CN107858423A - For diagnosing/predicting the kit of sudden cardiac death - Google Patents
For diagnosing/predicting the kit of sudden cardiac death Download PDFInfo
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- CN107858423A CN107858423A CN201711408648.2A CN201711408648A CN107858423A CN 107858423 A CN107858423 A CN 107858423A CN 201711408648 A CN201711408648 A CN 201711408648A CN 107858423 A CN107858423 A CN 107858423A
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Abstract
The present invention relates to a kind of kit for being used to diagnosing/predicting sudden cardiac death,Including the specific primer for detecting rs3917 insertion/deletions site on COL1A2 genes to (SEQ ID No.1 SEQ ID No.6),The specific primer in rs72014506 insertion/deletions site is to (SEQ ID No.7 SEQ ID No.12) on MIR155HG genes,On CTH genes on the specific primer (SEQ ID No.13 SEQ ID No.18) and DSG2 genes in rs113044851 insertion/deletions site the specific primer in No. rs397729601 insertion insertion/deletion site to the one or more in (SEQ ID No.19 SEQ ID No.24).Use above kit of the present invention passes through the polymorphic risk assessed individual and suffer from SCD of insertion and deletion, the genotype in four sites of Conjoint Analysis contributes to the diagnosis and prediction for SCD to provide support and help in terms of molecular genetics, SCD risk assess and diagnosis on great application value.The detection specificity of the kit is good, high sensitivity, accuracy are good.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of kit for being used to diagnosing/predicting sudden cardiac death.
Background technology
Sudden cardiac death (Sudden Cardiac Death, SCD) refers to paroxysmal non-pre- as caused by cardiac reasons
Material is dead, shows as the burst loss of consciousness and circulation, the respiratory arrest occurred in a short time.It is limited to during " short-term " defined, there is mesh
During the person of hitting<L hours, it is 24 hours during no witness.Epidemiological study shows that the Chinese population SCD incidences of disease are about 0.42 ‰,
Other people faciations work as (0.40 ‰) with Asia, slightly below Europe and Nortlz American population (0.5 ‰ -1.0 ‰).But due to Chinese population number
Measure huge, SCD cases are far more than other countries.SCD is because having morbidity is unexpected, disease progression is quick and death is hurried etc.
Feature, the health of the serious threat mankind is far-reaching to patient and home influence, is always a great public health problem.In recent years
The incidence of disease for carrying out SCD raises and turns into one of important death cause year by year, therefore SCD is always that medical field is special for a long time
It is the key subjects faced in Forensic Identification practice.
Conventional numerous studies show that Different types of etiopathogenises and hazards can cause SCD, and the cause of the death is mostly that coronary artery is athero- hard
The property changed heart disease or fatal arrhythmia.Epidemiology survey discovery, traditional heart disease risk factors such as age, the past heart
Flesh infarct, low left cardiac ejection fraction etc. is SCD high risk factor.Meanwhile found after observing a large amount of sudden cardiac death events,
SCD has strong genetic predisposition, and inherent cause is one of important determinant that SCD occurs.As full-length genome association is ground
Study carefully the extensive use of (GWAS) and two generation sequencing technologies, increasing genetic marker is proved to take part in SCD regulation and control.SCD
The continuous discovery of correlated inheritance mark helps to further elucidate the specific mechanism of SCD generations, also by doing for SCD people at highest risk
Pre- and prevention provides valuable theoretical foundation and evaluation index.With molecule dissection (Molecular autopsy) concept
Occur, the accurate diagnosis in medical jurisprudence to SCD is expected to be possibly realized.
The chain of NTx protein alpha 2 (Collagen type I alpha 2chain, COL1A2) closes as extracellular matrix
The part of key composition NTx albumen, its protein function or expression quantity are abnormal to can induce a series of lethal cardiac muscle diseases
Disease.MIR155HG (miR-155host gene) gene is miR-155 host gene, and miR-155 is a typical more work(
Energy miRNA, play an important roll in cardiac function regulation.Cystathionineγ lyase (cystathionine gamma-
Lyase, CTH) expression directly affect the production of Endogenous Hydrogen Sulfide, and then generation, development to angiocardiopathy with activity
Have an impact.Important composition albumen of the desmoglein (desmoglein, DSG2) as desmosome, its abnormal expression will be led
Cause desmosome connection imperfect and then influence myocardial function, research has proven to proarrhythmia type right ventricular cardiomyopathy
(arrhythmogenic right ventricular dysplasia, ARVC) and desmosome protein family abnormal gene expression
There is direct relation.
It is in the prior art, polymorphic to the insertion/deletions of above-mentioned four kinds of genes to be reported with the research of SCD correlations,
Also the phase of SCD neurological susceptibility and diagnosis SCD is predicted not over the insertion/deletion polymorphic site on four kinds of genes of joint-detection
Close report.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide a kind of examination for being used to diagnosing/predicting sudden cardiac death
Agent box, the kit can be used in assessing the neurological susceptibility that individual suffers from SCD, and accurate detection individual suffers from SCD risk.
The invention provides a kind of kit for being used to diagnosing/predicting sudden cardiac death, including for detecting COL1A2 bases
Because upper rs3917 insertion/deletions site specific primer to No. rs72014506 insertion on, MIR155HG genes/
The specific primer in deletion polymorphism site on, CTH genes rs113044851 insertion/deletions site it is special
Property primer pair and DSG2 genes on No. rs397729601 insertion insertion/deletion site specific primer centering one
Kind is several；Wherein
On COL1A2 genes the specific primer in rs3917 insertion/deletions site to SEQ ID No.1 and
SEQ ID No.2、SEQ ID No.3(Tm=52 DEG C of value) and SEQ ID No.4 (Tm=55 DEG C of value) or SEQ ID No.5 (TmValue
=56 DEG C) and SEQ ID No.6 (TmValue=56 DEG C) shown in sequence respectively as sense primer and antisense primer；
The specific primer in rs72014506 insertion/deletions site is to SEQ ID on MIR155HG genes
No.7 and SEQ ID No.8, SEQ ID No.9 (Tm=53 DEG C of value) and SEQ ID No.10 (Tm=55 DEG C of value) or SEQ ID
No.11(Tm=55 DEG C of value) and SEQ ID No.12 (TmValue=53 DEG C) shown in sequence draw respectively as sense primer and antisense
Thing；
The specific primer in rs113044851 insertion/deletions site is to SEQ ID No.13 on CTH genes
With SEQ ID No.14, SEQ ID No.15 (Tm=52 DEG C of value) and SEQ ID No.16 (Tm=55 DEG C of value) or SEQ ID
No.17(Tm=55 DEG C of value) and SEQ ID No.18 (TmValue=56 DEG C) shown in sequence draw respectively as sense primer and antisense
Thing；
The specific primer in rs397729601 insertion/deletions site is to SEQ ID on DSG2 genes
No.19 and SEQ ID No.20, SEQ ID No.21 (Tm=56 DEG C of value) and SEQ ID No.22 (Tm=52 DEG C of value) or SEQ ID
No.23(Tm=54 DEG C of value) and SEQ ID No.24 (TmValue=54 DEG C) shown in sequence draw respectively as sense primer and antisense
Thing.
Preferably, for diagnosing/predicting that the kit of sudden cardiac death is inserted/lacked including No. rs3917 on COL1A2 genes
The specific primer of pleomorphism site is lost to SEQ ID No.1 and SEQ ID No.2；No. rs72014506 on MIR155HG genes
The specific primer in insertion/deletion site is to SEQ ID No.7 and SEQ ID No.8；On CTH genes
The specific primer in rs113044851 insertion/deletions site is to SEQ ID No.13 and SEQ ID No.14；DSG2
The specific primer in rs397729601 insertion/deletions site is to SEQ ID No.19 and SEQ ID on gene
No.20。
Further, 5 ' ends of each specific primer pair are marked with fluorescent dye.
Further, the concentration of each specific primer pair is 0.2-1.0 μm of ol/ μ l.
Further, kit also includes 1U/ μ l-2.5U/ μ l hot resistant DNA polymerases.Preferably, hot resistant DNA polymerase
For Taq DNA polymerase.
Further, kit also include 2.5mM-10mM deoxyribonucleoside triphosphates mixed liquor (dNTP mixed liquors) and
25mM-50mM magnesium salt solutions.
Further, magnesium salts is magnesium chloride or magnesium sulfate.
Further, kit also includes 5 × PCR or 10 × PCR reaction buffers and water.
Further, the storage temperature of kit is -20 DEG C.
Further, the application method of mentioned reagent box comprises the following steps：
(1) gather sample and extract its genomic DNA；
(2) use mentioned reagent box in one pair of which or it is several to specific primer to PCR amplifying genom DNAs, obtain
Amplified production；
(3) amplified production is separated, carries out insertion/deletion genotyping.
Further, in step (1), the genomic DNA of peripheral blood is extracted.
Further, the PCR amplification system in step (2) is：1 μ l DNA profilings；200 μM of specific primers pair, have
Justice, each 0.04 μ l of antisense primer；The μ l of 2.5U/ μ lTaq archaeal dna polymerases 0.08；The μ l of 2.5mM dNTP mixed liquors 0.2；25mM magnesium salts
The μ l of the aqueous solution 0.6；The μ l of 10 × PCR reaction buffers 1；Deionized water complements to 10 μ l.
Preferably, in step (2) specific primer to for rs3917 insertion/deletions site on COL1A2 genes
Specific primer to SEQ ID No.1 and SEQ ID No.2；Rs72014506 insertion/deletions are more on MIR155HG genes
The specific primer in state property site is to SEQ ID No.7 and SEQ ID No.8；No. rs113044851 is inserted/lacked on CTH genes
The specific primer of pleomorphism site is lost to SEQ ID No.13 and SEQ ID No.14；No. rs397729601 on DSG2 genes
The specific primer in insertion/deletion site is to SEQ ID No.19 and SEQ ID No.20.
Further, the PCR amplification conditions in step (2) are：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 52 DEG C are moved back
Fiery 30s, 72 DEG C of extension 1min, 30 PCR cycles；Last 72 DEG C of extensions 5min.
Further, in step (3), capillary electrophoresis separation is carried out using the gene sequencers of ABI 3500.
Further, by the testing result of step (3) infer individual carry rs3917 deletion forms allele,
Rs72014506 insert types allele, rs113044851 deletion forms allele, rs397729601 deletion form allele
Any one or a few when, it, which suffers from SCD risk, significantly to increase.When four kinds of genotype meet, what individual suffered from SCD can
Can property up to 72%.
Molecular biology research shows：No. rs3917 on COL1A2 genes, No. rs72014506 on MIR155HG genes,
The polymorphic different allelotypes of rs397729601 insertion and deletions can No. rs113044851 on CTH genes, on DSG2 genes
Influence the transcriptional activity of respective gene：Rs3917 deletion forms allele makes COLI1A2 tables by being combined with miR-296-3p
Reduced up to level；Rs72014506 deletion form allele makes the significantly high expression of MIR155HG and miR-155 maturation bodies, carries
Individual its CTH protein expression level rise of rs113044851 insert type allele, and carry No. rs397729601 and lack
Its individual DSG2 protein level of mistake type allele reduces.In addition, sent out by the SCD case-control studies of certain scale crowd
Existing, No. rs3917, the polymorphic deletion form allele of rs397729601 insertion and deletions and SCD are proportionate；
Rs72014506 deletion forms, rs113044851 insert types allele and SCD generations are negatively correlated, even in adjustment year
After the factors such as age, sex, these correlations still have.Therefore these insertion and deletions are polymorphic can be used for assessment individual to suffer from SCD's
Neurological susceptibility.
By such scheme, the present invention at least has advantages below：
Inventor confirms that seven bases (GGACAAC) insertion and deletion in the 3'UTR of COL1A2 genes is more first
Four bases (AGAG) insertion and deletion in state (rs3917), MIR155HG gene intron 2s is polymorphic
(rs72014506), four bases (TCTG) insertion and deletion polymorphic (rs113044851), a DSG2 in the 3'UTR of CTH genes
Conspicuousness be present with suffering from SCD risk in two bases (TG) insertion and deletion polymorphic (rs397729601) on gene in 3'UTR
Association；And drawn by (I) SIS algorithms：The genotype detection individual of four insertion and deletion polymorphic sites of use in conjunction suffers from SCD wind
The rate of accuracy reached 72% of danger.
The different specific primers of the present invention are designed for the insertion and deletion site on different genes, can specificity
The fragment for including insertion/deletion is amplified, is held fluorochrome label the 5 ' of Oligonucleolide primers by fluorescent labelling techniques,
A chain of product carries the fluorescent dye of labeled primer after PCR amplifications.By detecting different length fragment in capillary electricity
Mobility in swimming identifies different genotype, finds, is detected on DNA COL1A2 genes with reference to case-control study
Rs3917 insertion and deletions site carries deletion form allele, the rs72014506 insertion and deletions on MIR155HG genes
Site carries insert type allele, the rs113044851 insertion and deletions site on CTH genes carries deletion form equipotential
It is the susceptible types of SCD that rs397729601 insertion and deletions site on gene, DSG2 genes, which carries deletion form allele,.Cause
This, the technology pass through on the COL1A2 genes of joint-detection individual No. rs3917, No. rs72014506 on MIR155HG genes,
No. rs113044851 on CTH genes, on DSG2 genes rs397729601 insertion and deletions site genotype, can play pre-
Survey individual SCD neurological susceptibility and then the effect diagnosed to SCD individuals.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Brief description of the drawings
Fig. 1 is the gene sequencing figure and PAGE gel electropherotyping of COL1A2 genes of the present invention；
Fig. 2 is the gene sequencing figure and PAGE gel electropherotyping of MIR155HG genes of the present invention；
Fig. 3 is the gene sequencing figure and PAGE gel electropherotyping of CTH genes of the present invention；
Fig. 4 is the gene sequencing figure and PAGE gel electropherotyping of DSG2 genes of the present invention.
Embodiment
With reference to the accompanying drawings and examples, the embodiment of the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
Embodiment 1
The a kind of of the present invention be used to diagnosing/predict the kit of sudden cardiac death, including fluorochrome label is used to examine
The specific primer in rs3917 insertion/deletions site on COL1A2 genes is surveyed on, MIR155HG genes
The specific primer in rs72014506 insertion/deletions site is to rs113044851 insertion/deletions on, CTH genes
The spy in No. rs397729601 insertion insertion/deletion site on the specific primer pair and DSG2 genes of pleomorphism site
Specific primer pair.Wherein
The specific primer in rs3917 insertion/deletions site is to SEQ ID No.1 institutes on COL1A2 genes
The sequence shown is used as antisense primer as sense primer using the sequence shown in SEQ ID No.2；
The specific primer in rs72014506 insertion/deletions site is to SEQ ID on MIR155HG genes
Sequence shown in No.7 is used as antisense primer as sense primer using the sequence shown in SEQ ID No.8；
The specific primer in rs113044851 insertion/deletions site is to SEQ ID No.13 on CTH genes
Shown sequence is used as antisense primer as sense primer using the sequence shown in SEQ ID No.14；
The specific primer in rs397729601 insertion/deletions site is to SEQ ID on DSG2 genes
Sequence shown in No.19 is used as antisense primer as sense primer using the sequence shown in SEQ ID No.20.Above sequence is specific
As shown in table 1：
1 each primer sequence of table
Fluorochrome label is in 5 ' ends of the Oligonucleolide primers of primer, and a chain of product carries mark after PCR amplifications
Remember the fluorescent dye of primer.Also include the general components of PCR amplifications and Capillary Electrophoresis in kit：Taq archaeal dna polymerases,
DNTP mixed liquors, MgCl2Solution, PCR reaction buffers and deionized water.The application method of above kit is as follows：
(1) extraction of DNA profiling
Use the genomic DNA of poba gene group DNA extraction systems (non-centrifugation column type) extraction peripheral blood.
(2) duplication of PCR reactions-purpose fragment
Using four species-specific primers pair in mentioned reagent box, the DNA profiling of PCR amplification steps (1), amplification production is obtained
Thing.Wherein, PCR reaction systems cumulative volume is 10ul, including：1 μ l DNA profilings；200 μM of specific primers pair, eight upper and lower
Swim each 0.01 μ l of primer；The μ l of 2.5U/ μ lTaq archaeal dna polymerases 0.08；The μ l of 2.5mM dNTP mixed liquors 0.2；25mM magnesium salts is water-soluble
The μ l of liquid 0.6；The μ l of 10 × PCR reaction buffers 1；Deionized water is supplied.In Eppendorf Mastercycler nexus PCR
Reacted on amplification instrument, reaction condition is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C extend
1min, 30 PCR cycles；Last 72 DEG C of extensions 5min.
(3) insertion and deletion genotyping
Amplification terminates rear product and carries out capillary electrophoresis separation using the gene sequencers of ABI 3500 so as to obtain detection
The genotype of body.
Using from SCD cases with area normal community's physical examination peripheral blood sample as a control group, it is desirable to itself and case
Sample sex matches, age-matched (± 5 years old), no SCD family histories.Control group is with SCD case groups on COLIA2 genes
The gene frequency difference in rs3917 insertion/deletions site is shown in Table 2 with risk OR values；Control group and SCD cases
The group gene frequency difference in rs72014506 insertion/deletions site and risk OR values on MIR155HG genes
It is shown in Table 3；Control group and the gene frequency in SCD case groups rs113044851 insertion/deletions site on CTH genes
Difference is shown in Table 4 with risk OR values；Control group and SCD case groups rs397729601 insertion/deletions on DSG2 genes are more
The gene frequency difference in state property site is shown in Table 5 with risk OR values.Rs3917 insertion and deletions position i.e. on COL1A2 genes
Its individual risk for suffering from SCD that point carries deletion form allele is 1.82 times of ordinary people.On MIR155HG genes
Rs72014506 insertion and deletions site carries insert type allele, the rs113044851 insertion and deletions on CTH genes
The individual that site carries deletion form allele suffers from SCD risk and is higher than ordinary people.No. rs397729601 on DSG2 genes is inserted
Enter deletion segment and carry 1.62 times that the risk that the individual of deletion form allele suffers from SCD is ordinary people.
Specifier closes that to state the normal human individual of insertion and deletion site parting be SCD people at highest risk, should be carried in daily life
Excessive risk is realized, and checks UP, prevents trouble before it happens on time.
Table 2rs3917 insertion/deletions and SCD risk relevances
Table 3rs72014506 insertion/deletions and SCD risk relevances
Table 4rs113044851 insertion/deletions and SCD risk relevances
Table 5rs397729601 insertion/deletions and SCD risk relevances
Note：aLogistic regression analyses correct age and sex factor；CI：Confidential interval；OR：Relative risk.
Embodiment 2
Be used to diagnose/predict the kit of sudden cardiac death in the embodiment of the present invention 1, application method with embodiment 1, its
It is as follows to diagnose SCD embodiments：
COLIA2 genes, MIR155HG genes, gene sequencing figure and the PAGE gel electricity of CTH genes and DSG2 genes
Parting of swimming diagram difference is as Figure 1-4.A is template strand sequencing result example in figure, and coding strand is corresponded to polymorphic at underscore
Insertion and deletion on site；B is from Different Individual (numbering 1-14, wherein numbering 1,2,3,8,9 and No. 10 samples are through legal medical expert
Pathology anatomical diagnosis is sudden cardiac death, and remaining sample is normal controls group) DNA sample using the present invention PCR expand
Increasing system obtains product electrophoresis schematic diagram.
Fig. 1 shows that numbering 1 and 8 is deletion form homozygote, and numbering 2,4,5,6,7,11,12,13 and 14 is that insert type is homozygous
Son, remaining is heterozygote, and the individual for illustrating to carry deletion form allele is the susceptible types of SCD.
Fig. 2 shows that numbering 5,7 and 12 is deletion form homozygote, and numbering 1,3,4,6,9,10 and 14 is insert type homozygote,
Remaining is heterozygote, illustrates that the risk that the individual for carrying deletion form allele suffers from SCD is relatively low.
Fig. 3 shows that numbering 1,2,4,7,8,10,12 and 14 is deletion form homozygote, and numbering 6 and 11 is that insert type is homozygous
Son, remaining is heterozygote, illustrates that the risk that the individual for carrying insert type allele suffers from SCD is relatively low.
Fig. 4 shows that numbering 1,2,7 and 11 is deletion form homozygote, and numbering 4,5,8,9,10,12 is insert type homozygote,
Remaining is heterozygote, and the individual for illustrating to carry deletion form allele is the susceptible types of SCD.
Comprehensive analysis, be diagnosed as Genotyping of SCD 6 samples on four insertion and deletion polymorphic sites with it is susceptible
Genotype is highly consistent, wherein No. 1 and No. 3 samples comply fully with excessive risk genotype combination, 2,8,9 and No. 10 sample coincidence rates
75%.Remaining control group sample highest coincidence rate is only 50%.Illustrate that the accuracy rate that this kit diagnoses for SCD is higher, should
Use Huge value.
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and
Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Sequence table
<110>University Of Suzhou
<120>For diagnosing/predicting the kit of sudden cardiac death
<141> 2017-12-22
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tctagatgat ggaagagtaa cc 22
Claims (10)
- A kind of 1. kit for being used to diagnosing/predicting sudden cardiac death, it is characterised in that：Including for detecting COL1A2 genes The specific primer in rs3917 insertion/deletions site is to rs72014506 insertion/deletions on, MIR155HG genes The specific primer of pleomorphism site draws to the specificity in rs113044851 insertion/deletions site on, CTH genes On thing pair and DSG2 genes one kind of the specific primer centering in No. rs397729601 insertion insertion/deletion site or It is several；WhereinOn the COL1A2 genes specific primer in rs3917 insertion/deletions site to SEQ ID No.1 and Sequence difference shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 or SEQ ID No.5 and SEQ ID No.6 As sense primer and antisense primer；The specific primer in rs72014506 insertion/deletions site is to SEQ ID on the MIR155HG genes Shown in No.7 and SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10 or SEQ ID No.11 and SEQ ID No.12 Sequence is respectively as sense primer and antisense primer；The specific primer in rs113044851 insertion/deletions site is to SEQ ID No.13 on the CTH genes With the sequence shown in SEQ ID No.14, SEQ ID No.15 and SEQ ID No.16 or SEQ ID No.17 and SEQ ID No.18 Row are respectively as sense primer and antisense primer；The specific primer in rs397729601 insertion/deletions site is to SEQ ID on the DSG2 genes No.19 and SEQ ID No.20, SEQ ID No.21 and SEQ ID No.22 or SEQ ID No.23 and SEQ ID No.24 institutes The sequence shown is respectively as sense primer and antisense primer.
- 2. the kit according to claim 1 for being used to diagnosing/predicting sudden cardiac death, it is characterised in that：Each specificity 5 ' ends of primer pair are marked with fluorescent dye.
- 3. the kit according to claim 1 for being used to diagnosing/predicting sudden cardiac death, it is characterised in that：Each specificity The concentration of primer pair is 0.2~1.0 μM/μ l.
- 4. the kit according to claim 1 for being used to diagnosing/predicting sudden cardiac death, it is characterised in that：The reagent Box also includes 1U/ μ L~2.5U/ μ L hot resistant DNA polymerases.
- 5. the kit according to claim 1 for being used to diagnosing/predicting sudden cardiac death, it is characterised in that：The reagent Box also includes 2.5mM~10mM deoxyribonucleoside triphosphates mixed liquor and 25mM~50mM magnesium salt solutions.
- 6. the kit according to claim 5 for being used to diagnosing/predicting sudden cardiac death, it is characterised in that：The magnesium salts For magnesium chloride or magnesium sulfate.
- 7. the kit according to claim 1 for being used to diagnosing/predicting sudden cardiac death, it is characterised in that：The reagent Box also includes 5 × PCR or 10 × PCR reaction buffers and water.
- 8. the kit for being used to diagnose/predict sudden cardiac death according to any one of claim 1-7, its feature exist In its application method comprises the following steps：(1) gather sample and extract its genomic DNA；(2) several specific primer is expanded using the one pair of which in the kit any one of claim 1-8 or to PCR Increase the genomic DNA, obtain amplified production；(3) amplified production is separated, carries out insertion/deletion genotyping.
- 9. the kit according to claim 8 for being used to diagnosing/predicting sudden cardiac death, it is characterised in that in step (2) In PCR amplification system be：1 μ l DNA profilings；200 μM of specific primers pair, there are justice, each 0.01 μ l of antisense primer；2.5U/μ The μ l of lTaq archaeal dna polymerases 0.08；The μ l of 2.5mM dNTP mixed liquors 0.2；The μ l of 25mM magnesium salt solutions 0.6；10 × PCR reactions are slow The μ l of fliud flushing 1；Deionized water complements to 10 μ l.
- 10. the kit according to claim 8 for being used to diagnosing/predicting sudden cardiac death, it is characterised in that in step (2) the PCR amplification conditions in are：94 DEG C of pre-degeneration 3min；94 DEG C denaturation 30s, 52 DEG C annealing 30s, 72 DEG C extension 1min, 30 PCR cycle；Last 72 DEG C of extensions 5min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711408648.2A CN107858423B (en) | 2017-12-22 | 2017-12-22 | Kit for predicting susceptibility to sudden cardiac death |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711408648.2A CN107858423B (en) | 2017-12-22 | 2017-12-22 | Kit for predicting susceptibility to sudden cardiac death |
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| CN107858423A true CN107858423A (en) | 2018-03-30 |
| CN107858423B CN107858423B (en) | 2020-06-30 |
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| CN201711408648.2A Active CN107858423B (en) | 2017-12-22 | 2017-12-22 | Kit for predicting susceptibility to sudden cardiac death |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029853A (en) * | 2020-10-12 | 2020-12-04 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on HSPA1B gene insertion deletion polymorphic site |
| CN112063711A (en) * | 2020-10-12 | 2020-12-11 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on STIM1 gene insertion deletion polymorphic site |
| CN112159841A (en) * | 2020-10-10 | 2021-01-01 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on COX10 gene insertion deletion polymorphic site |
| CN113684273A (en) * | 2021-09-08 | 2021-11-23 | 百世诺(北京)医学检验实验室有限公司 | Dilated cardiomyopathy detection kit based on mutant DSG2 gene |
| WO2022077539A1 (en) * | 2020-10-12 | 2022-04-21 | 苏州大学 | Susceptibility detection kit for sudden cardiac death based on insertion-deletion polymorphic site of stat5a gene |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112159841A (en) * | 2020-10-10 | 2021-01-01 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on COX10 gene insertion deletion polymorphic site |
| CN112159841B (en) * | 2020-10-10 | 2022-03-01 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on COX10 gene insertion deletion polymorphic site |
| WO2022073259A1 (en) * | 2020-10-10 | 2022-04-14 | 苏州大学 | Kit for detecting susceptibility of sudden cardiac death on basis of insertion and deletion polymorphic sites of cox10 gene |
| CN112029853A (en) * | 2020-10-12 | 2020-12-04 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on HSPA1B gene insertion deletion polymorphic site |
| CN112063711A (en) * | 2020-10-12 | 2020-12-11 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on STIM1 gene insertion deletion polymorphic site |
| CN112029853B (en) * | 2020-10-12 | 2022-03-01 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on HSPA1B gene insertion deletion polymorphic site |
| WO2022077540A1 (en) * | 2020-10-12 | 2022-04-21 | 苏州大学 | Sudden cardiac death susceptibility detection kit based on stim1 gene insertion and deletion polymorphic sites |
| WO2022077539A1 (en) * | 2020-10-12 | 2022-04-21 | 苏州大学 | Susceptibility detection kit for sudden cardiac death based on insertion-deletion polymorphic site of stat5a gene |
| CN113684273A (en) * | 2021-09-08 | 2021-11-23 | 百世诺(北京)医学检验实验室有限公司 | Dilated cardiomyopathy detection kit based on mutant DSG2 gene |
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