CN109666678A - Detect the kit of marfan's syndrome - Google Patents
Detect the kit of marfan's syndrome Download PDFInfo
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- CN109666678A CN109666678A CN201910084244.5A CN201910084244A CN109666678A CN 109666678 A CN109666678 A CN 109666678A CN 201910084244 A CN201910084244 A CN 201910084244A CN 109666678 A CN109666678 A CN 109666678A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- 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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- 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
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- 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
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The present invention provides a kind of mutated genes, it is characterised in that: it is that occur c.4460A > G mutation on the basis of mankind's FBN1 gene.The present invention also provides purposes of the related reagent of detection mankind FBN1 gene mutation site c.4460A > G in the screening agent for preparing marfan's syndrome.The variation in the FBN1 gene c.4460A site > G in the preparation applied to marfan's syndrome auxiliary diagnostic box, can be achieved the purpose that screening by the present invention.
Description
Technical field
The present invention relates to the fields SNP, in particular to SNP relevant to marfan's syndrome.
Background technique
Ma Fan (Marfan) syndrome, also there is congenital mesodermal dysplasia, Marchesani syndrome, arachodactylia
The title of sign, dolichostenomelia, it is characterized in that surrounding connective tissue is malnutritive, skeletal abnormality, interior eye diseases and Cardiovascular abnormality,
It is a kind of using connective tissue as the genetic disease of basic defect.Marfan's syndrome is to report 15 by Marfan (1896) earliest
Year girl, have it is special very thin and grow four limbs.To 1902, this sign was known as arachodactylia by Achard.Salle(1921)
Once 1 baby for suffering from this syndrome was dissected, discovery has acleistocardia.To 1931, Weve confirmed that this syndrome is dominant
Genetic disease simultaneously thinks to be caused by mesoderm tissues dysplasia.
Marfan's syndrome can not eradicate, and can only carry out limited alleviation to specific symptom by operation or drug.By
The most patients for the treatment of can survive to the middle age, usually die of aortic aneurysm rupture and heart failure.
Therefore, by correctly diagnosing, prevent newborn from suffering from this hereditary disease, it is particularly significant.
The mutation of FBN1 gene is one of the reason of leading to marfan's syndrome, and gene diagnosis also becomes that make a definite diagnosis horse at present all comprehensive
The important means of simulator sickness.However, be not any site of forementioned gene mutation it is all related to marfan's syndrome；Pass through detection
Existing minority SNP site diagnoses the testing result that marfan's syndrome also inevitably causes false negative.
Summary of the invention
The technical problem to be solved by the present invention is providing new FBN1 gene SNP site and they are all comprehensive in preparation horse
Purposes in simulator sickness kit for screening.
Firstly, the present invention provides a kind of mutated genes, it is characterised in that: it is gone out on the basis of mankind's FBN1 gene
Now following mutation obtains:
C.4460A > G: the 4460th bit base A of code area is mutated into G.
Secondly, the present invention also provides the related reagents of detection mankind FBN1 gene mutation site c.4460A > G to prepare
Purposes in the screening agent of marfan's syndrome.
Purposes as the aforementioned, the screening agent of the marfan's syndrome further include detecting FBN1 gene mutation site c.718C
The reagent of > T；C.718C the > T sports T by C for the 718th bit base of code area.
Purposes as the aforementioned, the reagent include detection mankind FBN1 gene mutation site c.4460A > G and
C.718C the related reagent of > T；It further include optional for expanding comprising the alkali of the gene coding region mankind FBN1 the 4460th, 718
The related reagent of the nucleic acid fragment of base.
Purposes as the aforementioned, c.4460A > G is related to c.718C > T's for the detection mankind FBN1 gene mutation site
Reagent is sequencing reagent.
Purposes as the aforementioned, c.4460A > G is related to c.718C > T's for the detection mankind FBN1 gene mutation site
Reagent is that quantitative fluorescent PCR reagent, restriction fragment length polymorphism method reagent or single-strand conformation polymorphism analysis are used
Reagent.
The present invention also provides a kind of kit for screening of marfan's syndrome, it is characterised in that: it includes optional is used for
Detect the related reagent of mankind FBN1 gene mutation site c.4460A > G.
Kit as the aforementioned, it further includes the reagent for detecting FBN1 gene mutation site c.718C > T；It is described c.718C
> T sports T by C for the 718th bit base of code area.
Kit as the aforementioned, the reagent include detection mankind FBN1 gene mutation site c.4460A > G and
C.718C the related reagent of > T；It further include optional for expanding comprising the alkali of the gene coding region mankind FBN1 the 4460th, 718
The related reagent of the nucleic acid fragment of base.
Kit as the aforementioned, the detection mankind FBN1 gene mutation site c.4460A > G and the c.718C phase of > T
Pass reagent is that sequencing reagent, quantitative fluorescent PCR reagent, restriction fragment length polymorphism method reagent or single stranded conformational are more
State property analyrical reagent.
Kit of the invention, by detect FBN1 gene SNP site c.4460A > G (and c.718C > T) come auxiliary
The screening of marfan's syndrome is helped, there is reliable specificity.Kit of the invention fails to detect in normal person described prominent
Become, and making a definite diagnosis in affected members in marfan's syndrome family detected, all detect the mutation, testing result has can
Repeatability.
The reagent of the SNP site c.4460A > G (and c.718C > T) of detection FBN1 gene provided by the present invention is being made
The purposes of standby marfan's syndrome kit for screening.And the detection reagent can be including sequencing reagent, quantitative fluorescent PCR examination
Agent, restriction fragment length polymorphism method reagent, single-strand conformation polymorphism analysis reagent or sequencing reagent etc. are a variety of
SNP detection reagent, the prospect for converting production are very wide.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
By the following examples, the specific embodiment of experimental example form is made above content of the invention further
It is described in detail.But this should not be interpreted as to the scope of the above subject matter of the present invention is limited to the following embodiments.It is all to be based on the present invention
The technology that above content is realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is marfan's syndrome pedigree chart: solid squares or round expression marfan's syndrome patient；Arrow expression is first demonstrate,proved
Person；Slash indicates late；A, B is corresponding in turn to 1, No. 2 family.
Fig. 2 is SNP site sequencer map of the present invention.
Specific embodiment
Embodiment kit of the invention and application method
Whole components, content and application method in kit of the present invention are as follows:
1.PCR amplifing reagent (50 person-portion):
PCR amplification reagent is used to expand the section of DNA sequence where SNP site, and composition is shown in Table 1.
1 PCR amplification reagent of table
| Component | Concentration | Volume |
| PCR mixed liquor | 2× | 1200μl |
| Primer pair | 10μM | 100μl |
| Pure water | 2ml |
PCR mixed liquor includes ingredient needed for the Standard PCRs such as Taq enzyme, dNTP, magnesium ion in table 1；Wherein primer pair information is such as
Shown in table 2.
2 FBN1 gene magnification the primer of table
2.FBN1 genetic mutation typing detection reagent (50 person-portion):
The reagent includes component as shown in table 3.
3 FBN1 genetic mutation typing detection reagent of table
| Component | Volume |
| Serum alkaline phosphatase | 100μl |
| Restricted excision enzyme | 6μl |
| Purification buffer | 6μl |
| Bigdye Mix | 15μl |
| 5×buffer | 120μl |
| ddH2O | 1ml |
| F primer | Each 100 μ l |
The F primer is sequencing (parting detection) the primer, wherein the c.4460A corresponding particular sequence in the site > G
(SEQ ID NO.5) are as follows: AGATTGGGCCCTGTTCTTTT；C.718C the corresponding particular sequence in the site > T (SEQ ID NO.6)
For GCATGATGGTTCCTGCTTTT.
3. standard DNA sample: with FBN1 gene c.4460A > G and the c.718C wild homozygote/heterozygosis in the site > T
Sub- DNA, each 50 μ l.
4. application method:
1) DNA is extracted
Patient whole blood (EDTA is anticoagulant) 2ml is taken, its genomic DNA is extracted.
2) contain the DNA fragmentation of SNP site detected by PCR amplification, each mutational site PCR amplification system is such as
Under:
| Component | Concentration | Volume |
| Sample DNA | 50ng/ μ l or more | 1μl |
| PCR reagent mixed liquor | 2× | 10μl |
| Primer pair | 10μM | 2μl |
| Pure water | 7μl |
Reaction condition (specific annealing temperature is shown in Table 2):
PCR product detection:
PCR product, the effect of observation PCR reaction are detected with 2% agarose gel electrophoresis, and determines that it exists as template
The amount being added in subsequent reactions.
3) Sanger sequencing detection
Step 1: PCR product purifies
System:
| Component | Volume |
| PCR product | 4μl |
| Serum alkaline phosphatase | 2μl |
| Restricted excision enzyme | 0.1μl |
| Purification buffer | 0.1μl |
Reaction condition:
Step 2: Sanger is sequenced
Using aforementioned typing detection reagent as sequencing amplifing reagent, the Sanger of the PCR product for first step purifying is surveyed
Sequence.
If sequencing result is mutated into G or the 718th bit base of code area in the 4460th bit base A of code area and is dashed forward by C
Become T, and it is bimodal heterozygosis occur, then illustrates the detection sample with marfan's syndrome.
The relationship in the mutational site and marfan's syndrome in order to further illustrate the present invention, and kit of the present invention
Effect provides following experimental example.
The verifying in experimental example mutational site
1. method
2 marfan's syndrome familys are recruited, complete orthopaedics, eyesight, angiocarpy have all been carried out to all family members
Systems inspection confirms it with marfan's syndrome.3 and 1 marfan's syndrome disease have been examined successively in family 1,2
People, according to family member's introduction, two familys respectively have 1 late member also to suffer from marfan's syndrome (family map is as shown in Figure 1).
In addition 1086 have been recruited and has not suffered from the people of marfan's syndrome as control.
The FBN1 gene of each member of aforementioned family and aforementioned control crowd are expanded, Sanger sequencing is carried out.
2. result
Sequencing result shows, the affected members that family 1,2 successively carry c.718C > T, c.4460A > G heterozygosis is prominent
Become；And non-diseased member and control crowd have no the mutation in aforementioned any site in family.Normal and mutational site sequencer map is such as
Shown in Fig. 2.
To sum up, the variation in FBN1 gene c.4460A site > G (and c.718C > T) is applied to all synthesis of horse by the present invention
In the preparation for levying auxiliary diagnostic box, it can achieve the purpose that screening.
SEQUENCE LISTING
<110>People's Hospital, Sichuan Prov.
<120>it is a kind of based on FBN1 gene c.4460A>the marfan's syndrome kit for screening in the site G
<130> GY008-2019P014909CC
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
agattgggcc ctgttctttt 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ttgggaataa ggtcccctct 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
gcatgatggt tcctgctttt 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
gcagtcagcg aaattgtgaa 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<400> 5
agattgggcc ctgttctttt 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence
<400> 6
gcatgatggt tcctgctttt 20
Claims (10)
1. a kind of mutated gene, it is characterised in that: it is that occur following mutation on the basis of mankind's FBN1 gene to obtain:
C.4460A > G: the 4460th bit base A of code area is mutated into G.
2. detecting the related reagent of mankind FBN1 gene mutation site c.4460A > G in the screening agent for preparing marfan's syndrome
In purposes.
3. purposes according to claim 2, which is characterized in that the screening agent of the marfan's syndrome further includes detection
The reagent of FBN1 gene mutation site c.718C > T；C.718C the > T sports T by C for the 718th bit base of code area.
4. purposes according to claim 3, it is characterised in that: the reagent includes detection mankind FBN1 gene mutation position
Put c.4460A > G and the c.718C related reagent of > T；It further include optional for expanding comprising the gene coding region mankind FBN1
4460th, the related reagent of the nucleic acid fragment of 718 bit bases.
5. purposes according to claim 4, it is characterised in that: the detection mankind FBN1 gene mutation site is c.4460A
> G and the c.718C related reagent of > T are sequencing reagent.
6. purposes according to claim 4, it is characterised in that: the detection mankind FBN1 gene mutation site is c.4460A
> G and the c.718C related reagent of > T be quantitative fluorescent PCR reagent, restriction fragment length polymorphism method reagent or
Single-strand conformation polymorphism analysis reagent.
7. a kind of kit for screening of marfan's syndrome, it is characterised in that: it includes optional for detecting mankind's FBN1 gene
The related reagent of mutational site c.4460A > G.
8. kit according to claim 7, it is characterised in that: the kit further includes detection FBN1 gene mutation position
Put the reagent of c.718C > T；C.718C the > T sports T by C for the 718th bit base of code area.
9. kit according to claim 8, it is characterised in that: the reagent includes detection mankind FBN1 gene mutation
Site c.4460A > G and the c.718C related reagent of > T；It further include optional for expanding comprising mankind FBN1 gene coding
Area the 4460th, 718 bit bases nucleic acid fragment related reagent.
10. kit according to claim 9, it is characterised in that: the detection mankind FBN1 gene mutation site
C.4460A > G and c.718C the related reagent of > T be sequencing reagent, quantitative fluorescent PCR reagent, restriction fragment length polymorphism
Property method reagent or single-strand conformation polymorphism analysis reagent.
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| CN201910084244.5A CN109666678A (en) | 2019-01-29 | 2019-01-29 | Detect the kit of marfan's syndrome |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484617A (en) * | 2019-09-03 | 2019-11-22 | 郑州大学第一附属医院 | A kind of FBN1 C2223A mutation and its application influencing the diagnosis and treatment of people's marfan's syndrome |
| CN110527686A (en) * | 2019-09-25 | 2019-12-03 | 百世诺（北京）医疗科技有限公司 | A kind of marfan's syndrome gene detecting kit |
| CN113980971A (en) * | 2021-11-02 | 2022-01-28 | 百世诺（北京）医疗科技有限公司 | Mutant Marfan syndrome pathogenic gene FBN1 and application thereof |
| CN114015768A (en) * | 2021-12-14 | 2022-02-08 | 中国医学科学院阜外医院 | Application of reagent for detecting FBN1 gene mutation site c.G3901T in preparation of kit for diagnosing Marfan syndrome |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484617A (en) * | 2019-09-03 | 2019-11-22 | 郑州大学第一附属医院 | A kind of FBN1 C2223A mutation and its application influencing the diagnosis and treatment of people's marfan's syndrome |
| CN110527686A (en) * | 2019-09-25 | 2019-12-03 | 百世诺（北京）医疗科技有限公司 | A kind of marfan's syndrome gene detecting kit |
| CN113980971A (en) * | 2021-11-02 | 2022-01-28 | 百世诺（北京）医疗科技有限公司 | Mutant Marfan syndrome pathogenic gene FBN1 and application thereof |
| CN113980971B (en) * | 2021-11-02 | 2022-05-06 | 百世诺（北京）医疗科技有限公司 | Mutant Marfan syndrome pathogenic gene FBN1 and application thereof |
| CN114015768A (en) * | 2021-12-14 | 2022-02-08 | 中国医学科学院阜外医院 | Application of reagent for detecting FBN1 gene mutation site c.G3901T in preparation of kit for diagnosing Marfan syndrome |
| CN114015768B (en) * | 2021-12-14 | 2023-10-10 | 中国医学科学院阜外医院 | Application of reagent for detecting FBN1 gene mutation site c.G3901T in preparation of kit for diagnosing Marfan syndrome |
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