CN113846158A - Composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof - Google Patents
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Abstract
The invention discloses a composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof, wherein the kit comprises primers for amplifying SNP sites of related loci of kidney diseases, type II diabetes and cerebral hemorrhage. Compared with the prior art, the invention has the following advantages: (1) the kit improves the SNP recognition capability of the primers through primer optimization, so that the primers can effectively recognize the difference of one base, the optimal annealing temperature range is enlarged, the false positive of a detection result caused by the accuracy requirement of the annealing temperature is avoided, and the detection accuracy and reliability are improved; (2) the kit combines an ARMS technology with a capillary electrophoresis method for the first time, simultaneously detects 10 SNP sites of three chronic diseases, and is the kit with the platform for detecting the most sites of the three chronic diseases; (3) the kit is convenient to use, the operation method is simple, and the detection time is shortened.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to a molecular marker and application thereof, in particular to a composite amplification kit for simultaneously detecting susceptibility genes of three chronic diseases and application thereof.
Background
The related disease risk gene detection is carried out on the population with family genetic history and the population with poor living habits, the risk of diseases is known in advance, and therefore the occurrence of the diseases is effectively avoided by various methods such as dietary nutrition adjustment, life style change, physical examination frequency increase, early diagnosis and treatment acceptance and the like. Therefore, the life quality of high risk people is guaranteed, and a large amount of medical expenses are saved for families and the society. Through disease susceptibility gene detection, genetic information evaluation is carried out, the sensitivity of an individual to the environmental influence is obtained, adverse environmental stimulation is avoided in time, professional prevention guidance and professional screening are accepted, and the occurrence of diseases can be prevented and delayed.
By consulting the literature, the SNP sites related to the kidney disease, cerebral hemorrhage and type II diabetes are investigated, and the selection of each site is introduced as follows:
the kidney disease site is selected according to the following steps: rs6495446 is located on MTHFS gene, and research shows that the locus is obviously related to the onset of the kidney disease, and the risk of the onset of the kidney disease is increased in people carrying C allele compared with people carrying T allele. Genome-wide association (GWAS) analysis indicated that PLA2R1 gene and HLA-DQA1 gene were closely associated with the risk of onset of renal disease. rs4664308 is located on PLA2R1 gene and carries an a allele which is at increased risk of renal disease compared to a G allele. rs2187668 is located on HLA-DQA1 gene, and has significantly increased risk of renal disease compared with C allele carrying human.
Cerebral hemorrhage site selection basis: MTHFR (methylene tetrahydrofolate reductase) mainly plays a role in reducing methylene tetrahydrofolate into methyl tetrahydrofolate in a folate metabolism pathway and further participates in the methylation process of homocysteine. When the rs1801133 locus of the gene is of an AA genotype, the MTHFR activity is reduced, the cysteine level is increased, cytotoxicity and vascular endothelial cell injury are caused, vascular smooth muscle cell proliferation is stimulated, an organism blood coagulation and fibrinolysis system is damaged, and the risk of cerebral hemorrhage is increased. Apolipoprotein E encoded by APOE gene is a multifunctional glycoprotein, is an important component of lipoproteins such as plasma very low density protein (VLDL), High Density Lipoprotein (HDL) and the like, can be combined with VLDL, influences lipoprotein metabolism through the transportation of plasma cholesterol and triglyceride, maintains blood lipid balance, and participates in the regulation of cardiovascular and cerebrovascular functions. The more common rs429358 allele T. If allele C and the same chromosome also contains the rs 7412C allele, the combination is called APOE- ε 4 allele. The APOE-e 4 allele has a large impact on the risk of alzheimer's disease.
Type II diabetes site selection basis: MTHFR (methylene tetrahydrofolate reductase) mainly plays a role in reducing methylene tetrahydrofolate into methyl tetrahydrofolate in a folate metabolism pathway and further participates in the methylation process of homocysteine. The rs1801133 locus has AA genotype, and the gene has low activity, so that the body is disordered in a plurality of basic biochemical processes, and further, a plurality of diseases are caused. The protein encoded by the CDKAL1 gene can specifically inhibit the activation of the cell cycle dependent protein kinase 5, and the protein kinase 5 has a key role in the formation of islet cells. The rs4712523 locus has GG genotype, CDKAL1 gene expression level is low, ATP generation is reduced, glucose channel response reaction is weakened, insulin secretion is reduced, too high blood sugar cannot be controlled, hyperglycemia is caused, too much blood sugar can be discharged only through urine, and diabetes is easily caused. Clinical preventive medicine studies find that the rs10811661 locus located on the CDKN2A/2B gene is associated with the onset of type II diabetes. The T allele at locus rs10811661 is a susceptible genotype, increasing the risk of type II diabetes compared to the C allele. The KCNJ11 gene codes for Kir6.2 subunit, and plays an important role in the secretion and action process of insulin. In people with TT gene type at rs5219 site, the content of Kir6.2 protein is low, and insulin secretion is reduced. The IGF2BP2 gene encodes insulin-like growth factor 2 binding protein 2, which is involved in impaired pancreatic islet beta cell function, insulin resistance, and glucose metabolism disorders. In the human with TT genotype at rs4402960 site, the function of islet beta cells is often damaged, so that the secretion of insulin is reduced, and diabetes is induced.
The ARMS-PCR technology is based on allele-specific extension reaction, and extension reaction can be carried out only when the 3' terminal base of a certain allele-specific primer is complementary with the base at the mutation site. The upstream and downstream primers used by the conventional PCR amplified DNA are completely matched with a target sequence, the allele PCR adopts two upstream primers with allele specificity, the two upstream primers are different in 3' end nucleotide, one is specific to wild allele, the other is specific to mutant allele, under the action of Taq DNA polymerase, the upstream primer which is not completely matched with the template can not anneal and can not generate PCR product, and the primer system matched with the template can amplify the product, and the existence of the amplified product can be easily distinguished through gel electrophoresis or qPCR, thereby determining SNP genotype.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art and obtain a method for comprehensively detecting 10 SNP sites of three chronic disease susceptibility genes, which is simple and low in detection cost, the invention provides a composite amplification kit for simultaneously detecting the three chronic disease susceptibility genes and application thereof, and particularly detects the SNP sites of the chronic disease susceptibility genes related to kidney diseases, type II diabetes and cerebral hemorrhage by an ARMS-PCR technology. The method specifically comprises the following steps: sites associated with three chronic diseases were selected: kidney diseases (MTHFS, HLA-DQA1, PLA2R1), type II diabetes (KCNJ11, IGF2BP2, CDKAL1, MTHFR, CDKN2A/2B), cerebral hemorrhage (MTHFR, APOE), and the susceptibility genes of three chronic diseases are detected by a CE-PCR detection platform, which is an effective and convenient genotyping method.
The technical scheme is as follows: a composite amplification kit for simultaneously detecting three chronic disease susceptibility genes, wherein the kit comprises primers for amplifying SNP loci of related loci of kidney diseases, type II diabetes and cerebral hemorrhage; each locus and its SNP site are: the loci and SNP loci of the kidney diseases are MTHFS, rs6495446, HLA-DQA1, rs2187668, PLA2R1 and rs 4664308; the loci and SNP loci of type II diabetes mellitus are KCNJ11, rs5219, IGF2BP2, rs4402960, CDKAL1, rs4712523, MTHFR, rs1801133 and CDKN2A/2B, rs 10811661; the locus and SNP locus of cerebral hemorrhage are MTHFR, rs1801133, APOE, rs7412 or rs 429358; wherein rs number represents the number of the site in dbSNP database.
Preferably, the primers include a common upstream primer and a specific downstream primer for each SNP site, the specific downstream primer introducing a mismatched base at position 1, 2 or 3 of the 3' end.
Preferably, the primers and the sequences thereof are as follows:
specific primers for the detection site MTHFS (rs6495446) were the following nucleotide sequences:
SEQ ID NO.1:MTHFS-F TGCCTAAAGTTACCATTCCTCA，
SEQ ID NO.2:MTHFS-W ATTAGCTAGAATTAAGAGACTAC，
SEQ ID NO.3:MTHFS-M ATTTGCTAGAATAAAGAGACTGC；
specific primers for detecting the locus HLA-DQA1(rs2187668) are the following nucleotide sequences:
SEQ ID NO.4:HLA-F ACATGCCCATTTTATTTGATTACTT，
SEQ ID NO.5:HLA-W TTACCACATGGACCTCACTT，
SEQ ID NO.6:HLA-M TTACCACATGGTCCTCAT；
specific primers for detecting the site PLA2R1(rs4664308) were the following nucleotide sequences:
SEQ ID NO.7:PLA-F TTGACCAAGAAGGTAAGAGCAT，
SEQ ID NO.8:PLA-W CAGGTAGAACAAGACCTTTCTTAT，
SEQ ID NO.9:PLA-M CAGGTAGAACAAGACCTTTCTTAC；
specific primers for detecting site KCNJ11(rs5219) were the following nucleotide sequences:
SEQ ID NO.10:KCN-F tCGAGGAATACGTGCTGACA，
SEQ ID NO.11:KCN-W GCACGGAACCTGGGCTC，
SEQ ID NO.12:KCN-M GCACGGTACCTGGGCTT；
specific primers for detecting the IGF2BP2 site (rs4402960) were the following nucleotide sequences:
SEQ ID NO.13:IGF-F TCTGCTTTGACCATTCCTTATCT，
SEQ ID NO.14:IGF-W TAAGGTAGGATGGACAGAAGATTG，
SEQ ID NO.15:IGF-M TAAGGAAGGATGGACAGTAGATTT；
specific primers for the detection site CDKAL1(rs4712523) were the following nucleotide sequences:
SEQ ID NO.16:CDK-F ATGGGTAAAGAGTCCAGGTTAGA，
SEQ ID NO.17:CDK-W CTCCTTCTGTTGCACCCA，
SEQ ID NO.18:CDK-M CTCCTTCTGTAGCACCCG；
the specific primer for detecting the site CDKN2A/2B (rs10811661) is one of the following nucleotide sequences:
SEQ ID NO.19:CDKN2A/2B-F AGGAGGAGCCAGAAGACAGAT，
SEQ ID NO.20:CDKN2A/2B-W CACCTCCAGCTTTAGATTTCCC，
SEQ ID NO.21:CDKN2A/2B-M CACCTCCAGCTTTAGTATTCTC；
specific primers for the detection site APOE (rs7412) were the following nucleotide sequences:
SEQ ID NO.22:APOE-F CCAGGCGCTCGCGGAT，
SEQ ID NO.23:APOE-W CCGATGACCTGCAGAAGC，
SEQ ID NO.24:APOE-M CCGTTGACCTGCAGAAGT；
specific primers for detecting the site APOE (rs429358) were the following nucleotide sequences:
SEQ ID NO.25:APOE-F1 GCAGCTCCTCGGTGCTCT，
SEQ ID NO.26:APOE-W1GGACATGGAGGACGTGC，
SEQ ID NO.27:APOE-M1 GGACATGGAGGACGTGT；
specific primers for the detection site MTHFR (rs1801133) were the following nucleotide sequences:
SEQ ID NO.28:MTHFR-F TCATCCCTATTGGCAGGTTA，
SEQ ID NO.29:MTHFR-W GCGAGATGATGAAATCGG，
SEQ ID NO.30:MTHFR-M GCGAGATGATGAAATCGA；
among them, underlined bases are introduced mismatched bases.
Preferably, when the detection site is MTHFS (rs6495446) wild-type site C, the primer combination is a common upstream primer of SEQ ID NO.1 and SEQ ID NO. 2; when the detection site is MTHFS (rs6495446) mutant site T, the primer combination is used as a common upstream primer SEQ ID NO.1 and SEQ ID NO. 3; when the detection site is HLA-DQA1(rs2187668) wild type site G, the primer combination is used as a common upstream primer SEQ ID NO.4 and SEQ ID NO. 5; when the detection site is HLA-DQA1(rs2187668) mutant site A, the primer combination is used as a common upstream primer SEQ ID NO.4 and SEQ ID NO. 6; when the detection site is PLA2R1(rs4664308) wild type site A, the primer combination is used as a common upstream primer SEQ ID NO.7 and SEQ ID NO. 8; when the detection site is PLA2R1(rs4664308) mutant site G, the primer combination is used as a common upstream primer SEQ ID NO.7 and SEQ ID NO. 9; when the detection site is KCNJ11(rs5219) wild type mutation site C, the primer combination is used as a common upstream primer SEQ ID NO.10 and SEQ ID NO. 11; when the detection site is KCNJ11(rs5219) mutant mutation site T, the primer combination is used as a common upstream primer SEQ ID NO.10 and SEQ ID NO. 12; when the detection site is IGF2BP2(rs4402960) wild type mutation site G, the primer combination is used as a common upstream primer of SEQ ID NO.13 and SEQ ID NO. 14; when the detection site is IGF2BP2(rs4402960) mutant type mutation site T, the primer combination is used as a common upstream primer of SEQ ID NO.13 and SEQ ID NO. 15; when the detection site is CDKAL1(rs4712523) wild type mutation site A, the primer combination is used as a common upstream primer of SEQ ID NO.16 and SEQ ID NO. 17; when the detection site is CDKAL1(rs4712523) mutant mutation site G, the primer combination is used as the common upstream primer of SEQ ID NO.16 and SEQ ID NO. 18; when the detection site is CDKN2A/2B (rs10811661) wild type mutation site C, the primer combination is used as a common upstream primer of SEQ ID NO.19 and SEQ ID NO. 20; when the detection site is CDKN2A/2B (rs10811661) wild type mutation site T, the primer combination is used as a common upstream primer of SEQ ID NO.19 and SEQ ID NO. 21; when the detection site is APOE (rs7412) wild-type mutation site C, the primer combination is used as a common upstream primer of SEQ ID NO.22 and SEQ ID NO. 23; when the detection site is APOE (rs7412) wild-type mutation site T, the primer combination is used as a common upstream primer SEQ ID NO.22 and SEQ ID NO. 24; when the detection site is APOE (rs429358) wild type mutation site G, the primer combination is used as a common upstream primer of SEQ ID NO.25 and SEQ ID NO. 26; when the detection site is APOE (rs429358) wild type mutation site A, the primer combination is used as a common upstream primer SEQ ID NO.25 and SEQ ID NO. 27; when detecting the wild-type mutation site C of the MTHFR (rs1801133), the primer combinations are the upstream primers SEQ ID NO.28 and SEQ ID NO.29, and when detecting the wild-type mutation site T of the MTHFR (rs1801133), the primer combinations are the upstream primers SEQ ID NO.28 and SEQ ID NO. 30.
Preferably, the primers are labeled with the fluorescent dyes FAM and HEX.
Preferably, the kit comprises an internal molecular weight standard and is labeled with the fluorescent dye SIZ.
Preferably, the kit comprises a reaction mixture, a hot start Taq enzyme, and sdH2O; wherein the reaction mixture comprises: MgCl2 7.5mM，Tris-HCl buffer 150mM，KCl 100mM，dNTPs 8.0mM，BSA 1.8mg/mL。
The composite amplification kit is applied to the simultaneous detection of 10 SNP sites of susceptibility genes of kidney diseases, type II diabetes and cerebral hemorrhage.
The application of any one of the composite amplification kits in prevention guidance, genetic information evaluation and screening of kidney diseases, type II diabetes and cerebral hemorrhage.
Has the advantages that: (1) the kit improves the SNP recognition capability of the primers through primer optimization, so that the primers can effectively recognize the difference of one base, the optimal annealing temperature range is enlarged, the false positive of a detection result caused by the accuracy requirement of the annealing temperature is avoided, and the detection accuracy and reliability are improved; (2) the kit combines an ARMS technology with a capillary electrophoresis method for the first time, simultaneously detects 10 SNP sites of three chronic diseases, and is the kit with the platform for detecting the most sites of the three chronic diseases; (3) the kit is convenient to use, the operation method is simple, and the detection time is shortened.
Drawings
FIG. 1 is a graph showing the results of typing on 9948 standard substance by the kit of the present invention;
FIG. 2 is a graph showing the result of typing the volunteer 353191 by the kit of the present invention;
FIG. 3 is a graph showing the result of typing the volunteer 409957 by the kit of the present invention.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The molecular biological test methods not specifically described in the following examples are performed with reference to molecular cloning, a laboratory manual (third edition) or according to a kit and a product manual; the kit biomaterials, if not specifically indicated, are commercially available. In the examples, the PCR instrument was purchased from Eppendorf, Germany, under the model Mastercycler nexus; ABI3500XL genetic analyzer was purchased from ABI corporation, usa; U-Taq DNA polymerase, Reaction mix and SIZ-500 are products of this company.
Example 1
A composite amplification kit for simultaneously detecting susceptibility genes of three chronic diseases comprises primers of 10 SNP locus loci, wherein the 10 SNP locus loci are as follows: kidney diseases (MTHFS, HLA-DQA1, PLA2R1), type II diabetes (KCNJ11, IGF2BP2, CDKAL1, MTHFR, CDKN2A/2B), cerebral hemorrhage (MTHFR, APOE); wherein MTHFR can detect kidney disease and cerebral hemorrhage simultaneously. MTHFS (rs6495446), HLA-DQA1(rs2187668), PLA2R1(rs4664308), KCNJ11(rs5219), IGF2BP2(rs4402960), CDKAL1(rs4712523), MTHFR (rs1801133), CDKN2A/2B (rs10811661), and APOE (rs7412, rs 429358). The rs number in brackets indicates the number of this site in the dbSNP database.
The primers comprise a common upstream primer and a specific downstream primer aiming at each SNP locus, and the specific downstream primer introduces mismatched bases at the 1 st, the 2 nd or the 3 rd position of the 3' end.
The primers and the sequences thereof are as follows:
specific primers for the detection site MTHFS (rs6495446) were the following nucleotide sequences:
SEQ ID NO.1:MTHFS-F TGCCTAAAGTTACCATTCCTCA，
SEQ ID NO.2:MTHFS-W ATTAGCTAGAATTAAGAGACTAC，
SEQ ID NO.3:MTHFS-M ATTTGCTAGAATAAAGAGACTGC；
specific primers for detecting the locus HLA-DQA1(rs2187668) are the following nucleotide sequences:
SEQ ID NO.4:HLA-F ACATGCCCATTTTATTTGATTACTT，
SEQ ID NO.5:HLA-W TTACCACATGGACCTCACTT，
SEQ ID NO.6:HLA-M TTACCACATGGTCCTCAT；
specific primers for detecting the site PLA2R1(rs4664308) were the following nucleotide sequences:
SEQ ID NO.7:PLA-F TTGACCAAGAAGGTAAGAGCAT，
SEQ ID NO.8:PLA-W CAGGTAGAACAAGACCTTTCTTAT，
SEQ ID NO.9:PLA-M CAGGTAGAACAAGACCTTTCTTAC；
specific primers for detecting site KCNJ11(rs5219) were the following nucleotide sequences:
SEQ ID NO.10:KCN-F tCGAGGAATACGTGCTGACA，
SEQ ID NO.11:KCN-W GCACGGAACCTGGGCTC，
SEQ ID NO.12:KCN-M GCACGGTACCTGGGCTT；
specific primers for detecting the IGF2BP2 site (rs4402960) were the following nucleotide sequences:
SEQ ID NO.13:IGF-F TCTGCTTTGACCATTCCTTATCT，
SEQ ID NO.14:IGF-W TAAGGTAGGATGGACAGAAGATTG，
SEQ ID NO.15:IGF-M TAAGGAAGGATGGACAGTAGATTT；
specific primers for the detection site CDKAL1(rs4712523) were the following nucleotide sequences:
SEQ ID NO.16:CDK-F ATGGGTAAAGAGTCCAGGTTAGA，
SEQ ID NO.17:CDK-W CTCCTTCTGTTGCACCCA，
SEQ ID NO.18:CDK-M CTCCTTCTGTAGCACCCG；
the specific primers for detecting site CDKN2A/2B (rs10811661) are the following nucleotide sequences:
SEQ ID NO.19:CDKN2A/2B-F AGGAGGAGCCAGAAGACAGAT，
SEQ ID NO.20:CDKN2A/2B-W CACCTCCAGCTTTAGATTTCCC，
SEQ ID NO.21:CDKN2A/2B-M CACCTCCAGCTTTAGTATTCTC；
specific primers for the detection site APOE (rs7412) were the following nucleotide sequences:
SEQ ID NO.22:APOE-F CCAGGCGCTCGCGGAT，
SEQ ID NO.23:APOE-W CCGATGACCTGCAGAAGC，
SEQ ID NO.24:APOE-M CCGTTGACCTGCAGAAGT；
specific primers for detecting the site APOE (rs429358) were the following nucleotide sequences:
SEQ ID NO.25:APOE-F1 GCAGCTCCTCGGTGCTCT，
SEQ ID NO.26:APOE-W1GGACATGGAGGACGTGC，
SEQ ID NO.27:APOE-M1 GGACATGGAGGACGTGT；
specific primers for the detection site MTHFR (rs1801133) were the following nucleotide sequences:
SEQ ID NO.28:MTHFR-F TCATCCCTATTGGCAGGTTA，
SEQ ID NO.29:MTHFR-W GCGAGATGATGAAATCGG，
SEQ ID NO.30:MTHFR-M GCGAGATGATGAAATCGA；
among them, underlined bases are introduced mismatched bases.
The primer is marked by fluorescent dyes FAM and HEX.
The kit comprises an internal molecular weight standard and is labeled with a fluorescent dye SIZ.
The kit comprises a reaction mixture, a hot start Taq enzyme and sdH2O; wherein the reaction mixture comprises: MgCl27.5mM，Tris-HCl buffer 150mM，KCl 100mM，dNTPs 8.0mM，BSA 1.8mg/mL。
Example 2
1. Material checking and collecting: the test material is 2 parts of oral swabs of volunteers.
2. Extracting template DNA: template DNA was extracted from 2 volunteers' buccal swab specimens by the Chelex-100 method.
3. And (3) PCR amplification:
(1) the used multiplex detection primers were:
(2) SNP locus re-amplification primer configuration table:
(3) the PCR amplification system is as follows:
PCR amplification by using the composite amplification primer
10 μ L system:
(4) the PCR amplification procedure was:
denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 15s, annealing at 58 deg.C for 55s, extension at 70 deg.C for 35s, performing 29 cycles, maintaining at 60 deg.C for 5min, and storing at 10 deg.C.
4. And (3) capillary electrophoresis detection:
taking SIZ-500 as a DNA molecular weight internal standard, mixing 1 mu L of PCR amplification product and 12 mu L of formamide internal standard SIZ-5001 mu L, adding into a 96-well plate, denaturing at 95 ℃ for 3min, immediately carrying out ice bath for 3min, centrifuging, and placing into an ABI 3130xl genetic analyzer for electrophoresis detection, wherein the detection result of genotyping of 2 volunteers is shown in figures 2 and 3.
5. And (3) detection results and suggestions:
(1) results of gene testing of volunteers 353191:
(2) results of gene testing of volunteers 409957:
sequence listing
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acatgcccat tttatttgat tactt 25
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ttaccacatg gacctcactt 20
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ttaccacatg gtcctcat 18
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<213> Artificial Sequence (Artificial Sequence)
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ttgaccaaga aggtaagagc at 22
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caggtagaac aagacctttc ttat 24
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caggtagaac aagacctttc ttac 24
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<213> Artificial Sequence (Artificial Sequence)
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taaggtagga tggacagaag attg 24
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<400> 16
atgggtaaag agtccaggtt aga 23
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ctccttctgt tgcaccca 18
<210> 18
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ctccttctgt agcacccg 18
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<211> 21
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<213> Artificial Sequence (Artificial Sequence)
<400> 19
aggaggagcc agaagacaga t 21
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Claims (9)
1. The composite amplification kit for simultaneously detecting three chronic disease susceptibility genes is characterized by comprising primers for amplifying SNP sites of loci related to renal diseases, type II diabetes and cerebral hemorrhage; each locus and its SNP site are: the loci and SNP loci of the kidney diseases are MTHFS, rs6495446, HLA-DQA1, rs2187668, PLA2R1 and rs 4664308; the loci and SNP loci of type II diabetes mellitus are KCNJ11, rs5219, IGF2BP2, rs4402960, CDKAL1, rs4712523, MTHFR, rs1801133 and CDKN2A/2B, rs 10811661; the locus and SNP locus of cerebral hemorrhage are MTHFR, rs1801133, APOE, rs7412 or rs 429358; wherein rs number represents the number of the site in dbSNP database.
2. The composite amplification kit for simultaneously detecting three chronic disease susceptibility genes as claimed in claim 1, wherein the primers comprise a common upstream primer and a specific downstream primer aiming at each SNP site, and the specific downstream primer introduces mismatched bases at the 1 st, 2 nd or 3 rd positions at the 3' end.
3. The composite amplification kit for simultaneously detecting three chronic disease susceptibility genes according to claim 1, wherein the primers and the sequences thereof are as follows:
specific primers for detection site rs6495446 were the following nucleotide sequences:
SEQ ID NO.1:MTHFS-F TGCCTAAAGTTACCATTCCTCA，
SEQ ID NO.2:MTHFS-W ATTAGCTAGAATTAAGAGACTAC，
SEQ ID NO.3:MTHFS-M ATTTGCTAGAATAAAGAGACTGC；
specific primers for detecting site rs2187668 are the following nucleotide sequences:
SEQ ID NO.4:HLA-F ACATGCCCATTTTATTTGATTACTT，
SEQ ID NO.5:HLA-W TTACCACATGGACCTCACTT，
SEQ ID NO.6:HLA-M TTACCACATGGTCCTCAT；
specific primers for detecting site rs4664308 are the following nucleotide sequences:
SEQ ID NO.7:PLA-F TTGACCAAGAAGGTAAGAGCAT，
SEQ ID NO.8:PLA-W CAGGTAGAACAAGACCTTTCTTAT，
SEQ ID NO.9:PLA-M CAGGTAGAACAAGACCTTTCTTAC；
specific primers for detecting site rs5219 are the following nucleotide sequences:
SEQ ID NO.10:KCN-F tCGAGGAATACGTGCTGACA，
SEQ ID NO.11:KCN-W GCACGGAACCTGGGCTC，
SEQ ID NO.12:KCN-M GCACGGTACCTGGGCTT；
specific primers for detecting the site rs4402960 are the following nucleotide sequences:
SEQ ID NO.13:IGF-F TCTGCTTTGACCATTCCTTATCT，
SEQ ID NO.14:IGF-W TAAGGTAGGATGGACAGAAGATTG，
SEQ ID NO.15:IGF-M TAAGGAAGGATGGACAGTAGATTT；
specific primers for detecting the locus rs4712523 are the following nucleotide sequences:
SEQ ID NO.16:CDK-F ATGGGTAAAGAGTCCAGGTTAGA，
SEQ ID NO.17:CDK-W CTCCTTCTGTTGCACCCA，
SEQ ID NO.18:CDK-M CTCCTTCTGTAGCACCCG；
specific primers for detecting site rs10811661 are the following nucleotide sequences:
SEQ ID NO.19:CDKN2A/2B-F AGGAGGAGCCAGAAGACAGAT，
SEQ ID NO.20:CDKN2A/2B-W CACCTCCAGCTTTAGATTTCCC，
SEQ ID NO.21:CDKN2A/2B-M CACCTCCAGCTTTAGTATTCTC；
the specific primers for detecting the locus rs7412 are the following nucleotide sequences:
SEQ ID NO.22:APOE-F CCAGGCGCTCGCGGAT，
SEQ ID NO.23:APOE-W CCGATGACCTGCAGAAGC，
SEQ ID NO.24:APOE-M CCGTTGACCTGCAGAAGT；
specific primers for detecting the locus rs429358 are the following nucleotide sequences:
SEQ ID NO.25:APOE-F1 GCAGCTCCTCGGTGCTCT，
SEQ ID NO.26:APOE-W1GGACATGGAGGACGTGC，
SEQ ID NO.27:APOE-M1 GGACATGGAGGACGTGT；
specific primers for detecting site rs1801133 are the following nucleotide sequences:
SEQ ID NO.28:MTHFR-F TCATCCCTATTGGCAGGTTA，
SEQ ID NO.29:MTHFR-W GCGAGATGATGAAATCGG，
SEQ ID NO.30:MTHFR-M GCGAGATGATGAAATCGA；
among them, underlined bases are introduced mismatched bases.
4. The multiplex amplification kit for simultaneously detecting three chronic disease susceptibility genes as claimed in claim 3, wherein when the detection site is rs6495446 wild type site C, the primer combination is a common upstream primer of SEQ ID No.1 and SEQ ID No. 2; when the detection site is rs6495446 mutant site T, the primer combination is used as a common upstream primer SEQ ID NO.1 and SEQ ID NO. 3; when the detection site is rs2187668 wild type site G, the primer combination is used as a common upstream primer SEQ ID NO.4 and SEQ ID NO. 5; when the detection site is rs2187668 mutant site A, the primer combination is a common upstream primer SEQ ID NO.4 and SEQ ID NO. 6; when the detection site is rs4664308 wild type site A, the primer combination is used as a common upstream primer SEQ ID NO.7 and SEQ ID NO. 8; when the detection site is rs4664308 mutant site G, the primer combination is used as a common upstream primer SEQ ID NO.7 and SEQ ID NO. 9; when the detection site is rs5219 wild type mutation site C, the primer combination is used as a common upstream primer SEQ ID NO.10 and SEQ ID NO. 11; when the detection site is rs5219 mutant mutation site T, the primer combination is used as a common upstream primer SEQ ID NO.10 and SEQ ID NO. 12; when the detection site is rs4402960 wild type mutation site G, the primer combination is a common upstream primer SEQ ID NO.13 and SEQ ID NO. 14; when the detection site is rs4402960 mutant type mutation site T, the primer combination is a common upstream primer SEQ ID NO.13 and SEQ ID NO. 15; when the detection site is rs4712523 wild type mutation site A, the primer combination is used as a common upstream primer SEQ ID NO.16 and SEQ ID NO. 17; when the detection site is rs4712523 mutant mutation site G, the primer combination is used as a common upstream primer of SEQ ID NO.16 and SEQ ID NO. 18; when the detection site is rs10811661 wild-type mutation site C, the primer combination is used as a common upstream primer SEQ ID NO.19 and SEQ ID NO. 20; when the detection site is rs10811661 wild-type mutation site T, the primer combination is used as a common upstream primer SEQ ID NO.19 and SEQ ID NO. 21; when the detection site is rs7412 wild type mutation site C, the primer combination is used as a common upstream primer SEQ ID NO.22 and SEQ ID NO. 23; when the detection site is rs7412 wild type mutation site T, the primer combination is used as a common upstream primer SEQ ID NO.22 and SEQ ID NO. 24; when the detection site is rs429358 wild type mutation site G, the primer combination is a common upstream primer SEQ ID NO.25 and SEQ ID NO. 26; when the detection site is rs429358 wild type mutation site A, the primer combination is a common upstream primer SEQ ID NO.25 and SEQ ID NO. 27; when the wild type mutation site C of the site rs1801133 is detected, the primer combinations are the upstream primers SEQ ID NO.28 and SEQ ID NO.29, and when the wild type mutation site T of the site rs1801133 is detected, the primer combinations are the upstream primers SEQ ID NO.28 and SEQ ID NO. 30.
5. The multiplex amplification kit for simultaneously detecting three chronic disease susceptibility genes as recited in claim 1, wherein the primers are labeled by fluorescent dyes FAM and HEX.
6. The multiplex amplification kit for simultaneously detecting three chronic disease susceptibility genes according to claim 1, wherein the kit comprises molecular weight internal standards and is labeled by a fluorescent dye SIZ.
7. The multiplex amplification kit for simultaneously detecting three chronic disease susceptibility genes as claimed in claim 1, wherein the kit comprises reaction mixture, hot start Taq enzyme, sdH2O; wherein the reaction mixture comprises: MgCl2 7.5mM，Tris-HCl buffer 150mM，KCl 100mM，dNTPs 8.0mM，BSA 1.8mg/mL。
8. The use of the multiplex amplification kit of any one of claims 1 to 7 for simultaneously detecting 10 SNP sites of susceptibility genes of renal disease, type II diabetes and cerebral hemorrhage.
9. Use of the multiplex amplification kit of any one of claims 1 to 7 for the prevention guidance, genetic information evaluation and screening of renal diseases, type II diabetes and cerebral hemorrhage.
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