends carry out TAMRA label.
Further, the component of the 2X PCR amplification liquid are as follows: the Tris-HCl, 20mM (NH that 50mM pH is 8.84)2SO4, 2.5mM MgSO4, 200 μM of dNTP, 0.05%-0.5%BSA, 0.01-0.05% gelatin, the poly- second two of 0.1%-0.5%
Alcohol octyl phenyl ether, 0.1-0.5M trehalose, 0.2-2M propylene glycol, 5% glycerol.
It is described to make the present invention also provides the application method of the kit for the detection of senile dementia risk genes
With method the following steps are included:
(1) blood collection: acquiring fresh blood, and heparin is added into blood to final concentration of 10U/ml；
(2) blood treatment: when carrying out PCR amplification, appropriate blood is taken to use 0.9% NaCl solution, according to the ratio of 1:8
Example will be stand-by after 9 times of blood progress of dilution；
(3) fluorescent quantitative PCR: taking 2X PCR amplification liquid, and treated whole blood is added thereto, is separately added into one kind
Probe pair and corresponding primer pair, remaining moisturizing carry out machine reaction；
(4) interpretation of result: judge whether senile dementia risk of dementia gene dashes forward according to fluorescent quantitative PCR result
Become.
The present invention also provides the kits for the detection of senile dementia risk genes in detection senile dementia wind
Application in dangerous gene loci.
The present invention is directed to Chinese group alzheimer's disease risk genes, with specific reference to KCNJ15 gene, GCH1 gene
And SORL1 gene, the kit containing specific primer and Taqman probe is designed, using the component in kit to coming from
The genomic DNA of the blood sample of people carries out fluorescent quantitative PCR, can be with according to the amount of the fluorescence of generation and the type of fluorescence
Know the genotype in site to be checked.
Kit provided by the invention is ingenious in design, without accurate and reliable, high sensitivity of extracting genome, testing result,
Senile dementia is vigilant in the reference that the result of genetic test is prevented as senile dementia, raising.
Specific embodiment
Embodiment 1
A kind of kit for the detection of senile dementia risk genes, comprising: the position rs72713460 of detection GCH1 gene
The probe pair and primer pair of point；Detect the probe pair and primer pair in the site rs928771 of KCNJ15 gene；Detect SORL1 gene
The site rs3824968 probe pair and primer pair；2X PCR amplification liquid；
Further, the probe pair in the site rs72713460 of the detection GCH1 gene are as follows: will be as shown in SEQ NO:1
Probe A and the probe B equal proportion as shown in SEQ NO:2 be mixed to get；The rs72713460 of the detection GCH1 gene
The primer pair in site is P1 and P2；The sequence of described P1, P2 are respectively as shown in SEQ NO:3 and SEQ NO:4.
Further, the 5 ' ends of the probe A carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe B
VIC label is carried out, 3 ' ends carry out TAMRA label.
Further, the probe pair in the site rs928771 of the detection KCNJ15 gene are as follows: will be as shown in SEQ NO:5
Probe C and the probe D equal proportion as shown in SEQ NO:6 be mixed to get；The rs928771 of the detection KCNJ15 gene
The primer pair in site is P3 and P4；The sequence of described P3, P4 are respectively as shown in SEQ NO:7 and SEQ NO:8.
Further, the 5 ' ends of the probe C carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe D
VIC label is carried out, 3 ' ends carry out TAMRA label.
Further, the probe pair in the site rs3824968 of the detection SORL1 gene are as follows: will be as shown in SEQ NO:9
Probe E and the probe F equal proportion as shown in SEQ NO:10 be mixed to get；The detection SORL1 gene
The primer pair in the site rs3824968 is P5 and P6；The sequence of described P5, P6 are respectively as shown in SEQ NO:11 and SEQ NO:12.
Further, the 5 ' ends of the probe E carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe F
VIC label is carried out, 3 ' ends carry out TAMRA label.
Further, the component of the 2X PCR amplification liquid are as follows: the Tris-HCl, 20mM (NH that 50mM pH is 8.84)2SO4, 2.5mM MgSO4, 200 μM of dNTP, 0.05%-0.5%BSA, 0.01-0.05% gelatin, the poly- second two of 0.1%-0.5%
Alcohol octyl phenyl ether, 0.1-0.5M trehalose, 0.2-2M propylene glycol, 5% glycerol.
Embodiment 2
A kind of application method of the kit for the detection of senile dementia risk genes, comprising the following steps:
(1) fresh blood is acquired, heparin is added into blood and is taken to final concentration of 10U/ml when carrying out PCR amplification
Appropriate blood uses 0.9% NaCl solution, will be stand-by after 9 times of blood progress of dilution according to the ratio of 1:8；
(2) PCR amplification: 25 μ L reaction systems are as follows: 2X PCR amplification liquid: 12.5 μ L；The 1 μ L of upstream primer of 10pmol；
1 μ L of 10pmol downstream primer；The probe pair of 10pmol: 0.5 μ L；Blood template: 1 μ L；Aqua sterilisa: 9 μ L；
The 2X PCR amplification liquid are as follows: the Tris-HCl of 50mM PH8.8,20mM (NH4)2SO4, 2.5mM MgSO4, 200 μ
The dNTP of M, 0.1% BSA, 0.02% gelatin, 0.1% Triton X-100, the trehalose of 0.2M, 0.2M the third two
Alcohol, 5% glycerol.
PCR amplification program is as follows:
Each probe pair and primer pair sequence are as shown in table 1:
Table 1
(3) interpretation of result:
As a result parting figure is analyzed, as a result as follows:
Reaction 1: for detecting the site rs72713460 of GCH1 gene, which examines site, and there are three types of genes in crowd
Type: GG, GT and TT, wherein carrying GG genotype is normal population, the crowd of remaining 2 kinds of genotype is compared with the people for carrying GG genotype
Group has higher risk to suffer from senile dementia；
Reaction 2: for detecting the site rs928771 of KCNJ15 gene, which examines site, and there are three types of genes in crowd
Type: GG, GT and TT, wherein carrying TT normal population, the crowd for carrying GT and GG genotype has higher risk to suffer from old be crazy about
It is slow-witted；
Reaction 3: for detecting the site rs3824968 of SORL1 gene, which examines site, and there are three types of genes in crowd
Type: AA, AT and TT, wherein carrying AA is normal population, the crowd for carrying AA and TT genotype, which should be noted, suffers from senile dementia
Risk；
According to above-mentioned testing result, it can be determined that whether above-mentioned inspected gene is normal, if subject's risk genes exist
Variation prompts the risk of possible illness, but whether illness also needs further to be checked, because senile dementia is by many factors
Influence, the prevention to senile dementia can be improved.If without gene unconventionality, substantially without the special specific aim of progress
Health control.
In conclusion senile dementia related locus detection method of the invention is ingenious in design, easy to operate, testing result is quasi-
It is really reliable, whether targeted health control and subsequent detection means further to be taken to provide reference frame.
It is above-mentioned to a kind of kit detected for senile dementia risk genes and its application method and to be answered referring to embodiment
It is illustrative without being restrictive with the detailed description carried out, several embodiments can be enumerated according to limited range,
Therefore the change and modification in the case where not departing from present general inventive concept, should belong within protection scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou Rui Chen Biotechnology Co., Ltd
<120>a kind of for the kit and its application method of the detection of senile dementia risk genes and application
<130> 1
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213>probe A
<400> 1
ctgatctacc taatttg 17
<210> 2
<211> 18
<212> DNA
<213>probe B
<400> 2
ctgatctacc taagttgg 18
<210> 3
<211> 26
<212> DNA
<213> P1
<400> 3
agataaaagt ggacagtgga tcagaa 26
<210> 4
<211> 17
<212> DNA
<213> P2
<400> 4
gggaccacag gcatgca 17
<210> 5
<211> 18
<212> DNA
<213>probe C
<400> 5
tgagatcaga agcatgga 18
<210> 6
<211> 16
<212> DNA
<213>probe D
<400> 6
atcagaagca gggact 16
<210> 7
<211> 28
<212> DNA
<213> P3
<400> 7
taaagcacac tgaagttaga gacacagt 28
<210> 8
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<212> DNA
<213> P4
<400> 8
gcacaaggta tgttggaatc ca 22
<210> 9
<211> 17
<212> DNA
<213>probe E
<400> 9
ccctctgctt cttgtgt 17
<210> 10
<211> 18
<212> DNA
<213>probe F
<400> 10
ccctctgcat cttgtgta 18
<210> 11
<211> 18
<212> DNA
<213> P5
<400> 11
cctggatgag gcccaaaa 18
<210> 12
<211> 27
<212> DNA
<213> P6
<400> 12
aaagatggga cctacctgta gtagaca 27
Claims (10)
1. a kind of kit for the detection of senile dementia risk genes, which is characterized in that the kit includes: detection GCH1
The probe pair and primer pair in the site rs72713460 of gene；It detects the probe pair in the site rs928771 of KCNJ15 gene and draws
Object pair；Detect the probe pair and primer pair in the site rs3824968 of SORL1 gene；2X PCR amplification liquid.
2. the kit according to claim 1 for the detection of senile dementia risk genes, which is characterized in that the detection
The probe pair in the site rs72713460 of GCH1 gene are as follows: the probe A as shown in SEQ NO:1 and as shown in SEQ NO:2
Probe B equal proportion is mixed to get；The primer pair in the site rs72713460 of the detection GCH1 gene is P1 and P2；It is described
The sequence of P1, P2 are respectively as shown in SEQ NO:3 and SEQ NO:4.
3. the kit according to claim 2 for the detection of senile dementia risk genes, which is characterized in that the probe
5 ' the ends of A carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe B carry out VIC label, and 3 ' ends carry out TAMRA
Label.
4. the kit according to claim 1 for the detection of senile dementia risk genes, which is characterized in that the detection
The probe pair in the site rs928771 of KCNJ15 gene are as follows: the probe C as shown in SEQ NO:5 and as shown in SEQ NO:6
Probe D equal proportion is mixed to get；The primer pair in the site rs928771 of the detection KCNJ15 gene is P3 and P4；It is described
The sequence of P3, P4 are respectively as shown in SEQ NO:7 and SEQ NO:8.
5. the kit according to claim 4 for the detection of senile dementia risk genes, which is characterized in that the probe
5 ' the ends of C carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe D carry out VIC label, and 3 ' ends carry out TAMRA
Label.
6. the kit according to claim 1 for the detection of senile dementia risk genes, which is characterized in that the detection
The probe pair in the site rs3824968 of SORL1 gene are as follows: the probe E as shown in SEQ NO:9 and as shown in SEQ NO:10
Probe F equal proportion is mixed to get；The primer pair in the site rs3824968 of the detection SORL1 gene is P5 and P6；It is described
The sequence of P5, P6 are respectively as shown in SEQ NO:11 and SEQ NO:12.
7. the kit according to claim 6 for the detection of senile dementia risk genes, which is characterized in that the probe
5 ' the ends of E carry out FAM label, and 3 ' ends carry out TAMRA label；5 ' the ends of the probe F carry out VIC label, and 3 ' ends carry out TAMRA
Label.
8. the kit according to claim 1 for the detection of senile dementia risk genes, which is characterized in that the 2X
The component of PCR amplification liquid are as follows: the Tris-HCl, 20mM (NH that 50mM pH is 8.84)2SO4, 2.5mM MgSO4, 200 μM of dNTP,
0.05%-0.5%BSA, 0.01-0.05% gelatin, 0.1%-0.5% Triton X-100,0.1-0.5M trehalose,
0.2-2M propylene glycol, 5% glycerol.
9. the user of the kit according to any one of claims 1 to 8 for the detection of senile dementia risk genes
Method, which is characterized in that the application method the following steps are included:
(1) blood collection: acquiring fresh blood, and heparin is added into blood to final concentration of 10U/ml；
(2) blood treatment: when carrying out PCR amplification, taking appropriate blood to use 0.9% NaCl solution, will according to the ratio of 1:8
It is stand-by after the dilution of 9 times of blood progress；
(3) fluorescent quantitative PCR: taking 2X PCR amplification liquid, and treated whole blood is added thereto, is separately added into a kind of probe
Pair and corresponding primer pair, remaining moisturizing carry out machine reaction；
(4) interpretation of result: judge whether senile dementia risk of dementia gene mutates according to fluorescent quantitative PCR result.
10. the kit according to any one of claims 1 to 8 for the detection of senile dementia risk genes is old in detection
Application in dementia risk genes site.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811575967 | 2018-12-22 | ||
| CN2018115759677 | 2018-12-22 |
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| Publication Number | Publication Date |
|---|---|
| CN109722475A true CN109722475A (en) | 2019-05-07 |
Family
ID=66300590
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| Application Number | Title | Priority Date | Filing Date |
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| CN201910126240.9A Pending CN109722475A (en) | 2018-12-22 | 2019-02-20 | A kind of kit and its application method and application for the detection of senile dementia risk genes |
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| CN113604583A (en) * | 2021-08-10 | 2021-11-05 | 河南省畜牧总站 | Method for detecting growth traits of goat KCNJ15 gene under assistance of CNV marker and special kit thereof |
| WO2024055378A1 (en) * | 2022-09-13 | 2024-03-21 | 北京湃德智健科技有限公司 | Protein-antigen combination for detecting autoantibodies in alzheimer's disease, and use thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113604583A (en) * | 2021-08-10 | 2021-11-05 | 河南省畜牧总站 | Method for detecting growth traits of goat KCNJ15 gene under assistance of CNV marker and special kit thereof |
| CN113604583B (en) * | 2021-08-10 | 2024-04-02 | 河南省畜牧总站 | Method for auxiliary detection of growth traits by goat KCNJ15 gene CNV markers and special kit thereof |
| WO2024055378A1 (en) * | 2022-09-13 | 2024-03-21 | 北京湃德智健科技有限公司 | Protein-antigen combination for detecting autoantibodies in alzheimer's disease, and use thereof |
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Application publication date: 20190507 |