CN103757091B - Sudden cardiac death rapid gene detection kit and detection method - Google Patents
Sudden cardiac death rapid gene detection kit and detection method Download PDFInfo
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Abstract
The invention discloses a kind of sudden cardiac death rapid gene detection kit, including 7 SNP polymorphic detections corresponding to detection KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B gene.Invention additionally discloses a kind of sudden cardiac death high-speed gene test method.Beneficial effects of the present invention is as follows: is detected by half-nest type AS PCR amplification and agarose gel electrophoresis, carries out Genotyping according to DNA electrophoretic band, filters out the individuality with potential sudden cardiac death risk, thus reaches to prevent the purpose of sudden cardiac death；The method is easy and simple to handle, and the time is short in detection, low cost, highly sensitive, and high specificity has good social benefit, has a extensive future.
Description
Technical field
The present invention relates to biological gene detection technique field, be specially a kind of sudden cardiac death rapid gene detection kit
And detection method.
Background technology
Refer to due to heart generation heart electrical disorder or mental and physical efforts in sudden cardiac death (sudden cardiac death, SCD)
The sudden death that exhaustion makes heart lose effective contraction and occurs.In recent years, SCD case takes place frequently, and its quantity is in going up year by year
The situation risen.At present, clinically the conventional detection means of SCD are mainly carried out examination to family history, personal history, carry out simultaneously
Physique and Electrocardioscopy, if there being positive findings, look into the dynamic heart in echocardiogram, exercise stress test and 24 hours the most further
Electrograph etc..But these detection means specific aims are not strong, and recall rate is low, has bigger limitation.
Research to sudden cardiac death both at home and abroad shows, the dead exists the phenomenon of some gene mutation in genomic level,
Mostly show as SNP or base deletion polymorphism.SCD has onset urgency, fast, the case fatality rate high that is in progress, and therefore, passes through
Early gene examination, risk assessment are evaded the generation of SCD and are just particularly important.
At present, sudden cardiac death makes SCD gene screening be possibly realized with gene pleiomorphism relevance achievement in research.Research table
Bright, KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B, 7 genes phase notable with sudden cardiac death
Close.In 7 genes, KCNE1, KCNH2, KCNJ11 belong to potassium-channel related gene, and SCN5A belongs to sodium-ion channel and is correlated with
Gene, CACNA1C, CACNB2B belong to calcium channel related gene.These ion channels are at myocardial action potential different times
Playing an important role, its gene mutation would generally affect the 26S Proteasome Structure and Function of ion channel.NUP155 is that a coding nucleopore is combined
The gene of thing component, its major function is to control inhereditary material mRNA by nucleus to cytoplasmic transhipment, is one to be in and compare
The situation monitoring gene of upstream, it is possible to other a lot of genes and protein expression are regulated and controled, thus causes atrial fibrillation and send out
Raw, generally threaten the health of Young crowd.Some trickle changes of NUP155 gene, it is also possible to increase common distributing
Property atrial fibrillation occur danger.
In China Han sudden cardiac death crowd, it is widely present KCNE1, KCNH2, KCNJ11 SNP and changes
Becoming, thus cause the sudden death of Ventricular Tachycardia, therefore, the sudden death that potassium-channel related gene causes belongs to high-risk gene and dashes forward
Modification.Clinical study results shows, the sudden death that sodium-ion channel related gene SCN5A sudden change causes, sudden cardiac death crowd
In account for 1-5 ‰；The sudden cardiac death that calcium channel related gene CACNA1C, CACNB2B sudden change causes then accounts for 0.1-0.3 ‰,
Belong to low degree of hazard saltant type；Coding nuclear Pore Complex component gene NUP155 gene be in Chinese han population find with
The peculiar gene of sudden cardiac death significant correlation, therefore its sudden change of examination has special meaning for Chinese population sudden cardiac death
Justice.
Summary of the invention
It is an object of the invention to based on above-mentioned prior art, it is provided that a kind of operation is simple, testing cost is low, to heart source
Property sudden death can play the sudden cardiac death rapid gene detection kit of prevention effect.
Another object of the present invention is to provide a kind of sudden cardiac death high-speed gene test method.
In order to realize the object of the invention, the present invention adopts the technical scheme that: sudden cardiac death rapid gene detection reagent
Box, the minimum primer mixture including detecting KCNE1, KCNH2, KCNJ11 gene polymorphic site.
Wherein, the primer mixture in detection KCNE1 gene polymorphic site is:
SEQ NO. 1: 5 '-GTGCCTGGGAAGTTTGAGC-3 ' (sequence 1)；
SEQ NO.2: 5 '-GCAGGTCCCCCCGCAGAA-3 ' (sequence 2)；
SEQ NO.3: 5 '-GCAGGTCCCCCCGCAGAG-3 ' (sequence 3)；
SEQ NO.4: 5 '-CCGTTCTTTCCCAGTCTCAT-3 ' (sequence 4)；
And mole ratio is SEQ NO.1:SEQ NO.2/ SEQ NO.3:SEQ NO.4=1:1:1；When detection, dense
Degree is 0.3ul-0.7ul for the consumption of the above-mentioned primer (SEQ NO. 1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) of 10uM.
The primer mixture in detection KCNH2 gene polymorphic site is:
SEQ NO.5:5 '-GCCGTGCTGTTCTTGCTC-3 ' (sequence 5)；
SEQ NO.6: 5 '-AGGACAAGTATGTGACGGCTC-3 ' (sequence 6)；
SEQ NO.7: 5 '-AGGACAAGTATGTGACGGCTT-3 ' (sequence 7)；
SEQ NO.8:5 '-GATTGGGGATCTGGTGGAA-3 ' (sequence 8).
And mole ratio is SEQ NO.5:SEQ NO.6/ SEQ NO.7:SEQ NO.8=1:1:1, when detection, dense
Degree is 0.3ul-for the consumption of the above-mentioned primer (SEQ NO. 5, SEQ NO.6, SEQ NO.7 and SEQ NO.8) of 10uM
0.7ul；
The primer mixture in detection KCNJ11 gene polymorphic site is:
SEQ NO.9:5 '-CTCATCGTGCAGAACATCGTG-3 ' (sequence 9)；
SEQ NO.10: 5 '-CGCAAGAGCATGATCATCCA-3 ' (sequence 10)；
SEQ NO.11: 5 '-CGCAAGAGCATGATCATCCG-3 ' (sequence 11)；
SEQ NO.12: 5 '-TGGCCGGGCTACATACCA-3 ' sequence 12)；
And mole ratio is SEQ NO.9:SEQ NO.10/ SEQ NO.11:SEQ NO.12=1:1:1；In detection
Time, concentration is the consumption of the above-mentioned primer (SEQ NO. 91, SEQ NO.10, SEQ NO.11 and SEQ NO.12) of 10uM
For 0.3ul-0.7ul；
Also include the primer mixture detecting SCN5A gene polymorphic site, wherein, detection SCN5A gene polymorphic site
Primer mixture is:
SEQ NO.13: 5 '-ACAGTGATGCTGGCTGGAAG-3 ' (sequence 13)；
SEQ NO.14: 5 '-GAAGTACTTCTTCTCCCCGTC-3 ' (sequence 14)；
SEQ NO.15:5 '-GAAGTACTTCTTCTCCCCGTT-3 ' (sequence 15)；
SEQ NO.16:5 '-ATGTTGATGAGGCTTATCTGGTT-3 ' (sequence 16)；
And mole ratio is SEQ NO.13:SEQ NO.14/ SEQ NO.15:SEQ NO.16=0.2-0.5:1:1；
Detection time, concentration be the consumption of the above-mentioned SEQ NO.13 primer of 10uM be 0.1ul-0.3ul, concentration is the above-mentioned of 10uM
SEQ NO.14, SEQ NO.15 and SEQ NO.16 primer consumption are 0.3ul-0.7ul.
And also include the primer mixture detecting CACNA1C, CACNB2B gene polymorphic site, wherein, detect CACNA1C
The primer mixture in gene polymorphic site is:
SEQ NO.17:5 '-GCCTGCAATAGCTTGAAATAAGG-3 ' (sequence 17)；
SEQ NO.18:5 '-GGTGGGGATGTGCTCAGGTA-3 ' (sequence 18)；
SEQ NO.19: 5 '-GTGGGGATGTGCTCAGGTG-3 ' (sequence 19)；
SEQ NO.20:5 '-AGAAGGGAAAGAAGGGAATGAG-3 ' (sequence 20)；
And mole ratio is SEQ NO.17:SEQ NO.18/ SEQ NO.19:SEQ NO.20=1:0.8-1.2:1；?
During detection, concentration is the consumption of the above-mentioned primer (SEQ NO. 17, SEQ NO.18, SEQ NO.19 and SEQ NO.20) of 10uM
For 0.3ul-0.7ul；
The primer mixture in detection CACNB2B gene polymorphic site is:
SEQ NO.21:5 '-GCACTTGCTCTGGGACAT-3 ' (sequence 21)；
SEQ NO.22:5 '-CCCAGCACCGCTCTTCCGC-3 ' (sequence 22)；
SEQ NO.23:5 '-TCCCAGCACCGCTCTTCCGT-3 ' (sequence 23)；
SEQ NO.24:5 '-TGATTCTGGCTTGTTGGATA-3 ' (sequence 24)；
And mole ratio is SEQ NO.21:SEQ NO.22/ SEQ NO.23:SEQ NO.24=0.125:1:1；In inspection
During survey, concentration be the consumption of the above-mentioned SEQ NO.21 primer of 10uM be 0.05ul-0.2ul, concentration is the above-mentioned primer of 10uM
SEQ NO.22, SEQ NO.23 and SEQ NO.24 consumption are 0.6ul-1ul.
Also include detecting NUP155 gene polymorphic site, wherein, the primer mixture in detection NUP155 gene polymorphic site
For:
SEQ NO.25: 5 '-GGAGAATCGCTTGAACCC-3 ' (sequence 25)；
SEQ NO.26: 5 '-CTGACGCTGGTTCATGTCAA-3 ' (sequence 26)；
SEQ NO.27: 5 '-CTGACGCTGGTTCATGTCAG-3 ' (sequence 27)；
SEQ NO.28: 5 '-TATTGCGTGGTGCTAAGGTT-3 ' (sequence 28)；
And mole ratio is SEQ NO.25:SEQ NO.26/ SEQ NO.27:SEQ NO.28=0.4:1:1；In inspection
During survey, concentration be the consumption of the above-mentioned SEQ NO.25 primer of 10uM be 0.1ul-0.3ul, concentration is the above-mentioned primer of 10uM
The consumption of SEQ NO.26, SEQ NO.27 and SEQ NO.28 is 0.3ul-0.7ul.
Described sudden cardiac death rapid gene detection kit also includes that amplifing reagent, described amplifing reagent include that PCR delays
Rush liquid, dNTPs mixture, hot start Taq polymerase and ultra-pure water；
Preferably, described amplifing reagent includes 10 times of PCR buffer solutions (wherein, Tris-HCl pH8.3 100mM； KCl
500 mM； MgCl215mM), each concentration of component is the dNTPs mixture of 2.5mM, the hot start Taq polymerase (Taq of 5U/ μ l
HS) consumption is 0.1-0.125 μ l.
Wherein, described PCR buffer solution (wherein, Tris-HCl pH8.3 100mM; KCl 500 mM; MgCl2
15mM), each concentration of component of described dNTPs mixture is 2.5mM, and hot start Taq polymerase (Taq HS) is 5U/ μ l.
Described kit also includes the Genotyping reagent after amplification, and the Genotyping reagent after described amplification includes
Agarose, TAE buffer solution, DNA molecular amount standard (DNA Marker), DNA non-toxic dye and DNA sample-loading buffer.Described DNA
Non-toxic dye can be Biotium Gelred non-toxic dye.
Preferably, amplifing reagent is made up of the component of volumes below:
For the primer corresponding to above-mentioned KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B
In, SEQ NO.1, SEQ NO.5, SEQ NO.9, SEQ NO.13, SEQ NO.17, SEQ NO.21, SEQ NO.25 are on internal reference
Trip primer, SEQ NO.2, SEQ NO.6, SEQ NO.10, SEQ NO.14, SEQ NO.18, SEQ NO.22, SEQ NO.26,
SEQ NO.3, SEQ NO.7, SEQ NO.11, SEQ NO.15, SEQ NO.19, SEQ NO.23, SEQ NO.27 are that polymorphism is drawn
Thing, SEQ NO.4, SEQ NO.8, SEQ NO.12, SEQ NO.16, SEQ NO.20, SEQ NO.24, SEQ NO.28 are downstream
Primer.The concentration of described internal reference upstream primer is 10uM, and the concentration of described polymorphism primer is 10uM, described downstream primer
Concentration is 10uM.For described KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B, described internal reference
The consumption of upstream primer, polymorphism primer and downstream primer presses the above.
Sudden cardiac death high-speed gene test method, its step includes:
(1), the DNA of sample genome extracts；
(2), amplification and the detection analysis of amplified production；
Wherein, PCR amplifing reagent is made up of the component of volumes below:
For the primer corresponding to above-mentioned KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B
In, SEQ NO.1, SEQ NO.5, SEQ NO.9, SEQ NO.13, SEQ NO.17, SEQ NO.21, SEQ NO.25 are on internal reference
Trip primer, SEQ NO.2, SEQ NO.6, SEQ NO.10, SEQ NO.14, SEQ NO.18, SEQ NO.22, SEQ NO.26,
SEQ NO.3, SEQ NO.7, SEQ NO.11, SEQ NO.15, SEQ NO.19, SEQ NO.23, SEQ NO.27 polymorphism are drawn
Thing, SEQ NO.4, SEQ NO.8, SEQ NO.12, SEQ NO.16, SEQ NO.20, SEQ NO.24, SEQ NO.28 are downstream
Primer.The concentration of described internal reference upstream primer is 10uM, and the concentration of described polymorphism primer is 10uM, described downstream primer
Concentration is 10uM.For described KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B, described internal reference
The consumption of upstream primer, polymorphism primer and downstream primer presses the above.
Wherein, described PCR buffer solution (wherein, Tris-HCl pH8.3 100mM; KCl 500 mM; MgCl2
15mM), each concentration of component of described dNTPs mixture is 2.5mM, and hot start Taq polymerase (Taq HS) is 5U/ μ l；
PCR amplification program is as follows:
(3) agarose gel electrophoresis analysis, is done.
Described amplification use AS-PCR(ApoE gene) carry out, then use agarose gel electrophoresis and
Gel imaging system carries out polymorphism parting detection.
Wherein, the human gene group DNA detected is for using Chelex method, magnetic bead extraction method or phenol/chloroform extraction method to coming
Source sample carries out processing the DNA obtained；Described source sample is to derive from the filter paper blood cake/buccal swab sample of the mankind, FTA
Card blood cake or blood.
Wherein, primer SEQ NO.1, SEQ NO.5, SEQ NO.9, SEQ NO.13, SEQ NO.17, SEQ NO.21,
SEQ NO.25 is internal reference upstream primer；SEQ NO.2、SEQ NO.6、SEQ NO.10、 SEQ NO.14、SEQ NO.18、SEQ
NO.22、SEQ NO.26、SEQ NO.3、SEQ NO.7、SEQ NO.11、SEQ NO.15、SEQ NO.19、SEQ NO.23、SEQ
3 ' end second the bases of NO.27 are respectively base mismatch, for detection upstream primer；SEQ NO.4、SEQ NO.8、SEQ
NO.12, SEQ NO.16, SEQ NO.20, SEQ NO.24, SEQ NO.28 is internal reference and the shared downstream primer of detection.
By AS-PCR and agarose gel electrophoresis specific detection sudden cardiac death gene；Primer for genetic test
For: SEQ NO.1, SEQ NO.5, SEQ NO.9, SEQ NO.13, SEQ NO.17, SEQ NO.21, SEQ NO.25, SEQ
NO.2、SEQ NO.6、SEQ NO.10、SEQ NO.14、SEQ NO.18、SEQ NO.22、SEQ NO.26、SEQ NO.3、SEQ
NO.7、SEQ NO.11、SEQ NO.15、SEQ NO.19、SEQ NO.23、SEQ NO.27、SEQ NO.4、SEQ NO.8、SEQ
NO.12, SEQ NO.16, SEQ NO.20, SEQ NO.24, SEQ NO.28, the amplification of described gene uses polymerase chain reaction
Should realize, use agarose gel electrophoresis to detect.
The present invention chooses KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B, 7 and heart source property
The polymorphic site of the gene of sudden death significant correlation is as examination target spot, and application AS-PCR technology realizes quickly detection, examination and carries
The potential sudden death person at risk in SCD risk gene polymorphic site, thus reach to prevent the purpose of SCD generation.
The present invention detects KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B7 gene simultaneously
SNP(SNP) polymorphic site.The detection of each SNP need 4 PCR primers (a pair upstream and downstream primer, two
The polymorphic primer of bar), need altogether to design 28 primers.The detection of each SNP needs to be two pipe PCR amplifications, 7 SNP, adds a pipe cloudy
Control, is 15 pipe PCR altogether.Owing to 15 pipe pcr amplification reactions synchronize to carry out, in order to realize heart source property in same PCR instrument
The high efficiency of sudden death detection, specifically, it is desirable to the Tm value (annealing temperature) of 36 PCR primer is close, for this setting in PCR primer
Meter needs to do substantial amounts of DAN sequence alignment software analysis work, and the optimization of Tm value needs to determine design by substantial amounts of experimental result
Reasonability.Based on above reason, the detection of this kit each gene SNP polymorphism is the most relatively independent but also connects each other,
Indivisible.
Beneficial effects of the present invention is as follows: detected by half-nest type AS-PCR amplification and agarose gel electrophoresis, according to
DNA electrophoretic band carries out Genotyping, filters out the individuality with potential sudden cardiac death risk, reaches to prevent sudden cardiac death
Purpose；The method is easy and simple to handle, and the time is short in detection, low cost, highly sensitive, and high specificity has good social benefit,
Have a extensive future.
Accompanying drawing explanation
Fig. 1 is sudden cardiac death rapid gene detection kit Genotyping electrophoretogram 1 of the present invention；
Fig. 2 is that sudden cardiac death rapid gene detection kit Genotyping electrophoretogram 2(of the present invention accepts Fig. 1)；
Fig. 3 is that sudden cardiac death rapid gene detection kit of the present invention is to each typing gene polymorphisms electrophoresis
7,22,13, No. 35 sequencer maps that sample is corresponding in figure.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail explanation.
Sudden cardiac death rapid gene detection kit, including the Genotyping reagent after amplifing reagent and amplification；
Described amplifing reagent includes PCR buffer solution, dNTPs mixture, hot start Taq polymerase, primer mixture and ultra-pure water；
Genotyping reagent after described amplification: reagent agarose, TAE buffer solution, DNA molecular amount standard, DNA non-toxic dye and
DNA sample-loading buffer.Described DNA sample-loading buffer (DNA loading buffer, 6X), is a kind of through suitably improvement
The DNA sample-loading buffer of six times of conventional concentrations, with bromophenol blue as indicator, after being diluted to 1X, proportion is the biggest, during loading
Easily sinking, and color is high-visible, this DNA sample-loading buffer is commercially commercially available.
Described amplifing reagent includes that amplifing reagent includes 10 times of PCR buffer solutions (wherein, Tris-HCl pH8.3 100mM;
KCl 500 mM; MgCl215mM), each concentration of component is the dNTPs mixture of 2.5mM, the hot start Taq polymerase of 5U/ μ l
(Taq HS) consumption is 0.1-0.125 μ l.Preferably, hot start Taq polymerase (Taq HS) consumption of 5U/ μ l is 0.125 μ l.Its
In, dNTP mixture is triphosphoric acid guanine deoxyribonucleoside acid dGTP, triphosphoric acid adenyl-deoxyribonucleotide dATP, three phosphorus
Acid thymidylic acid dTTP, triphosphoric acid deoxycytidylic acid dCTP tetra-nucleotide forms.
Sudden cardiac death rapid gene detection kit, also include KCNE1, KCNH2, SCN5A, KCNJ11, NUP155,
Primer mixture corresponding to CACNA1C, CACNB2B.
Wherein, the primer mixture in detection KCNE1 gene polymorphic site is:
SEQ NO.1: 5 '-GTGCCTGGGAAGTTTGAGC-3 '；
SEQ NO.2: 5 '-GCAGGTCCCCCCGCAGAA-3 '；
SEQ NO.3: 5 '-GCAGGTCCCCCCGCAGAG-3 '；
SEQ NO.4: 5 '-CCGTTCTTTCCCAGTCTCAT-3 '；
And mole ratio is SEQ NO.1:SEQ NO.2/ SEQ NO.3:SEQ NO.4=1:1:1；Or, in detection
Time, concentration be the consumption of the above-mentioned primer (SEQ NO. 1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) of 10uM be 0.3ul-
0.7ul。
The primer mixture in detection KCNH2 gene polymorphic site is:
SEQ NO.5: 5 '-GCCGTGCTGTTCTTGCTC-3 '；
SEQ NO.6: 5 '-AGGACAAGTATGTGACGGCTC-3 '；
SEQ NO.7: 5 '-AGGACAAGTATGTGACGGCTT-3 '；
SEQ NO.8: 5 '-GATTGGGGATCTGGTGGAA-3 '；
And mole ratio is SEQ NO.5:SEQ NO.6/ SEQ NO.7:SEQ NO.8=1:1:1, or, in detection
Time, concentration be the consumption of the above-mentioned primer (SEQ NO. 5, SEQ NO.6, SEQ NO.7 and SEQ NO.8) of 10uM be 0.3ul-
0.7ul。
The primer mixture in detection SCN5A gene polymorphic site is:
SEQ NO.9: 5 '-ACAGTGATGCTGGCTGGAAG-3 '；
SEQ NO.10: 5 '-GAAGTACTTCTTCTCCCCGTC-3 '；
SEQ NO.11: 5 '-GAAGTACTTCTTCTCCCCGTT-3 '；
SEQ NO.12: 5 '-ATGTTGATGAGGCTTATCTGGTT-3 '.
And mole ratio is SEQ NO.9:SEQ NO.10/ SEQ NO.11:SEQ NO.12=0.2-0.5:1:1；Or
Person, detection time, concentration be the consumption of the above-mentioned SEQ NO.9 primer of 10uM be 0.1ul-0.3ul, concentration is the above-mentioned of 10uM
Primer SEQ NO.10, SEQ NO.11 and SEQ NO.12 consumption is 0.3ul-0.7ul.
The primer mixture in detection KCNJ11 gene polymorphic site is:
SEQ NO.13: 5 '-CTCATCGTGCAGAACATCGTG-3 '；
SEQ NO.14: 5 '-CGCAAGAGCATGATCATCCA-3 '；
SEQ NO.15: 5 '-CGCAAGAGCATGATCATCCG-3 '；
SEQ NO.16: 5 '-TGGCCGGGCTACATACCA-3 '.
And mole ratio is SEQ NO.13:SEQ NO.14/ SEQ NO.15:SEQ NO.16=1:1:1；Or,
During detection, concentration is the consumption of the above-mentioned primer (SEQ NO. 13, SEQ NO.14, SEQ NO.15 and SEQ NO.16) of 10uM
For 0.3ul-0.7ul.
The primer mixture in detection CACNA1C gene polymorphic site is:
SEQ NO.17: 5 '-GCCTGCAATAGCTTGAAATAAGG-3 '；
SEQ NO.18: 5 '-GGTGGGGATGTGCTCAGGTA-3 '；
SEQ NO.19: 5 '-GTGGGGATGTGCTCAGGTG-3 '；
SEQ NO.20: 5 '-AGAAGGGAAAGAAGGGAATGAG-3 '；
And mole ratio is SEQ NO.17:SEQ NO.18/ SEQ NO.19:SEQ NO.20=1:0.8-1.2:1；Or
Person, when detection, concentration is the above-mentioned primer (SEQ NO. 17, SEQ NO.18, SEQ NO.19 and SEQ NO.20) of 10uM
Consumption is 0.3ul-0.7ul.
The primer mixture in detection CACNB2B gene polymorphic site is:
SEQ NO.21: 5 '-GCACTTGCTCTGGGACAT-3 '；
SEQ NO.22:5 '-CCCAGCACCGCTCTTCCGC-3 '；
SEQ NO.23: 5 '-TCCCAGCACCGCTCTTCCGT-3 '；
SEQ NO.24: 5 '-TGATTCTGGCTTGTTGGATA-3 '；
And mole ratio is SEQ NO.21:SEQ NO.22/ SEQ NO.23:SEQ NO.24=0.125:1:1；Or
Person, detection time, concentration be the consumption of the above-mentioned SEQ NO.21 primer of 10uM be 0.05ul-0.2ul, concentration is the upper of 10uM
Stating primer SEQ NO.22, SEQ NO.23 and SEQ NO.24 consumption is 0.6ul-1ul.
The primer mixture in detection NUP155 gene polymorphic site is:
SEQ NO.25: 5 '-GGAGAATCGCTTGAACCC-3 '；
SEQ NO.26:5 '-CTGACGCTGGTTCATGTCAA-3 '；
SEQ NO.27: 5 '-CTGACGCTGGTTCATGTCAG-3 '；
SEQ NO.28:5 '-TATTGCGTGGTGCTAAGGTT-3 '.
And mole ratio is SEQ NO.25:SEQ NO.26/ SEQ NO.27:SEQ NO.28=0.4:1:1；Or,
Detection time, concentration be the consumption of the above-mentioned SEQ NO.25 primer of 10uM be 0.1ul-0.3ul, concentration is that the above-mentioned of 10uM is drawn
Thing SEQ NO.26, the consumption of SEQ NO.27 and SEQ NO.28 is 0.3ul-0.7ul.
Preferably, detection KCNE1 gene polymorphic site primer mixture SEQ NO. 1, SEQ NO.2, SEQ NO.3 and
The consumption of SEQ NO.4 is respectively 0.3ul；The primer mixture SEQ NO. 5, SEQ in detection KCNH2 gene polymorphic site
The consumption of NO.6, SEQ NO.7 and SEQ NO.8 is respectively 0.3ul；The primer mixture in detection KCNJ11 gene polymorphic site
The consumption of SEQ NO. 9, SEQ NO.10, SEQ NO.11 and SEQ NO.12 is respectively 0.3ul；Detection SCN5A gene polymorphic position
In the primer mixture of point, the consumption of SEQ NO.13 primer is 0.1ul, and SEQ NO.14, SEQ NO.15 and SEQ NO.16 draws
Thing consumption is respectively 0.3ul；The primer mixture SEQ NO.17 in detection CACNA1C gene polymorphic site, SEQ NO.18, SEQ
The consumption of NO.19 and SEQ NO.20 is respectively 0.3ul；The primer mixture SEQ in detection CACNB2B gene polymorphic site
The consumption of NO.21 primer is 0.05ul, SEQ NO.22, SEQ NO.23 and SEQ NO.24 consumption is respectively 0.6ul；Detection
The consumption of the primer mixture SEQ NO.25 primer in NUP155 gene polymorphic site is 0.1ul, SEQ NO.26, SEQ NO.27
It is respectively 0.3ul with the consumption of SEQ NO.28.
Preferably, detection KCNE1 gene polymorphic site primer mixture SEQ NO. 1, SEQ NO.2, SEQ NO.3 and
The consumption of SEQ NO.4 is respectively 0.7ul；The primer mixture SEQ NO. 5, SEQ in detection KCNH2 gene polymorphic site
The consumption of NO.6, SEQ NO.7 and SEQ NO.8 is respectively 0.7ul；The primer mixture in detection KCNJ11 gene polymorphic site
The consumption of SEQ NO.9, SEQ NO.10, SEQ NO.11 and SEQ NO.12 is respectively 0.7ul；Detection SCN5A gene polymorphic position
In the primer mixture of point, the consumption of SEQ NO.13 primer is 0.3ul, and SEQ NO.14, SEQ NO.15 and SEQ NO.16 draws
Thing consumption is respectively 0.7ul；The primer mixture SEQ NO.17 in detection CACNA1C gene polymorphic site, SEQ NO.18, SEQ
The consumption of NO.19 and SEQ NO.20 is respectively 0.7ul；The primer mixture SEQ in detection CACNB2B gene polymorphic site
The consumption of NO.21 primer is 0.2ul, SEQ NO.22, SEQ NO.23 and SEQ NO.24 consumption is respectively 1ul；Detection
The consumption of the primer mixture SEQ NO.25 primer in NUP155 gene polymorphic site is 0.3ul, SEQ NO.26, SEQ NO.27
It is respectively 0.7ul with the consumption of SEQ NO.28.
The detection of the SNP site corresponding to each gene needs 4 primers to complete, due in a PCR amplification system
Need to expand internal reference band and polymorphism purpose band simultaneously, therefore, PCR competitive reaction can be produced, in order to improve internal reference bar
Band and the resolution ratio of polymorphic bands, need by experiment by the molal quantity of the molal quantity/volume of internal reference primer and polymorphic primer/
The ratio of volume is adjusted to optimal proportion, to improve inspection time measurement resolution.
Embodiment 1
Amplifing reagent is made up of the component of volumes below:
Described internal reference upstream primer is SEQ NO.1, SEQ NO.5, SEQ NO.9, SEQ NO.13, SEQ NO.17, SEQ
NO.21, SEQ NO.25, polymorphism primer is SEQ NO.2, SEQ NO.6, SEQ NO.10, SEQ NO.14, SEQ NO.18,
SEQ NO.22、SEQ NO.26、SEQ NO.3、SEQ NO.7、SEQ NO.11、SEQ NO.15、SEQ NO.19、SEQ
NO.23, SEQ NO.27, downstream primer is SEQ NO.4, SEQ NO.8, SEQ NO.12, SEQ NO.16, SEQ NO.20,
SEQ NO.24, SEQ NO.28, the concentration of described internal reference upstream primer is 10uM, and the concentration of described polymorphism primer is 10uM,
The concentration of described downstream primer is 10uM.
PCR amplification program is as follows:
Embodiment 2
Amplifing reagent is made up of the component of volumes below:
Embodiment 3
Amplifing reagent is made up of the component of volumes below:
In order to verify above-mentioned information, can by amplified production detection analysis on ABI3130 gene genetic analyzer, with
The checking above-mentioned testing result of the present invention.
Formed loading mixture by deionized formamide and molecular weight internal standard, 10ul loading mixture is produced with 1ul amplification
Thing or the mixing of alleles analysis reference material, it is to avoid produce bubble, 95 DEG C of sex change 5min, do electrophoresis, use ABI3130 gene genetic
Analyzer detection analysis verification analysis result.
Described amplification uses AS-PCR(ApoE gene) carry out, use agarose gel electrophoresis to carry out many
State property parting detects.
The human gene group DNA wherein detected is for using Chelex method, magnetic bead extraction method or phenol/chloroform extraction method to source
Sample carries out processing the DNA obtained；Described source sample is to derive from the filter paper blood cake/buccal swab sample of the mankind, FTA card
Blood cake or blood.
By AS-PCR and agarose gel electrophoresis specific detection sudden cardiac death gene, the primer of KCNE1 gene is
SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, the primer of KCNH2 gene are SEQ NO.5, SEQ NO.6, SEQ
The primer of NO.7, SEQ NO.8, SCN5A gene is SEQ NO.13, SEQ NO.14, SEQ NO.15, SEQ NO.16, KCNJ11
The primer of gene is SEQ NO.9, the primer of SEQ NO.10, SEQ NO.11, SEQ NO.12, NUP155 gene is SEQ
The primer of NO.25, SEQ NO.26, SEQ NO.27, SEQ NO.28, CACNA1C gene is SEQ NO.17, SEQ NO.18,
SEQ NO.19, SEQ NO.20, the primer of CACNB2B gene are SEQ NO.21, SEQ NO.22, SEQ NO.23, SEQ
NO.24, the amplification of described gene uses PCR to realize, and uses agarose gel electrophoresis to detect.
Embodiment 4
The kit of the present invention detects 50 individual DNA samples of sudden death.
1, sample to be tested 50 parts, all use the technical method of the above " DNA extracts PCR amplification order-checking " to gene
Carry out order-checking detection.Wherein No. 7, No. 13, No. 22, No. 35 sample detection positive findingses.
2, the extracting genome DNA of sample
Chelex extraction method: cut 1～3mm blood cake and be placed in 1.5ml centrifuge tube, adds sdH2O 1ml, vibration is centrifugal,
Abandon supernatant, repeat step twice, abandon supernatant, quickly draw with the rifle head cutting head after 5%Chelex-100 vibration is suspended
200ul adds in centrifuge tube, vibrates the several seconds, after 56 DEG C of water bath heat preservation 30min, vibrates the several seconds, 95 DEG C of boiling water bath 10min, gently
The micro oscillation several seconds.2000rpm is centrifuged 5min, and supernatant is the DNA extracted.
3, amplification and the detection analysis of amplified production
3.1 PCR amplification system:
3.1 PCR amplification system:
Described internal reference upstream primer is SEQ NO.1, SEQ NO.5, SEQ NO.9, SEQ NO.13, SEQ NO.17, SEQ
NO.21, SEQ NO.25, polymorphism primer is SEQ NO.2, SEQ NO.6, SEQ NO.10, SEQ NO.14, SEQ NO.18,
SEQ NO.22、SEQ NO.26、SEQ NO.3、SEQ NO.7、SEQ NO.11、SEQ NO.15、SEQ NO.19、SEQ
NO.23, SEQ NO.27, downstream primer is SEQ NO.4, SEQ NO.8, SEQ NO.12, SEQ NO.16, SEQ NO.20,
SEQ NO.24, SEQ NO.28, the concentration of described internal reference upstream primer is 10uM, and the concentration of described polymorphism primer is 10uM,
The concentration of described downstream primer is 10uM.
3.2 PCR amplification programs:
Described amplification uses AS-PCR(ApoE gene) carry out, use agarose gel electrophoresis and gel
Imaging system carries out polymorphism parting detection.
4, amplified production detection analysis verification on ABI3130 gene genetic analyzer
By deionized formamide and molecular weight internal standard form loading mixture (0.2ul molecular weight internal standard × sample introduction book+
9.8ul deionized formamide × sample introduction number).By 10ul loading mixture and 1ul amplified production or alleles analysis reference material
Mixing, it is to avoid produce bubble.95 DEG C of sex change 5min, and electrophoresis as early as possible, analyze with the detection of ABI3130 gene genetic analyzer.
5, conclusion
Above-described embodiment parting is completely clear, and result is as depicted in figs. 1 and 2.Wherein, with M.DL1000 DNA molecular amount mark
Accurate；1-6 swimming lane is the pcr amplification product of KCNE1 gene；7-10 swimming lane is the pcr amplification product of KCNH2 gene；11-16 swimming lane
Pcr amplification product for SCN5A gene；17-20 swimming lane is the pcr amplification product of KCNJ11 gene；21-24 swimming lane is
The pcr amplification product of CACNA1C gene;25-28 swimming lane is the pcr amplification product of CACNB2B gene;29-32 swimming lane is
The pcr amplification product of NUP155 gene.7,8,11,12,25,26 swimming lane Genotypings are CC type；9,10,13,14,27,28 swimming
Dao gene parting is CT type；15,16 swimming lane Genotypings are TT type；1,2 swimming lane Genotypings are AA type；3,4,19,20,23,
24,31,31 swimming lane Genotypings are AG type；5,6,17,18,21,22,29,30 swimming lane Genotypings are GG type.
In Fig. 1 sudden cardiac death Genotyping electrophoretogram, the reference clip size of KCNE1 gene is 894bp, purpose bar
Band size is 754bp;The reference clip size of KCNH2 gene is 760bp, and purpose stripe size is 579bp；KCNJ11 gene
Being 795bp with reference to clip size, purpose stripe size is 624bp;The reference clip size of SCN5A gene is 764bp, purpose bar
Band size is 698bp;The reference clip size of CACNA1C gene is 851bp, and purpose stripe size is 203bp;CACNB2B base
The reference clip size of cause is 914bp, and purpose stripe size is 750bp;The reference clip size of NUP155 gene is 610bp,
Purpose stripe size is 321bp.
As in figure 2 it is shown, the genotype of normal person is: KCNE1 gene GG type, KCNH2 gene C c-type, KCNJ11 gene GG
Type, SCN5A gene C c-type, CACNA1C gene GG type, CACNB2B gene C c-type, NUP155 gene GG type.Other bases in figure
The positive sample electrophoresis result of site-directed mutagenesis technique clone is used because type is this laboratory.
As it is shown on figure 3, in Fig. 1: No. 7 sample sudden cardiac death Genotyping electrophoretograms, A swimming lane and G swimming lane 700bp position
Left and right occurs that purpose expands once band, and size is coincide with reason purpose band theoretical value 698bp, examines as AG heterozygosis, with sequencing result one
Cause, show that this individuality exists higher sudden death risk.
In FIG, in No. 22 sample sudden cardiac death Genotyping electrophoretograms, there is purpose about position in A swimming lane 700bp
Expanding once band, size is coincide with reason purpose band theoretical value 698bp, and G swimming lane occurs without purpose band, examines and isozygotys for AA, with survey
Sequence result is consistent, shows that this individuality exists the highest sudden death risk.
In FIG, in No. 13 sample sudden cardiac death Genotyping electrophoretograms, below C swimming lane and T swimming lane 700bp position
Occurring that purpose expands once band, size is coincide with reason purpose band theoretical value 579bp, examines as CT heterozygosis, consistent with sequencing result, table
There is higher sudden death risk in this individuality bright.
In FIG, in No. 35 sample sudden cardiac death Genotyping electrophoretograms, about A swimming lane and G swimming lane 300bp position
Occurring that purpose expands once band, size is coincide with reason purpose band theoretical value 321bp, examines as AG heterozygosis, consistent with sequencing result, table
There is higher sudden death risk in this individuality bright.
Taq thermal starting polymerase used in above example and other reagent and consumptive material are commercially available prod.
The preferred embodiment of the present invention described in detail above, it will be appreciated that the ordinary skill of this area is without wound
The property made work just can make many modifications and variations according to the design of the present invention.Therefore, all technical staff in the art
According to present inventive concept on the basis of prior art by logic analysis, reasoning or according to the limited available technology of experiment
Scheme, all should be among by protection domain determined by the claims.
SEQUENCE LISTING
<110>the Guangzhou Research Institute of Physical Culture
<120>sudden cardiac death rapid gene detection kit and detection method
<160> 28
<140> 201310419777.7
<141> 2013-09-13
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>
<400>1
GTGCCTGGGA AGTTTGAGC
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400>2
GCAGGTCCCC CCGCAGAA
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400>3
GCAGGTCCCC CCGCAGAG
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400>4
CCGTTCTTTC CCAGTCTCAT
<210> 5
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400>5
GCCGTGCTGT TCTTGCTC
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 6
AGGACAAGTA TGTGACGGCT C
<210> 7
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 7
AGGACAAGTA TGTGACGGCT T
<210> 8
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 8
GATTGGGGAT CTGGTGGAA
<210> 9
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 9
CTCATCGTGC AGAACATCGT G
<210> 10
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 10
CGCAAGAGCA TGATCATCCA
<210> 11
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 11
CGCAAGAGCA TGATCATCCG
<210> 12
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 12
TGGCCGGGCT ACATACCA
<210> 13
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 13
ACAGTGATGC TGGCTGGAAG
<210> 14
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 14
GAAGTACTTC TTCTCCCCGT C
<210> 15
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 15
GAAGTACTTC TTCTCCCCGT T
<210> 16
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 16
ATGTTGATGA GGCTTATCTG GTT
<210> 17
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 17
GCCTGCAATA GCTTGAAATA AGG
<210> 18
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 18
GGTGGGGATG TGCTCAGGTA
<210> 19
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 19
GTGGGGATGT GCTCAGGTG
<210> 20
<211> 22
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 20
AGAAGGGAAA GAAGGGAATG AG
<210> 21
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 21
GCACTTGCTC TGGGACAT
<210> 22
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 22
CCCAGCACCG CTCTTCCGC
<210> 23
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 23
TCCCAGCACC GCTCTTCCGT
<210> 24
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 24
TGATTCTGGC TTGTTGGATA
<210> 25
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 25
GGAGAATCGC TTGAACCC
<210> 26
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 26
CTGACGCTGG TTCATGTCAA
<210> 27
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 27
CTGACGCTGG TTCATGTCAG
<210> 28
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 28
TATTGCGTGG TGCTAAGGTT
Claims (6)
1. sudden cardiac death rapid gene detection kit, it is characterised in that minimum including detects KCNE1, KCNH2, KCNJ11
Gene polymorphic site,
Wherein, the primer mixture in detection KCNE1 gene polymorphic site is:
SEQ NO.1: 5 '-GTGCCTGGGAAGTTTGAGC-3 '；
SEQ NO.2: 5 '-GCAGGTCCCCCCGCAGAA-3 '；
SEQ NO.3: 5 '-GCAGGTCCCCCCGCAGAG-3 '；
SEQ NO.4: 5 '-CCGTTCTTTCCCAGTCTCAT-3 '；
The primer mixture in detection KCNH2 gene polymorphic site is:
SEQ NO.5: 5 '-GCCGTGCTGTTCTTGCTC-3 '；
SEQ NO.6: 5 '-AGGACAAGTATGTGACGGCTC-3 '；
SEQ NO.7: 5 '-AGGACAAGTATGTGACGGCTT-3 '；
SEQ NO.8: 5 '-GATTGGGGATCTGGTGGAA-3 '；
The primer mixture in detection KCNJ11 gene polymorphic site is:
SEQ NO.9: 5 '-CTCATCGTGCAGAACATCGTG-3 '；
SEQ NO.10: 5 '-CGCAAGAGCATGATCATCCA-3 '；
SEQ NO.11: 5 '-CGCAAGAGCATGATCATCCG-3 '；
SEQ NO.12: 5 '-TGGCCGGGCTACATACCA-3 '.
Sudden cardiac death rapid gene detection kit the most according to claim 1, it is characterised in that also include detection
SCN5A gene polymorphic site, wherein the primer mixture in detection SCN5A gene polymorphic site is:
SEQ NO.13: 5 '-ACAGTGATGCTGGCTGGAAG-3 '；
SEQ NO.14: 5 '-GAAGTACTTCTTCTCCCCGTC-3 '；
SEQ NO.15: 5 '-GAAGTACTTCTTCTCCCCGTT-3 '；
SEQ NO.16: 5 '-ATGTTGATGAGGCTTATCTGGTT-3 '.
Sudden cardiac death rapid gene detection kit the most according to claim 2, it is characterised in that also include detection
CACNA1C, CACNB2B gene polymorphic site, wherein, the primer mixture in detection CACNA1C gene polymorphic site is:
SEQ NO.17:5 '-GCCTGCAATAGCTTGAAATAAGG-3 '；
SEQ NO.18: 5 '-GGTGGGGATGTGCTCAGGTA-3 '；
SEQ NO.19: 5 '-GTGGGGATGTGCTCAGGTG-3 '；
SEQ NO.20: 5 '-AGAAGGGAAAGAAGGGAATGAG-3 '；
The primer mixture in detection CACNB2B gene polymorphic site is:
SEQ NO.21: 5 '-GCACTTGCTCTGGGACAT-3 '；
SEQ NO.22: 5 '-CCCAGCACCGCTCTTCCGC-3 '；
SEQ NO.23: 5 '-TCCCAGCACCGCTCTTCCGT-3 '；
SEQ NO.24: 5 '-TGATTCTGGCTTGTTGGATA-3 ' '.
Sudden cardiac death rapid gene detection kit the most according to claim 3, it is characterised in that also include detection
NUP155 gene polymorphic site, wherein, the primer mixture in detection NUP155 gene polymorphic site is:
SEQ NO.25: 5 '-GGAGAATCGCTTGAACCC-3 '；
SEQ NO.26: 5 '-CTGACGCTGGTTCATGTCAA-3 '；
SEQ NO.27: 5 '-CTGACGCTGGTTCATGTCAG-3 '；
SEQ NO.28: 5 '-TATTGCGTGGTGCTAAGGTT-3 '.
Sudden cardiac death rapid gene detection kit the most according to claim 1, it is characterised in that also include amplification examination
Genotyping reagent after agent and amplification, described amplifing reagent includes PCR buffer solution, dNTPs mixture, hot start Taq polymerase
And ultra-pure water；Genotyping reagent after described amplification includes that agarose, TAE buffer solution, DNA molecular amount standard, DNA are nontoxic
Dyestuff and DNA sample-loading buffer.
Sudden cardiac death rapid gene detection kit the most according to claim 5, it is characterised in that described amplifing reagent
Including 10 times of PCR buffer solutions: wherein, Tris-HCl pH8.3 100mM; KCl 500 mM; MgCl215mM, each component is dense
Degree is the dNTPs mixture of 2.5mM, and the hot start Taq polymerase consumption of 5U/ μ l is 0.1-0.125 μ l.
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| CN106868126A (en) * | 2017-02-20 | 2017-06-20 | 深圳美因临床检验所有限公司 | Fluorescent quantificationally PCR detecting kit and detection method |
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| CN106460062A (en) * | 2014-05-05 | 2017-02-22 | 美敦力公司 | Methods and compositions for SCD, CRT, CRT-D, or SCA therapy identification and/or selection |
| CN104561310B (en) * | 2015-01-04 | 2019-02-05 | 西安百思达生物科技有限公司 | Sudden cardiac death mutated gene detection kit |
| CN108517357B (en) * | 2018-03-12 | 2022-05-24 | 山西医科大学 | Kit for detecting sudden cardiac death-related SNP (single nucleotide polymorphism) on SCN5A gene related to sudden cardiac death and detection method thereof |
| CN109457019B (en) * | 2018-12-31 | 2021-10-15 | 山西医科大学 | KCNH2 gene SCD related SNP detection kit and detection method |
| 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 |
| CN112111572B (en) * | 2020-10-12 | 2021-10-08 | 苏州大学 | STAT5A gene insertion deletion polymorphism site-based sudden cardiac death susceptibility detection kit |
| 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 |
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