CN108531584A - A kind of primer and detection method for detecting the relevant SNP site of headstroke neurological susceptibility - Google Patents
A kind of primer and detection method for detecting the relevant SNP site of headstroke neurological susceptibility Download PDFInfo
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Abstract
The present invention provides a kind of primer and detection method for detecting the relevant SNP site of headstroke neurological susceptibility, which includes primer, the primer positioned at the sites rs266729, the primer positioned at the sites rs822391 and the primer positioned at the sites rs822396 positioned at the sites rs2230500.Using the method for the relevant SNP site polymorphism of primer detection headstroke neurological susceptibility, include the following steps：(1) it acquires sample and extracts DNA；(2) PCR amplification of target gene, glue recycling are carried out using above-mentioned primer respectively；(3) concentration for measuring glue recovery product, carries out fluorescent marker PCR reactions, and purify to reaction product；(4) machine in purified product is sequenced, and SNP testing results is analyzed.The primer specificity is good, high sensitivity, accuracy are good, and detection method is simple, and predictable subject suffers from the risk of headstroke.
Description
Technical field
The invention belongs to biotechnologies, and in particular to one kind is for detecting the relevant SNP site of headstroke neurological susceptibility
Primer and detection method.
Background technology
Headstroke is also referred to as cerebral apoplexy or cerebrovascular disease, be due to brain inside the unexpected rupture haemorrhage of blood vessel or because of blood vessel
Blocking cause cerebrum ischemia, anoxic and cause.Clinical manifestation is to happen suddenly the disturbance of consciousness or facial paralysis, hemiplegia, ability to speak not
Clearly, cognitive disorder is main feature.Palsy includes Ischemic Stroke (transient ischemic attack, atherosclerotic thrombus
Property cerebral infarction, lacunar infarction, cerebral embolism) with hemorrhagic apoplexy (cerebral hemorrhage, subarachnoid hemorrhage).According to " the Chinese heart
The data that cerebrovascular disease epidemiology cooperating research group " is delivered, Ischemic Stroke accounts for 62.4% in China's apoplexy patient, cerebral hemorrhage
27.5% is accounted for, subarachnoid hemorrhage accounts for 1.8%.With the change of China human mortality aging and people life mode, brain in recent years
The incidence of palsy is in significant ascendant trend.It is shown according to epidemiological survey data：There are about 7,000,000 cerebral apoplexies trouble at present for China
Person, annual new hair 2,000,000 people of cerebral apoplexy, the patient for dying of apoplexy every year about 1,500,000, just have an example newly to send out within average every 15 seconds
Patient, an every 21 seconds dead people.With the great change that life style occurs, cardiovascular and cerebrovascular disease rapidly rises, wherein cerebrovascular disease
Tumour and coronary heart disease are alreadyd exceed, is at the first place in national dead, disabling disease, currently, incidence is still annual to approach
9% speed rises.International comparative studies prompt：The morbidity and mortality of Chinese population cerebrovascular disease are higher than international average
It is horizontal.Cerebrovascular disease is given with its high incidence, high relapse rate, high disability rate, high mortality and higher and higher diagnosis and treatment expense
State and society causes huge economic loss, it has also become seriously affects the important public hygiene problem of China's national economy, prevents
It is extremely urgent to control demand, it is necessary to attract great attention.
Pass through detection and the relevant SNP of headstroke neurological susceptibility, it will be able to help headstroke Susceptible population to understand it and suffer from brain
The risk of wind, carries out prevention work in advance, avoids the generation of headstroke, the more early discovery of headstroke and timely diagnosis and treatment, cures chance
It is bigger.
It, not only can be from molecular genetic level by detecting the polymorphism with the relevant SNP site of headstroke neurological susceptibility
Upper understanding cell carcinogenesis mechanism, and be of great significance to the prediction, prevention, genetic counselling etc. of headstroke.
Invention content
For the above-mentioned problems in the prior art, the present invention provides a kind of relevant for detecting headstroke neurological susceptibility
The primer and detection method of SNP site, primer specificity is good, and detection method accuracy is high, and the risk that can be used for headstroke is commented
Estimate, guidance is provided for the disease prevention of headstroke.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is：
A kind of primer for detecting the relevant SNP site of headstroke neurological susceptibility, these primers include being located at PRKCH genes
The sites rs2230500 primer, positioned at the primer in the sites rs266729 of ADIPOQ genes, positioned at ADIPOQ genes
The primer in the sites rs822391 and primer positioned at the sites rs822396 of ADIPOQ genes；
Wherein, the primer in the sites rs2230500 is F：5′-ACTGTTTGGGTTGAAGTAAG-3′(SEQ ID No：1) and
R：5′-GTCTCAGCTCTGTAAGAAAAG-3′(SEQ ID No：2)；
The primer in the sites rs266729 is F：5′-GCCTAATGTGACTTCTCTTG-3′(SEQ ID No：And R 3)：5′-
CCTGTTTTTCCAGTTCCTT-3′(SEQ ID No：4)；
The primer in the sites rs822391 is F：5′-CAAGTCTCAGGTTCTCCTAC-3′(SEQ ID No：And R 5)：5′-
CCCAGAAATCACCTCAC-3′(SEQ ID No：6)；
The primer in the sites rs822396 is F：5′-TTACAATCAGAGTCCGTTC-3′(SEQ ID No：And R 7)：5′-
TAAACTTCCTCACTGCTTG-3′(SEQ ID No：8).
Using the method for the relevant SNP site polymorphism of above-mentioned primer detection headstroke neurological susceptibility, include the following steps：
(1) it acquires sample and extracts its genomic DNA；
(2) it carries out the PCR amplification of target gene to genomic DNA respectively using above-mentioned primer, and amplified production is carried out
Glue recycles；
(3) concentration mensuration is carried out to glue recovery product, then calculation template amount carries out PCR amplification (fluorescent marker reaction)
And it purifies；
(4) by the purified product in step (3) in the full-automatic sequenator of 3730 types (U.S. Applied
Biosystems companies) loading, SNP partings are analyzed with Chromas softwares.
Further, PCR amplification system is in step (2)：DNA profiling content is 100-150ng, a concentration of 5pmol/ μ L
Forward primer 3.0 μ L, a concentration of 5pmol/ μ L 3.0 μ L, Prime STAR MAX of reverse primer, 25.0 μ L, mended with ddH2O
Sufficient volume is to 50 μ L.
Further, pcr amplification reaction condition is in step (2)：95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s, 55 DEG C are moved back
Fiery 15s, 72 DEG C of extension 15s carry out 30 cycles, last 72 DEG C of extensions 5min.
Further, PCR amplification system is in step (3)：DTCS Master Mix 2.5 μ L, a concentration of 5pmol/ μ L
Reverse primer 1.0 μ L, DNA profiling 20ng use ddH2O supplies volume to 10 μ L.
Further, pcr amplification reaction condition is in step (3)：94 DEG C of pre-degeneration 30s, 94 DEG C of denaturation 25s, 55 DEG C are moved back
Fiery 25s, 60 DEG C of extension 3min, 30 cycles, last 60 DEG C of extensions 20min.
Provided by the present invention for detect the relevant SNP site of headstroke neurological susceptibility primer and detection method, have with
Lower advantageous effect：
(1) the present invention provides the primer for detecting headstroke tumor susceptibility gene related locus, which has specificity
Advantage high, accuracy is good, realizes the detection of the relevant SNP site of headstroke neurological susceptibility, improves detection efficiency.
(2) the present invention also provides a kind of method of the detection relevant SNP site of headstroke neurological susceptibility, this method is direct
The advantages that PCR sequencing PCR, this detection method is simple, and testing result also has specific good, high sensitivity, and accuracy is good, Ke Yiwei
The disease prevention of headstroke provides guidance.
Description of the drawings
Fig. 1 is the agarose that different subject's blood DNA samples carry out PCR amplification products therefrom under 4 kinds of different primers
Gel electrophoresis figure；
Fig. 2 is the sequencing result figure of rs2230500 loci polymorphisms detection；
Fig. 3 is the sequencing result figure of rs266729 loci polymorphisms detection；
Fig. 4 is the sequencing result figure of rs822391 loci polymorphisms detection；
Fig. 5 is the sequencing result figure of rs822396 loci polymorphisms detection；
Fig. 6 is the fluorescence proof diagram of rs2230500 loci polymorphism testing results；
Fig. 7 is the fluorescence proof diagram of rs266729 loci polymorphism testing results；
Fig. 8 is the fluorescence proof diagram of rs822391 loci polymorphism testing results；
Fig. 9 is the fluorescence proof diagram of rs822396 loci polymorphism testing results.
Specific implementation mode
The primer of the design amplification SNP site of embodiment 1
A large amount of primer is devised for the relevant SNP site of headstroke neurological susceptibility, passes through the optimization of primer reaction condition
With compare, filter out the good four pairs of primers of specificity, including：Positioned at the sites rs2230500 of PRKCH genes primer, be located at
The primer in the sites rs266729 of ADIPOQ genes, positioned at the sites rs822391 of ADIPOQ genes primer and be located at
The primer in the sites rs822396 of ADIPOQ genes；
Wherein, the primer in the sites rs2230500 is F：5′-ACTGTTTGGGTTGAAGTAAG-3′(SEQ ID No：1) and
R：5′-GTCTCAGCTCTGTAAGAAAAG-3′(SEQ ID No：2)；
The primer in the sites rs266729 is F：5′-GCCTAATGTGACTTCTCTTG-3′(SEQ ID No：And R 3)：5′-
CCTGTTTTTCCAGTTCCTT-3′(SEQ ID No：4)；
The primer in the sites rs822391 is F：5′-CAAGTCTCAGGTTCTCCTAC-3′(SEQ ID No：And R 5)：5′-
CCCAGAAATCACCTCAC-3′(SEQ ID No：6)；
The primer in the sites rs822396 is F：5′-TTACAATCAGAGTCCGTTC-3′(SEQ ID No：And R 7)：5′-
TAAACTTCCTCACTGCTTG-3′(SEQ ID No：8).
The rs codes sequence such as SEQ ID No of rs2230500：Shown in 9, the rs codes sequence such as SEQ ID No of rs266729：10
It is shown, the rs codes sequence such as SEQ ID No of rs822391：Shown in 11, the rs codes sequence such as SEQ ID No of rs822396：12 institutes
Show.
PCR amplification, PCR are carried out respectively using the primer pair testing gene group DNA of the relevant SNP site of headstroke neurological susceptibility
Amplification system is：DNA profiling x μ L make its content be 100-150ng, deionized water 19-x μ L, the forward direction of a concentration of 5pmol/ μ L
3.0 μ L, Primer STAR MAX of reverse primer, the 25.0 μ L of primer 3.0 μ L, a concentration of 5pmol/ μ L；Amplification reaction condition
For：95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 15s, 72 DEG C of extension 15s, 30 cycles, last 72 DEG C extend
In 4 DEG C of preservations after 5min；By amplified production into row agarose gel electrophoresis, the results are shown in Figure 1, wherein 1 hole is
The primer amplification band (360bp) in the sites rs2230500；2 holes are the primer amplification band (467bp) in the sites rs266729；3 holes
For the primer amplification band (433bp) in the sites rs822391；4 holes are the primer amplification band (437bp) in the sites rs822396.
As shown in Figure 1, purpose band is clear, no miscellaneous band, and clip size and design is in the same size, illustrates drawing for design
Object specificity is good.
The detection of embodiment 2 and the relevant SNP site polymorphism of headstroke neurological susceptibility
(1) extraction of genomic DNA
Subject peripheral blood 5mL is acquired with EDTA anticoagulant tubes, the extracting method of genomic DNA is with reference to poba gene group
The specification of DNA extraction kit (be purchased from Beijing Tiangeng biochemical technology Co., Ltd), the operating procedure in by specification carry out
Extraction.The complete genome DNA of acquisition using ultramicron nucleic acid-protein analyzer (cypress essence BioDrop μ Lite) detect its concentration with
And purity, record initial data.The DNA that concentration and purity are all reached to requirement is placed in -20 DEG C of refrigerators and saves backup.
(2) PCR amplification of target gene, electrophoresis and glue recycling
PCR amplification：With above-mentioned rs2230500, rs266729, rs822391, the primer in the sites rs822396 is respectively to carrying
The DNA taken carries out PCR amplification.Reaction system is 50 μ L, and specific as shown in table 1, amplification program is as shown in table 2.PCR reactions are completed
Afterwards, 4 DEG C of PCR product is taken out to preserve and (- 20 DEG C of placement is needed to freeze overnight).
1 PCR reaction systems of table
| Reagent | Volume (μ L) |
| DNA | a(100-150ng) |
| ddH2O | 19-a |
| Primer F | 3.0 |
| Primer R | 3.0 |
| Primer STAR MAX | 25.0 |
| total | 50.0 |
2 PCR amplification program of table
After PCR amplification, 50 μ L amplified productions are taken, using 2% agarose gel electrophoresis, electrophoretic parameters are voltage
120V, electric current 400mA, time 30min, specific band nothing but in electrophoresis result；In gel electrophoresis images point after the completion of electrophoresis
The gel containing target fragment is cut under analyzer, gel piece is fitted into EP pipes, and the method for glue recycling is with reference to TaKaRa
The specification of MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 kits, finally to glue recycling
Product carries out concentration mensuration, and 4 DEG C save backup.
(3) it marks, purify and is sequenced
Label：A clean 0.2mL PCR pipe is taken, is sequentially added：DTCS Master Mix, sequencing primer, sterilizing are super
The dosage of pure water, template DNA, wherein template DNA and sterilizing ultra-pure water is determined by the DNA concentration that previous step is measured, and is added
Template DNA amount be 20ng, reaction system is 10 μ L, and specific then to carry out fluorescent marker PCR as shown in table 3, amplification program is shown in
Table 4；
3 PCR reaction systems of table
| Reagent | Volume (μ L) |
| DTCS Master Mix | 2.5 |
| Primer R | 1.0 |
| ddH2O | 6.5-b |
| Template DNA | b(20ng) |
| It amounts to | 10.0 |
4 PCR amplification program of table
Purifying：By a concentration of 3mol/L, sodium-acetate buffer, 0.1mol/L of the pH value for 5.2, pH value is 8.0
Na2Edta buffer liquid and Glycogen are with volume ratio for 2:2:1 prepares terminate liquid, then takes 5 μ L terminate liquids that above-mentioned fluorescence is added
It is mixed well in label reaction PCR product, centrifuges, liquid is transferred in a new 1.5mL EP pipe, 50 μ L are then added
Absolute ethyl alcohol is pre-chilled, mixes well, is put into after -20 DEG C of freezing 10min of refrigerator and centrifuges 5min in 12000r/min, discard supernatant,
150 μ L are added into EP pipes again, 70% ethyl alcohol is pre-chilled, be put into supercentrifuge centrifugation, 12000r/min centrifuges 2min, discards
Clearly, it slightly centrifuging, blots remaining liquid in EP pipes with pipettor, open EP pipe lids, room temperature is dried to white precipitate bleach, then
25 μ L SLS (Sample Loading Solution) dissolving DNA is added into EP pipes, stands 8min, brief centrifugation；
Sequencing：The DNA of dissolving is added in 96 hole sample plate holes, bubble cannot be generated during addition, is then added again
Enter appropriate mineral oil；One piece of 96 new orifice plate is taken, 10 drop dissociating buffers are added in corresponding aperture, ultra-pure water is added into glue groove
To liquid close to index line, finally 96 hole sample panels, 96 hole buffer solutions and glue groove loading 3730 sequenators of ABI are sequenced.
Pass through sequence analysis software (GenomeLabTMABI 3730 (Genetic Analysis System)), it will be sequenced
As a result it is compared with standard sequence, finds SNP site, by the type for analyzing base at SNP site, so that it may to obtain SNP
The genotype in site.
2-1,2-2 and 2-3 are the sequencing result figure of rs2230500 loci polymorphisms detection in Fig. 2, and genotype is followed successively by
AA、AG、GG；Wherein AA is the non-susceptible genotype in the sites rs2230500, and the easy sensillary base that GG and AG is the sites rs2230500
Because of type.
3-1,3-2 and 3-3 are the sequencing result figure of rs266729 loci polymorphisms detection in Fig. 3, and genotype is followed successively by
GG、CG、CC；Wherein GG is the non-susceptible genotype in the sites rs266729, and CG and CC is the susceptible of the sites rs266729 site
Genotype.
4-1,4-2 and 4-3 are the sequencing result figure of rs822391 loci polymorphisms detection in Fig. 4, and genotype is followed successively by
CC、TC、TT；Wherein CC is the non-susceptible genotype in the sites rs822391, and the tumor susceptibility gene that TT and TC is the sites rs822391
Type.
5-1,5-2 and 5-3 are the sequencing result figure of rs822396 loci polymorphisms detection in Fig. 5, and genotype is followed successively by
GG、AG、AA；Wherein GG is the non-susceptible genotype in the sites rs822396, and the tumor susceptibility gene that AG and AA is the sites rs822396
Type.
Above-mentioned sequencing result figure without set peak, background peaks, miscellaneous peak, float peak phenomena such as, illustrate using PCR sequencing PCR detection SNP
Point polymorphism, specificity is good, accuracy is high.Therefore, testing agency can according to the polymorphism of above-mentioned SNP site, assess by
Inspection person suffers from the risk of headstroke, and guidance is provided for the disease prevention of headstroke.
The verification of embodiment 3 and the relevant SNP site polymorphic detection result of headstroke neurological susceptibility
Above-mentioned testing result is verified using fluorescence probe method, result is as Figure 6-9.
Wherein, in Fig. 6 6-1,6-2 and 6-3 be rs2230500 loci polymorphism testing results fluorescence proof diagram, base
Because type is followed successively by AA, AG, GG；Wherein AA is the non-susceptible genotype in the sites rs2230500, and GG and AG is rs2230500
The susceptible genotype of point.
7-1,7-2 and 7-3 are the fluorescence proof diagram of rs266729 loci polymorphism testing results in Fig. 7, genotype according to
Secondary is GG, CG, CC；Wherein GG is the non-susceptible genotype in the sites rs266729, and CG and CC is the sites rs266729 site
Susceptible genotype.
8-1,8-2 and 8-3 are the fluorescence proof diagram of rs822391 loci polymorphism testing results in Fig. 8, genotype according to
Secondary is CC, TC, TT；Wherein CC is the non-susceptible genotype in the sites rs822391, and TT and TC is the susceptible of the sites rs822391
Genotype.
9-1,9-2 and 9-3 are the fluorescence proof diagram of rs822396 loci polymorphism testing results in Fig. 9, genotype according to
Secondary is GG, AG, AA；Wherein GG is the non-susceptible genotype in the sites rs822396, and AG and AA is the susceptible of the sites rs822396
Genotype.
Above-mentioned fluorescence probe method testing result is consistent with PCR sequencing PCR testing result 100%, further demonstrates using sequencing
Method detects the specificity and accuracy of SNP site polymorphism.
Sequence table
<110>Co., Ltd of medical test institute of Chengdu Zhong Chuanqing sections
<120>A kind of primer and detection method for detecting the relevant SNP site of headstroke neurological susceptibility
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tttgaggatt ttgtatactt tttttttttt gagacagagt cttgctctgt cacccaggtt 780
ggagtgcagc tcaatgcaaa ttccgcctcc caggctcaag caattcttat gtctcagcct 840
cctgagtacc tgggattaca ggcaggcacc accaccccca gctaattttt ttgtattttc 900
agtagagacg gggttttgcc atattggcca ggctggtctt gaactcctga cttcatgtga 960
cccacccatc tcagcctccc aaagtgctga gatgacaggt g 1001
<210> 12
<211> 1001
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
gctgacatgg gtgagggttc cttggagtat cattttcatg tggcattttc aaaacttatt 60
ttacctaatc ttcccaaagc cctgcttttg actctaatgt gtctcctgag acttggagag 120
cgcaagatgc tagcgacaga gcaagactcc atctccagat aaataaataa gtaaaataaa 180
aaagaacaca aataattttg aaaatttttt tgaaaattag gcacgtttgc actgaccttc 240
aattgttatt aattgctggt ttcccaccca gaattaagtt ggaatgcaac tttcttttac 300
aatcagagtc cgttcttggt cttggaaact tctgaggctc ctgtgctaat cacactcttg 360
tatttttggc acctctaccc cgtgccactg tcatggaacc caggctgatc gcacctatta 420
gtggagaaat ctgtccataa tactgaagtt tggggacaaa cagtgttccc ttagggtagg 480
agaaagagat ctttattttt racaaagggg gaggagccag aaaactccag agacccctga 540
gtttgccctc tctccaaggt ttggggtaag ccccccgtca ccctttatct ctggggcttt 600
cacatattct ggattctctc ctcctgtttc ccagcagaaa aggatggagc ctcacagatt 660
cttcccattt ctggagaaaa acatgcatgg agctcaaagt tcttctcagg agttttattg 720
ccaaagccat aataagaaag ggtggaggtg acaagcagtg aggaagttta aagatgcatg 780
aaatctgtaa agtctcagaa caagaattct cctaaaatgc aaaaggggct ttgctggtct 840
ccccttggct tctcatgtag ctcacctctt ttttcttatc ttgagactag tcaaacctaa 900
gctgtttctc attttatttc cagaagctat tgagaacact ctcctgaatt cttcaaattc 960
agtagagggc gacaaatgta catataaatg atggtagtgg g 1001
Claims (6)
1. a kind of primer for detecting the relevant SNP site of headstroke neurological susceptibility, which is characterized in that the primer includes being located at
The primer in the sites rs2230500 of PRKCH genes, positioned at the sites rs266729 of ADIPOQ genes primer, be located at ADIPOQ
The primer in the sites rs822391 of gene and primer positioned at the sites rs822396 of ADIPOQ genes；
Wherein, the primer in the sites rs2230500 is F：5 '-ACTGTTTGGGTTGAAGTAAG-3 ' and R：5′-
GTCTCAGCTCTGTAAGAAAAG-3′；
The primer in the sites rs266729 is F：5 '-GCCTAATGTGACTTCTCTTG-3 ' and R：5′-
CCTGTTTTTCCAGTTCCTT-3′；
The primer in the sites rs822391 is F：5 '-CAAGTCTCAGGTTCTCCTAC-3 ' and R：5′-CCCAGAAATCACCTCAC-
3′；
The primer in the sites rs822396 is F：5 '-TTACAATCAGAGTCCGTTC-3 ' and R：5′-
TAAACTTCCTCACTGCTTG-3′。
2. using the method for the relevant SNP site polymorphism of primer detection headstroke neurological susceptibility described in claim 1, feature
It is, includes the following steps：
(1) it acquires sample and extracts its genomic DNA；
(2) it carries out the PCR amplification of target gene to genomic DNA respectively using the primer, and glue is carried out to amplified production and is returned
It receives；
(3) concentration mensuration is carried out to glue recovery product, then calculation template amount carries out fluorescent marker PCR reactions, and produced to reaction
Object is purified；
(4) machine in the purified product in step (3) is sequenced, and SNP testing results is analyzed.
3. the method for the detection relevant SNP site polymorphism of headstroke neurological susceptibility according to claim 2, feature exist
In PCR amplification system is in step (2)：DNA profiling content is 100-150ng, 3.0 μ of forward primer of a concentration of 5pmol/ μ L
3.0 μ L, Prime STAR MAX of reverse primer, the 25.0 μ L of L, a concentration of 5pmol/ μ L, volume is supplied to 50 μ L with ddH2O.
4. the method for the detection relevant SNP site polymorphism of headstroke neurological susceptibility according to claim 2, feature exist
In pcr amplification reaction condition is in step (2)：95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 15s, 72 DEG C extend
15s carries out 30 cycles, last 72 DEG C of extensions 5min.
5. the method for the detection relevant SNP site polymorphism of headstroke neurological susceptibility according to claim 2, feature exist
In PCR amplification system is in step (3)：DTCS Master Mix 2.5 μ L, 1.0 μ of reverse primer of a concentration of 5pmol/ μ L
L, DNA profiling 20ng, uses ddH2O supplies volume to 10 μ L.
6. the method for the detection relevant SNP site polymorphism of headstroke neurological susceptibility according to claim 2, feature exist
In pcr amplification reaction condition is in step (3)：94 DEG C of pre-degeneration 30s, 94 DEG C of denaturation 25s, 55 DEG C of annealing 25s, 60 DEG C extend
3min, 30 cycles, last 60 DEG C of extensions 20min.
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Cited By (1)
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| CN113767179A (en) * | 2018-12-13 | 2021-12-07 | 国立研究开发法人国立循环器病研究中心 | Method for predicting risk of developing cerebral infarction |
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