CN110129426B - A kit for rapid detection of SNP loci associated with treatment of hypertension and prognosis - Google Patents
A kit for rapid detection of SNP loci associated with treatment of hypertension and prognosis Download PDFInfo
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Abstract
本发明公开了一种用于快速检测高血压治疗与预后相关的SNP位点的试剂盒，包括检测KLKB1/F12/SIGLEC12/AGT基因所对应的4个SNP多态性位点的四对引物。本发明的试剂盒基于nsSNP基因分型的Multi‑AS‑PCR引物设计，通过实时荧光定量PCR扩增，根据熔解曲线特征峰进行高血压治疗与预后风险基因SNP分型，为高血压病人个性化诊疗提供依据。本发明的试剂盒具有操作简单、费用低、耗时短和结果可靠等特点，应用前景广阔。
The invention discloses a kit for rapidly detecting SNP sites related to hypertension treatment and prognosis, including four pairs of primers for detecting four SNP polymorphism sites corresponding to KLKB1/F12/SIGLEC12/AGT gene. The kit of the invention is based on the design of Multi-AS-PCR primers for nsSNP genotyping, through real-time fluorescence quantitative PCR amplification, and carries out hypertension treatment and prognosis risk gene SNP typing according to the characteristic peaks of the melting curve, so as to be personalized for hypertensive patients Provide the basis for diagnosis and treatment. The kit of the invention has the characteristics of simple operation, low cost, short time consumption, reliable results and the like, and has broad application prospects.
Description
Technical Field
The invention belongs to the field of molecular biological detection and the field of cardiovascular and cerebrovascular diseases, and particularly relates to a kit for quickly detecting SNP (single nucleotide polymorphism) sites related to hypertension treatment and prognosis.
Background
In 2014, 2.9 million cardiovascular patients are estimated to exist in the whole country, wherein 2.7 million hypertension patients, at least 700 million stroke patients and 250 million myocardial infarction patients exist. Through research on the occurrence and development mechanism of cardiovascular disease complications, genetic factors are found to play an important role. Analysis of the different symptomatic cases, which are prone, prevalent and clinical, has revealed that Single Nucleotide Polymorphisms (SNPs) are closely linked to the occurrence and progression of the disease, especially non-synonymous SNPs (nssnps).
Nejatizadeh et al (2008) found through case-control studies that a common SNP (G-6A, rs5051) and two nsSNPs (T174M, rs 4762; M235T, rs699) existing in the promoter and gene coding region of the angiotensinogen gene AGT are associated with hypertension, and the existence of these polymorphisms results in the change of the transcription level and protein function of the gene, and the renin-angiotensin system is broken down, thereby causing the dysfunction of the vascular endothelium. Verweij ET al (2013) found that SNPs (C/T and T/C) in plasma kallikrein-releasing gene KLKB1 and gene F12 of coagulation factor XII resulted in elevated plasma levels of proadrenomedullin midpeptide (MR-pro-ADM) and C-terminal endothelin-1 (CT-pro-ET-1), revealing the potential of kallikrein in plasma to be a new drug target. McDonough et al (2013) comparatively analyzed functional genotyping of cardiovascular prognosis association of verapamil sustained release agent (calcium channel blocker) and trandolapril (beta receptor blocker) after treatment of different hypertensive individuals, and found that nsSNP exists in SiGLEC12 gene of sialic acid binding immunoglobulin-like lectin 12, which results in amino acid mutation at position 29 as a stop codon, and has association with cardiovascular prognosis. Those carriers of the stop codon experienced the same risk of all-cause death, non-fatal myocardial infarction and non-fatal stroke when treated with verapamil as a slow release formulation and trandolapril. However, the risk of all-cause death, non-fatal myocardial infarction and non-fatal stroke in those patients with the Gln/Gln (G/G) genotype treated with verapamil sustained release agent is significantly higher than in patients treated with trandolapril.
The discovery of the nsSNPs provides a theoretical basis for diagnosing the genotyping of the hypertension patients and enabling the hypertension patients to avoid cardiovascular prognosis caused by side effects caused by treatment of calcium channel blockers and beta receptor blockers, and has important significance for individual treatment selection of hypertension. Therefore, in order to meet the requirements of high throughput, high precision and low cost in clinic, a proper gene typing technology for the hypertension patient is established, and the individual diagnosis and treatment of the hypertension patient in China are very urgent.
Disclosure of Invention
Aiming at the problems in the prior art, the invention utilizes Multi-real time-AS-PCR technology (Multi-target real-time quantitative allele specific PCR) according to the existing nsSNPs data information associated with hypertension diseases and cardiovascular prognosis of antihypertensive treatment, aims to establish a kit for rapidly detecting SNP sites of genes related to hypertension treatment and prognosis, and provides guarantee for individual treatment drug selection of hypertension in China and reduction of cardiovascular prognosis risk caused by treatment.
The first aspect of the invention provides a kit for rapidly detecting SNP sites related to prognosis of hypertension treatment, which comprises four pairs of primers respectively used for detecting 4 SNP polymorphic sites corresponding to KLKB1/F12/SIGLEC12/AGT genes:
(1) a first primer pair for detecting rs4253238 site of KLKB1 gene, comprising:
an upstream primer: 5'-TTCTGTGCTGCAACCAGGTATC-3' (SEQ ID NO: 3);
a downstream primer: 5'-ATCCATGCACGAAAATAACTTC-3' (SEQ ID NO: 4);
(2) a second primer pair for detecting rs2731672 site of F12 gene, comprising:
an upstream primer: 5'-GGCTCATTTGTTAGGAATGCTA-3' (SEQ ID NO: 5);
a downstream primer: 5'-ACAGAGCCAGACTCTATCTCAA-3' (SEQ ID NO: 6);
(3) a third primer pair for detecting rs16982743 locus of SIGLEC12 gene, comprising:
an upstream primer: 5'-GAGGATTACCAGCTGAAGACTC-3' (SEQ ID NO: 7);
a downstream primer: 5'-GAGCATCCCGGTCTGTATGG-3' (SEQ ID NO: 8);
(4) the fourth primer pair is used for detecting the rs2004776 locus of the AGT gene and comprises the following components:
an upstream primer: 5'-ACCACAGGAGCATGAGATG-3' (SEQ ID NO: 9);
a downstream primer: 5'-TGAGGCAGAGACTGTCGTTTGT-3' (SEQ ID NO: 10).
According to the present invention, preferably, the molar ratio of the first primer pair, the second primer pair, the third primer pair and the fourth primer pair is 20 to 22: 1-1.5: 1-1.5: 1.
according to the present invention, further, the kit comprises: positive standard plasmid KFSA-native; the positive standard plasmid KFSA-native is used for simultaneously detecting SNP sites rs4253238/rs2731672/rs16982743/rs2004776, wherein the sequences of KLKB1/F12/SIGLEC12/AGT gene fragments corresponding to the SNP sites rs4253238/rs2731672/rs16982743/rs2004776 are shown as SEQ ID NO: 1 is shown.
Preferably, the vector of the positive standard plasmid KFSA-native is a pUC57 vector plasmid, SEQ ID NO: the sequence shown in 1 is synthesized by adopting a whole gene and then cloned into a pUC57 vector plasmid to form a positive standard plasmid KFSA-native.
According to the present invention, further, the kit comprises: and the allelic standard plasmid KFSA-allele is used for simultaneously detecting the variation of the SNP sites rs4253238/rs2731672/rs16982743/rs2004776, wherein the sequence of the KLKB1/F12/SIGLEC12/AGT gene fragment corresponding to the variation of the SNP sites rs4253238/rs2731672/rs16982743/rs2004776 is shown as SEQ ID NO: 2, respectively.
Preferably, the vector of the allelic standard plasmid KFSA-allele is pUC57 vector plasmid, SEQ ID NO: 2, the sequence is synthesized by adopting a whole gene and then cloned into a pUC57 vector plasmid to form an allelic standard plasmid KFSA-allele.
According to the invention, the kit may further comprise at least one of the following amplification reagents: fluorescent PCR amplification reaction buffer, fluorescent Dye and ultrapure water, for example, 2 XqPCR SYBR Green Master Mix, 50 XROX Reference Dye 1. The amount of each component may be determined as required, and the present invention is not particularly limited thereto.
The rapid detection kit is applied to a rapid detection method for the SNP locus polymorphism of a gene related to hypertension treatment and prognosis, and comprises the following steps:
(1) collecting a blood sample and extracting genome DNA of the blood sample;
(2) adopting the primer mixture of the KLKB1/F12/SIGLEC12/AGT gene polymorphic site to amplify the target fragment of the genome DNA by a real-time fluorescent quantitative PCR method;
(3) and analyzing the result of the SNP site according to the melting curve.
Further, the PCR amplification system in step (2) may be: 2 XqPCR SYBR Green Master Mix 10. mu.L, 50 XROX Reference Dye 10.4. mu.L, primer Mix 2-4. mu. L, DNA template 0.5-10ng, in ddH2O make up the volume to 20. mu.L.
Further, the PCR amplification reaction conditions in step (2) may be: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10s, annealing at 60.5 ℃ for 20s, and extension at 72 ℃ for 32s, for 30 cycles; melting curves were collected at 95 ℃ for 10s, 65 ℃ for 1min, and 95 ℃ for 15 s.
And (4) judging a result: according to the melting curve, the state that the peak type characteristic appears at the corresponding position is judged as a Native state, otherwise, the state is judged as a mutant state.
The invention has the beneficial effects that: the kit provided by the invention is designed based on a Multi-AS-PCR primer of nsSNP genotyping, carries out hypertension treatment and prognosis risk gene SNP genotyping according to a melting curve characteristic peak through real-time fluorescent quantitative PCR amplification, and can provide a basis for individual diagnosis and treatment of hypertension patients. The kit has the characteristics of simple operation, low cost, short time consumption, reliable result and the like, and has wide application prospect.
Drawings
FIG. 1 shows the 2.5% agarose gel electrophoresis result of PCR products obtained by PCR amplification of a primer at the rs4253238 site of KLKB1 gene, a primer at the rs2731672 site of F12 gene, a primer at the rs16982743 site of SIGLEC12 gene and a primer at the rs2004776 site of AGT gene in the kit by taking positive standard plasmid KFSA-native as a DNA template.
FIG. 2 shows the 2.5% agarose gel electrophoresis result of PCR products obtained by PCR amplification of a primer at the rs4253238 site of KLKB1 gene, a primer at the rs2731672 site of F12 gene, a primer at the rs16982743 site of SIGLEC12 gene and a primer at the rs2004776 site of AGT gene in the kit by using an allelic standard plasmid KFSA-allele as a DNA template.
FIG. 3 shows the melting curve result obtained by real-time fluorescence quantitative PCR amplification using positive standard plasmid KFSA-native as template and the kit of the present invention.
FIG. 4 shows the melting curve result obtained by real-time fluorescence quantitative PCR amplification using the kit of the present invention and using blood DNA of a hypertensive as a template.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples.
In the following examples of the present invention,
(1) the first primer pair (or primer of rs4253238 locus of KLKB1 gene) is used for detecting rs4253238 locus of KLKB1 gene and comprises the following components:
an upstream primer: 5'-TTCTGTGCTGCAACCAGGTATC-3' (SEQ ID NO: 3);
a downstream primer: 5'-ATCCATGCACGAAAATAACTTC-3' (SEQ ID NO: 4);
(2) a second primer pair (or primer of rs2731672 site of F12 gene) for detecting rs2731672 site of F12 gene, comprising:
an upstream primer: 5'-GGCTCATTTGTTAGGAATGCTA-3' (SEQ ID NO: 5);
a downstream primer: 5'-ACAGAGCCAGACTCTATCTCAA-3' (SEQ ID NO: 6);
(3) a third primer pair (or primer of rs16982743 site of SIGLEC12 gene) for detecting rs16982743 site of SIGLEC12 gene, comprising:
an upstream primer: 5'-GAGGATTACCAGCTGAAGACTC-3' (SEQ ID NO: 7);
a downstream primer: 5'-GAGCATCCCGGTCTGTATGG-3' (SEQ ID NO: 8);
(4) a fourth primer pair (or called primer of AGT gene rs2004776 site) for detecting AGT gene rs2004776 site, comprising:
an upstream primer: 5'-ACCACAGGAGCATGAGATG-3' (SEQ ID NO: 9);
a downstream primer: 5'-TGAGGCAGAGACTGTCGTTTGT-3' (SEQ ID NO: 10).
The positive standard plasmid KFSA-native contains SEQ ID NO: 1, SEQ ID NO: the sequence shown in 1 is synthesized by adopting a whole gene and then cloned into a pUC57 vector plasmid to form a positive standard plasmid KFSA-native.
The allelic standard plasmid KFSA-allele contains SEQ ID NO: 2, SEQ ID NO: 2, the sequence is synthesized by adopting a whole gene and then cloned into a pUC57 vector plasmid to form an allelic standard plasmid KFSA-allele.
Example 1 working Effect detection and elimination of false positives for primer design
1. Preparing a plasmid mini-drawer kit containing the nucleotide sequence of SEQ ID NO: 1 and a positive standard plasmid KFSA-native containing a sequence shown in SEQ ID NO: 2, detecting the concentration and purity of the allelic standard plasmid KFSA-allele by using an ultra-micro protein nucleic acid analyzer, recording original data, and storing the DNA with the concentration and purity meeting the requirements in a refrigerator at the temperature of-20 ℃ for later use.
2. The working efficiency of a primer at the site rs4253238 of the KLKB1 gene, a primer at the site rs2731672 of the F12 gene, a primer at the site rs16982743 of the SIGLEC12 gene and a primer at the site rs2004776 of the AGT gene is respectively detected by adopting a conventional PCR method. The reaction system is as follows:
the reaction conditions are as follows: pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 60.5 ℃ for 20s, and extension at 72 ℃ for 32s for 20 cycles; further extension at 72 deg.C for 7 min; treating at 4 deg.C for 1 min; the reaction was completed. After the reaction was completed, electrophoresis was performed using 2.5% agarose gel to obtain electrophoresis pictures 1 and 2.
And (4) analyzing results:
FIG. 1 shows that when a designed primer of KLKB1 gene rs4253238 site, a designed primer of F12 gene rs2731672 site, a designed primer of SIGLEC12 gene rs16982743 site and a designed primer of AGT gene rs2004776 site are added, when a positive standard plasmid KFSA-native is used as a DNA template, an amplification product is obtained and the visual detection level is reached after 20 cycles of reaction; when no primer is added in the reaction system and other components are unchanged (blank group), no detectable amplification product is generated in the reaction; the results show that the primers work well.
FIG. 2 shows that when primers at site rs4253238 of KLKB1 gene, site rs2731672 of F12 gene, site rs16982743 of SIGLEC12 gene and site rs2004776 of AGT gene are added, and allele standard plasmid KFSA-allele is used as a DNA template, no detectable amplification product is generated in the reaction after 20 cycles of reaction; when no primer is added in the reaction system and other components are unchanged (blank group), the reaction does not produce detectable amplification products; the results can exclude false positives with primers that result when using the allelic standard plasmid KFSA-allele as DNA template.
Example 2 establishment of Standard reaction System and conditions for the kit of the invention and the basis for the determination of the results
1. Preparation of Positive Standard plasmid KFSA-native
Adopting a plasmid miniprep kit, and operating the operation process according to the kit specification to prepare a plasmid containing SEQ ID NO: 1 sequence, detecting the concentration and purity of the positive standard plasmid KFSA-native by using an ultra-micro protein nucleic acid analyzer, recording original data, and storing the DNA with the concentration and purity meeting the requirements in a refrigerator at the temperature of-20 ℃ for later use.
2. Real-time quantitative PCR amplification of target genes
The method comprises the following steps of (1) using a primer of a KLKB1 gene rs4253238 locus, a primer of a F12 gene rs2731672 locus, a primer of a SIGLEC12 gene rs16982743 locus and a primer of an AGT gene rs2004776 locus according to the molar ratio of 20-22: 1-1.5: 1-1.5: 1, obtaining a primer mixture, and carrying out real-time quantitative PCR amplification.
The PCR amplification system is as follows: 2 × qPCR SYBR Green Master Mix 10 μ L, 50 × ROX Reference Dye 10.4 μ L, primer Mix 2.4 μ L, DNA template (positive standard plasmid KFSA-native)1 μ L with ddH2O is complementaryThe volume was 20. mu.L.
The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10s, annealing at 60.5 ℃ for 20s, and extension at 72 ℃ for 32s, for 30 cycles; melting curves were collected at 95 ℃ for 10s, 65 ℃ for 1min, and 95 ℃ for 15 s. The results are shown in FIG. 3.
And (4) analyzing results:
the melting curve in fig. 3 is the amplification condition of KLKB1, F12, SIGLEC12 and AGT genes in positive standard plasmid KFSA-native, the characteristic peak Tm corresponding to the melting curve at site rs4253238 of KLKB1 gene is 75.48 ℃, the characteristic peak Tm corresponding to the melting curve at site rs2731672 of F12 gene is 80.32 ℃, the characteristic peak Tm corresponding to the melting curve at site rs16982743 of SIGLEC12 gene is 83.65 ℃, and the characteristic peak Tm corresponding to the melting curve at site rs2004776 of AGT gene is 87.71 ℃. In this example, 4 characteristic peaks of KLKB1, F12, SIGLEC12 and AGT gene were detected simultaneously in positive KFSA-native plasmid, which indicates that KFSA-native plasmid has no nsSNP mutation at site rs4253238 of KLKB1 gene, site rs2731672 of F12 gene, site rs16982743 of SIGLEC12 gene and site rs2004776 of AGT gene.
Example 3 SNP site analysis of hypertensive patients Using the kit of the present invention
1. Sample collection and whole genome DNA extraction
Randomly collecting 3mL of peripheral Blood of a hypertensive by using an EDTA anticoagulant tube, extracting the whole genome DNA of a detected person by using a Fastpure Blood DNA Isolation Mini Kit of Vazyme company, operating the operation process according to the Kit specification, detecting the concentration and purity of the obtained whole genome DNA by using an ultra-micro protein nucleic acid analyzer, recording original data, and storing the DNA with the concentration and purity meeting the requirements in a refrigerator at the temperature of-20 ℃ for later use.
2. Real-time quantitative PCR amplification of target genes
The method comprises the following steps of (1) using a primer of a KLKB1 gene rs4253238 locus, a primer of a F12 gene rs2731672 locus, a primer of a SIGLEC12 gene rs16982743 locus and a primer of an AGT gene rs2004776 locus according to the molar ratio of 20-22: 1-1.5: 1-1.5: 1, obtaining a primer mixture, and carrying out real-time quantitative PCR amplification.
PCR amplificationThe system is as follows: 2 XqPCR SYBR Green Master Mix 10. mu.L, 50 XROX Reference Dye 10.4. mu.L, primer Mix 2.4. mu. L, DNA template (subject whole genomic DNA) 1. mu.L, in ddH2O make up the volume to 20. mu.L.
The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10s, annealing at 60.5 ℃ for 20s, and extension at 72 ℃ for 32s, for 30 cycles; melting curves were collected at 95 ℃ for 10s, 65 ℃ for 1min, and 95 ℃ for 15 s. The results are shown in FIG. 4.
And (4) analyzing results:
the melting curve in fig. 4 is the amplification condition of KLKB1, F12, SIGLEC12 and AGT gene of hypertension patient, the Tm of characteristic peak corresponding to the melting curve of rs4253238 site of KLKB1 gene is 76.37 ℃, the Tm of characteristic peak corresponding to the melting curve of rs2731672 site of F12 gene is 81.32 ℃, according to the determination basis established in example 2, it can be confirmed that there is no nsSNP mutation at rs4253238 site of KLKB1 gene and rs2731672 site of F12 gene of hypertension patient; mutations may exist at site rs16982743 of SIGLEC12 gene and site rs2004776 of AGT gene.
The foregoing description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the illustrated embodiments.
Sequence listing
<110> Jiangsu medical profession college
<120> kit for rapidly detecting SNP (single nucleotide polymorphism) sites related to hypertension treatment and prognosis
<130> 1900312
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1271
<212> DNA
<213> Homo sapiens
<400> 1
ttctgtgctg caaccaggtg gcataacctc tcacctgatt ccttagctct agtgaagtta 60
ttttcgtgca tggatggctc atttgttagg aatgtgacaa cctccatgga aagagaaggc 120
ctctagcttt ataattccag caaaaatcct ggggctgcct ctcactggtt cgatgtgggt 180
taggtgttct tttttttttt cttttgagat agagtctggc tctgttccat atgtgcttcc 240
ccagaccgcc caggtttgct gaatgtgaag cccattgaag acattcaaga caacctgcta 300
caagccctgg agctccagct gaagctgaac caccctgagt cctcacagct gtttgccaag 360
ctgctccaga aaatgacaga cctcagacag attgtctgga agtctggagt ctccaggtgg 420
gcctggcacg tcagcgtcac attggccaag ggtttcagca gtgattcgga ctctgcccac 480
aggctgggct gcgtctcata aactgcaagg ggtggctggg atcagctcag tgccacagag 540
acagggctcc cccccggcct gggcccacca ttcccagacc tcacccctgc actcacatat 600
ggctgcttct gtcactgggc cccaggtgac acctgcggag acagccgagg attaccagct 660
gaagatgcag aagtccgtga cggtgcagga gggcctgtgt gtctctgtgc tttgctcctt 720
ttcctacccc caaaatggct ggaatgactc cgatccagtt catggctact ggttctgggc 780
agggtcccat acagaccggg atgctccagt ggccacaaac aacccagccc agctgctgct 840
gtccacggtg gtgggcgtgt tcacagcccc aggcctgcac ctgaagcagc cgtttgtgca 900
gggcctggct ctctataccc ctgtggtcct cccacgctct ctggacttca cagaactgga 960
tgttgctgct gagaagattg acaggttcat gcaggctgtg acaggatgga agactggcac 1020
cacaggagca tgagccgtca tcttcacctt tagcattcag cccgggagaa gtagggagac 1080
atagaagggg caggtgctgg ccaagaggca ggggcaggag aggagaaggc ggaggggcac 1140
tcagggcgag ggtgtcaggc ccgccacccc agagcaccat tactcccagg acgcggctgc 1200
gtgcagacct ggaaccagcc tagggagcag ccgcagatca caactgagaa caaacgacag 1260
tctctgcctc a 1271
<210> 2
<211> 1271
<212> DNA
<213> Homo sapiens
<400> 2
ttctgtgctg caaccaggtg gtataacctc tcacctgatt ccttagctct agtgaagtta 60
ttttcgtgca tggatggctc atttgttagg aatgtggcaa cctccatgga aagagaaggc 120
ctctagcttt ataattccag caaaaatcct ggggctgcct ctcactggtt cgatgtgggt 180
taggtgttct tttttttttt cttttgagat agagtctggc tctgttccat atgtgcttcc 240
ccagactgcc caggtttgct gaatgtgaag cccattgaag acattcaaga caacctgcta 300
caagccctgg agctccagct gaagctgaac caccctgagt cctcacagct gtttgccaag 360
ctgctccaga aaatgacaga cctcagacag attgtctgga agtctggagt ctccaggcgg 420
gcctggcacg tcagcgtcac attggccaag ggtttcagca gtgattcgga ctctgcccac 480
aggctgggct gcgtctcata aactgcaagg ggtggctggg atcagctcag tgccacagag 540
acagggctcc cccccggcct gggcccacca ttcccagacc tcacccctgc actcacatat 600
ggctgcttct gtcactgggc cccaggtgac acctgcggag acagccgagg attaccagct 660
gaagatgtag aagtccgtga cggtgcagga gggcctgtgt gtctctgtgc tttgctcctt 720
ttcctacccc caaaatggct ggaatgactc cgatccagtt catggctact ggttctgggc 780
agggtcccat acagaccggg atgctccagt ggccacaaac aacccagccc agctgctgct 840
gtccatggtg gtgggcgtgt tcacagcccc aggcctgcac ctgaagcagc cgtttgtgca 900
gggcctggct ctctataccc ctgtggtcct cccacgctct ctggacttca cagaactgga 960
tgttgctgct gagaagattg acaggttcat gcaggctgtg acaggatgga agactggcac 1020
cacaggagca tgagccatca tcttcacctt tagcattcag cccgggagaa gtagggagac 1080
atagaagggg caggtgctgg ccaagaggca ggggcaggag aggagaaggc ggaggggcac 1140
tcagggcgag ggtgtcaggc ccgccacccc agagcaccat tactcccagg acgcggctgc 1200
gtgcagacct ggaaccagcc tagggagcag ccgcagatca caactgagaa caaacgacag 1260
tctctgcctc a 1271
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttctgtgctg caaccaggta tc 22
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atccatgcac gaaaataact tc 22
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggctcatttg ttaggaatgc ta 22
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acagagccag actctatctc aa 22
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaggattacc agctgaagac tc 22
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gagcatcccg gtctgtatgg 20
<210> 9
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
accacaggag catgagatg 19
<210> 10
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tgaggcagag actgtcgttt gt 22
Claims (3)
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468782A (en) * | 2012-06-06 | 2013-12-25 | 解码（上海）生物医药科技有限公司 | Essential hypertension gene noninvasive detection kit |
| CN109082464A (en) * | 2017-06-14 | 2018-12-25 | 合肥中科普瑞昇生物医药科技有限公司 | A kind of primer sets and kit detecting hypertension drug metabolism related gene |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468782A (en) * | 2012-06-06 | 2013-12-25 | 解码（上海）生物医药科技有限公司 | Essential hypertension gene noninvasive detection kit |
| CN109082464A (en) * | 2017-06-14 | 2018-12-25 | 合肥中科普瑞昇生物医药科技有限公司 | A kind of primer sets and kit detecting hypertension drug metabolism related gene |
Non-Patent Citations (4)
| Title |
|---|
| Genome-Wide Association Study on Plasma Levels of Midregional-Proadrenomedullin and C-Terminal-Pro-Endothelin-1;Niek Verweij et al;《Hypertension》;20131231;第602-608页 * |
| Hypertension pharmacogenomics: in search of personalized treatment approaches;Rhonda M.Cooper-DeHoff et al;《Nat Rev Nephrol.》;20161231;第12卷(第2期);第110-122页 * |
| Reevaluation of the association of seven candidate genes with blood pressure and hypertension: a replication study and meta-analysis with a larger sample size;Fumihiko Takeuchi et al;《Hypertension Research》;20121231;第35卷;第825-831页 * |
| 激肽释放酶-激肽系统与高血压;杨剑辉等;《国外医学泌尿系统分册》;20031231;第23卷(第6期);第765-768页 * |
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