CN113846158B - Composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof - Google Patents
Composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof Download PDFInfo
- Publication number
- CN113846158B CN113846158B CN202111403363.6A CN202111403363A CN113846158B CN 113846158 B CN113846158 B CN 113846158B CN 202111403363 A CN202111403363 A CN 202111403363A CN 113846158 B CN113846158 B CN 113846158B
- Authority
- CN
- China
- Prior art keywords
- seq
- site
- primer
- detection site
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 28
- 208000017667 Chronic Disease Diseases 0.000 title claims abstract description 21
- 238000012197 amplification kit Methods 0.000 title claims abstract description 15
- 239000002131 composite material Substances 0.000 title claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 85
- 208000017169 kidney disease Diseases 0.000 claims abstract description 18
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims abstract description 15
- 206010008111 Cerebral haemorrhage Diseases 0.000 claims abstract description 14
- 238000011144 upstream manufacturing Methods 0.000 claims description 47
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 30
- 108010009392 Cyclin-Dependent Kinase Inhibitor p16 Proteins 0.000 claims description 17
- 102100031739 5-formyltetrahydrofolate cyclo-ligase Human genes 0.000 claims description 9
- 108020005541 5-formyltetrahydrofolate cyclo-ligase Proteins 0.000 claims description 9
- 102100029392 Secretory phospholipase A2 receptor Human genes 0.000 claims description 9
- 102200088972 rs1801133 Human genes 0.000 claims description 9
- 101001126009 Homo sapiens Secretory phospholipase A2 receptor Proteins 0.000 claims description 7
- 239000011541 reaction mixture Substances 0.000 claims description 7
- 102220539361 ATP-sensitive inward rectifier potassium channel 11_E23K_mutation Human genes 0.000 claims description 6
- 239000007850 fluorescent dye Substances 0.000 claims description 6
- 102210002203 rs2187668 Human genes 0.000 claims description 6
- 102200017290 rs429358 Human genes 0.000 claims description 6
- 102220000965 rs4402960 Human genes 0.000 claims description 6
- 102210008728 rs4712523 Human genes 0.000 claims description 6
- 102200017284 rs7412 Human genes 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102100028966 HLA class I histocompatibility antigen, alpha chain F Human genes 0.000 claims description 3
- 101000986080 Homo sapiens HLA class I histocompatibility antigen, alpha chain F Proteins 0.000 claims description 3
- 102000009508 Cyclin-Dependent Kinase Inhibitor p16 Human genes 0.000 claims 4
- 238000000034 method Methods 0.000 abstract description 12
- 238000000137 annealing Methods 0.000 abstract description 5
- 238000005251 capillar electrophoresis Methods 0.000 abstract description 3
- 238000005457 optimization Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 33
- 108700028369 Alleles Proteins 0.000 description 19
- 108010030837 Methylenetetrahydrofolate Reductase (NADPH2) Proteins 0.000 description 17
- 102100029684 Methylenetetrahydrofolate reductase Human genes 0.000 description 17
- 230000035772 mutation Effects 0.000 description 15
- 101150037123 APOE gene Proteins 0.000 description 13
- 102100029470 Apolipoprotein E Human genes 0.000 description 13
- 102100024458 Cyclin-dependent kinase inhibitor 2A Human genes 0.000 description 13
- 101000737828 Homo sapiens Threonylcarbamoyladenosine tRNA methylthiotransferase Proteins 0.000 description 10
- 102100035310 Threonylcarbamoyladenosine tRNA methylthiotransferase Human genes 0.000 description 9
- 102100036243 HLA class II histocompatibility antigen, DQ alpha 1 chain Human genes 0.000 description 8
- 108010086786 HLA-DQA1 antigen Proteins 0.000 description 8
- 101000599779 Homo sapiens Insulin-like growth factor 2 mRNA-binding protein 2 Proteins 0.000 description 8
- 102100037919 Insulin-like growth factor 2 mRNA-binding protein 2 Human genes 0.000 description 8
- 101000614701 Homo sapiens ATP-sensitive inward rectifier potassium channel 11 Proteins 0.000 description 7
- 102000017792 KCNJ11 Human genes 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- -1 rs6495446 Proteins 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000003914 insulin secretion Effects 0.000 description 3
- TZBGSHAFWLGWBO-ABLWVSNPSA-N (2s)-2-[[4-[(2-amino-4-oxo-5,6,7,8-tetrahydro-1h-pteridin-6-yl)methylamino]benzoyl]amino]-5-methoxy-5-oxopentanoic acid Chemical compound C1=CC(C(=O)N[C@@H](CCC(=O)OC)C(O)=O)=CC=C1NCC1NC(C(=O)NC(N)=N2)=C2NC1 TZBGSHAFWLGWBO-ABLWVSNPSA-N 0.000 description 2
- QYNUQALWYRSVHF-ABLWVSNPSA-N 5,10-methylenetetrahydrofolic acid Chemical compound C1N2C=3C(=O)NC(N)=NC=3NCC2CN1C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 QYNUQALWYRSVHF-ABLWVSNPSA-N 0.000 description 2
- 101710084687 Cyclin-dependent kinase 2 homolog Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 102000015779 HDL Lipoproteins Human genes 0.000 description 2
- 108010010234 HDL Lipoproteins Proteins 0.000 description 2
- 101150038552 HLA-DQA1 gene Proteins 0.000 description 2
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 2
- 101150054719 PLA2R1 gene Proteins 0.000 description 2
- 101710122046 Secretory phospholipase A2 receptor Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229940014144 folate Drugs 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 235000019152 folic acid Nutrition 0.000 description 2
- 239000011724 folic acid Substances 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 102100021177 ATP-sensitive inward rectifier potassium channel 11 Human genes 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 101710095339 Apolipoprotein E Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 101001076292 Homo sapiens Insulin-like growth factor II Proteins 0.000 description 1
- 101150104609 IGF2BP2 gene Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 102100025947 Insulin-like growth factor II Human genes 0.000 description 1
- 101150090219 Kcnj11 gene Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 101150074563 MTHFS gene Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000009084 cardiovascular function Effects 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000008455 cerebrovascular function Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 208000022602 disease susceptibility Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000018914 glucose metabolism disease Diseases 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000008604 lipoprotein metabolism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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 loci of related loci of kidney disease, type II diabetes and cerebral hemorrhage. Compared with the prior art, the invention has the following advantages: (1) According to the kit, through primer optimization, the identification capability of the primer to SNP is improved, so that the primer can effectively identify the difference of one base, the optimal annealing temperature range is enlarged, false positive of a detection result caused by the accurate requirement of the annealing temperature is avoided, and the detection accuracy and reliability are improved; (2) The kit combines ARMS technology with capillary electrophoresis method for the first time, and simultaneously detects 10 SNP loci of three chronic diseases, thus being the most kit for three chronic disease detection loci by the platform; (3) The kit provided by the invention is convenient to use, simple in operation method and short in detection time.
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 crowd with family genetic history and the crowd with bad life habit, and the risk of the disease is known in advance, so that the occurrence of the disease is effectively avoided by various methods such as adjusting diet nutrition, changing life style, increasing physical examination frequency, accepting early diagnosis and treatment, and the like. Thus, the life quality of high-risk groups is ensured, and great medical expenses are saved for families and society. Through disease susceptibility gene detection, genetic information evaluation is carried out, the sensitivity of individuals to environmental influence is obtained, adverse environmental stimulation is timely avoided, professional prevention guidance is received, and the occurrence of diseases can be prevented and delayed through professional screening.
By consulting the literature, SNP loci associated with kidney diseases, cerebral hemorrhage and type II diabetes are investigated, and the basis of selection of each locus is presented as follows:
kidney disease site selection was based on: rs6495446 is located on the MTHFS gene, and researches show that the locus has obvious association with the onset of kidney disease, and the locus carries a C allele which is higher than a T allele of a human body, so that the onset risk of kidney disease is increased. Whole genome association (GWAS) analysis showed that PLA2R1 gene and HLA-DQA1 gene are closely related to the risk of kidney disease. rs4664308 is located on the PLA2R1 gene and increases the risk of renal disease in humans carrying a allele compared to G allele. rs2187668 is located on HLA-DQA1 gene, and the risk of onset of kidney disease is obviously increased for people carrying T allele rather than C allele.
Cerebral hemorrhage site selection was based on: MTHFR (methylene tetrahydrofolate reductase) has the main function of reducing methylene tetrahydrofolate to methyl tetrahydrofolate in the folate metabolic pathway and is further involved in the homocysteine methylation process. When the rs1801133 locus of the gene is AA genotype, MTHFR activity is reduced, cysteine level is increased, cytotoxicity is caused, vascular endothelial cells are damaged, vascular smooth muscle cell proliferation is stimulated, and the body coagulation and fibrinolysis system is destroyed, so that the risk of cerebral hemorrhage is increased. APOE gene encoded apolipoprotein E is a multifunctional glycoprotein, is an important component of lipoproteins such as very low density proteins (VLDL) and High Density Lipoproteins (HDL) in blood plasma, can be combined with VLDL, influences lipoprotein metabolism through transportation of plasma cholesterol and triglyceride, maintains blood lipid balance, and participates in regulation of cardiovascular and cerebrovascular functions. More common is the rs429358 allele T. If allele C and the same chromosome also contains the rs 7412C allele, then the combination is referred to as the APOE- ε4 allele. The APOE-epsilon 4 allele has a large impact on the risk of alzheimer's disease.
Type II diabetes locus selection basis: MTHFR (methylene tetrahydrofolate reductase) has the main function of reducing methylene tetrahydrofolate to methyl tetrahydrofolate in the folate metabolic pathway and is further involved in the homocysteine methylation process. The rs1801133 locus has AA genotype, and the gene has low activity, which leads to disorder of a plurality of basic biochemical processes of the organism and further causes a plurality of symptoms. The CDKAL1 gene encodes a protein that specifically inhibits activation of the cell cycle dependent protein kinase 5, and the protein kinase 5 has a key role in islet cell formation. The rs4712523 locus has the GG genotype, the CDKAL1 gene expression level is low, the generation of ATP is reduced, the response reaction of glucose channels is weakened, the insulin secretion is reduced, the excessively high blood sugar can not be controlled, the hyperglycemia is caused, the excessive blood sugar can only be discharged through urine, and diabetes is easily caused. Clinical preventive medicine research shows that the site of rs10811661 on CDKN2A/2B gene is related to the pathogenesis of type II diabetes. The T allele at position rs10811661 is a susceptibility genotype that increases the risk of type II diabetes compared to the C allele. The KCNJ11 gene encodes kir6.2 subunit and plays an important role in insulin secretion and action. The rs5219 locus has TT genotype, and the Kir6.2 protein content is low, so that insulin secretion is reduced. The IGF2BP2 gene encodes insulin-like growth factor 2 binding protein 2, which is associated with impaired islet beta cell function, insulin resistance, glucose metabolism disorders, and the like. The rs4402960 locus has TT genotype, so that the functions of islet beta cells are often damaged, and further, the secretion of insulin is reduced, and diabetes is induced.
ARMS-PCR technology is based on allele-specific extension reactions, which can only be performed when the 3' -terminal base of a certain allele-specific primer is complementary to the base at the mutation site. The upstream primer and the downstream primer used for amplifying DNA by conventional PCR 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 a wild type allele, the other is specific to a mutant allele, the upstream primers which are incompletely matched with a template cannot anneal and cannot generate PCR products under the action of Taq DNA polymerase, a primer system matched with the template can amplify the products, and the existence of the amplified products can be easily distinguished by gel electrophoresis or qPCR, so that the SNP genotype is determined.
Disclosure of Invention
The technical problems to be solved are as follows: in order to overcome the defects of the prior art, a method for comprehensively detecting 10 SNP loci of three chronic disease susceptibility genes is obtained, which is simple and low in detection cost, and in view of the fact, the invention provides a composite amplification kit for simultaneously detecting the three chronic disease susceptibility genes and application thereof, and particularly detects the SNP loci of the chronic disease susceptibility genes related to kidney disease, type II diabetes and cerebral hemorrhage by an ARMS-PCR technology. The method comprises the following steps: sites associated with three chronic diseases were selected: kidney disease (MTHFS, HLA-DQA1, PLA2R 1), type II diabetes (KCNJ 11, IGF2BP2, CDKAL1, MTHFR, CDKN 2A/2B), cerebral hemorrhage (MTHFR, APOE), and detecting susceptibility genes of three chronic diseases 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 disease, type II diabetes and cerebral hemorrhage; each locus and SNP locus thereof are: the gene loci and SNP loci of kidney diseases are MTHFS, rs6495446, HLA-DQA1, rs2187668 and PLA2R1, rs4664308; the loci and SNP loci of type II diabetes are KCNJ11, rs5219, IGF2BP2, rs4402960, CDKAL1, rs4712523, MTHFR, rs1801133 and CDKN2A/2B, rs10811661; the locus of cerebral hemorrhage and SNP locus are MTHFR, rs1801133, APOE, rs7412 or rs429358; wherein, rs represents the number of the site in the dbSNP database.
Preferably, the primers include a common upstream primer for each SNP site and a specific downstream primer that introduces a mismatched base at position 1, position 2 or position 3 of the 3' end.
Preferably, the primer and the sequence thereof are as follows:
specific primers for the detection site MTHFS (rs 6495446) are 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 the detection site HLA-DQA1 (rs 2187668) 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 detection site PLA2R1 (rs 4664308) 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 detection site KCNJ11 (rs 5219) 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 the detection site IGF2BP2 (rs 4402960) 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 (rs 4712523) 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；
the specific primer for detecting the site CDKN2A/2B (rs 10811661) 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 detection of the site APOE (rs 7412) 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 detection of site APOE (rs 429358) 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 (rs 1801133) 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；
wherein the underlined base is the introduced mismatched base.
Preferably, when the detection site is the wild type site C of MTHFS (rs 6495446), the combination of primers is used as the common upstream primer SEQ ID NO.1 and SEQ ID NO.2; when the detection site is MTHFS (rs 6495446) mutant type site T, the common upstream primer SEQ ID NO.1 and SEQ ID NO.3 are used as primer combinations; when the detection site is HLA-DQA1 (rs 2187668) wild type site G, the common upstream primer SEQ ID NO.4 and SEQ ID NO.5 are used as primer combinations; when the detection site is HLA-DQA1 (rs 2187668) mutant site A, the common upstream primer SEQ ID NO.4 and SEQ ID NO.6 are used as primer combinations; when the detection site is PLA2R1 (rs 4664308) wild type site A, the common upstream primer SEQ ID NO.7 and SEQ ID NO.8 are used as the primer combination; when the detection site is PLA2R1 (rs 4664308) mutant site G, the common upstream primer SEQ ID NO.7 and SEQ ID NO.9 are used as primer combinations; when the detection site is a KCNJ11 (rs 5219) wild mutation site C, the common upstream primer SEQ ID NO.10 and SEQ ID NO.11 are used as primer combinations; when the detection site is a KCNJ11 (rs 5219) mutant mutation site T, the common upstream primer SEQ ID NO.10 and SEQ ID NO.12 are used as primer combinations; when the detection site is IGF2BP2 (rs 4402960) wild type mutation site G, the common upstream primer SEQ ID NO.13 and SEQ ID NO.14 are used as primer combinations; when the detection site is IGF2BP2 (rs 4402960) mutant mutation site T, the common upstream primer SEQ ID NO.13 and SEQ ID NO.15 are used as primer combinations; when the detection site is a CDKAL1 (rs 4712523) wild mutation site A, the common upstream primer SEQ ID NO.16 and SEQ ID NO.17 are used as primer combinations; when the detection site is CDKAL1 (rs 4712523) mutant mutation site G, the common upstream primer SEQ ID NO.16 and SEQ ID NO.18 are used as primer combinations; when the detection site is CDKN2A/2B (rs 10811661) wild mutation site C, the common upstream primer SEQ ID NO.19 and SEQ ID NO.20 are used as primer combinations; when the detection site is CDKN2A/2B (rs 10811661) wild mutation site T, a primer combination is used to form a common upstream primer SEQ ID NO.19 and SEQ ID NO.21; when the detection site is an APOE (rs 7412) wild-type mutation site C, the common upstream primer SEQ ID NO.22 and SEQ ID NO.23 are used as primer combinations; when the detection site is an APOE (rs 7412) wild-type mutation site T, a common upstream primer SEQ ID NO.22 and SEQ ID NO.24 are used as primer combinations; when the detection site is an APOE (rs 429358) wild-type mutation site G, the common upstream primer SEQ ID NO.25 and SEQ ID NO.26 are used as primer combinations; when the detection site is an APOE (rs 429358) wild-type mutation site A, the common upstream primer SEQ ID NO.25 and SEQ ID NO.27 are used as primer combinations; when the wild type mutation site C of the MTHFR (rs 1801133) is detected, the upstream primer SEQ ID NO.28 and SEQ ID NO.29 are used as the primer combination, and when the wild type mutation site T of the MTHFR (rs 1801133) is detected, the upstream primer SEQ ID NO.28 and SEQ ID NO.30 are used as the primer combination.
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, sdH 2 O; wherein the reaction mixture comprises: mgCl 2 7.5mM，Tris-HCl buffer 150mM，KCl 100mM，dNTPs 8.0mM，BSA 1.8mg/mL。
The application of the composite amplification kit in simultaneously detecting 10 SNP loci of susceptibility genes of kidney diseases, type II diabetes and cerebral hemorrhage.
The composite amplification kit is applied to prevention guidance, genetic information evaluation and screening of kidney diseases, type II diabetes and cerebral hemorrhage.
The beneficial effects are that: (1) According to the kit, through primer optimization, the identification capability of the primer to SNP is improved, so that the primer can effectively identify the difference of one base, the optimal annealing temperature range is enlarged, false positive of a detection result caused by the accurate requirement of the annealing temperature is avoided, and the detection accuracy and reliability are improved; (2) The kit combines ARMS technology with capillary electrophoresis method for the first time, and simultaneously detects 10 SNP loci of three chronic diseases, thus being the most kit for three chronic disease detection loci by the platform; (3) The kit provided by the invention is convenient to use, simple in operation method and short in detection time.
Drawings
FIG. 1 is a graph of the typing results of the kit of the present invention on a standard 9948;
FIG. 2 is a graph showing the typing results of the kit of the present invention on volunteers 353191;
FIG. 3 is a graph showing the typing results of the kit of the present invention on volunteers 409957.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Molecular biological assay methods not specifically described in the examples below are all described in reference to the guidelines for molecular cloning experiments (third edition) or according to the instructions of the kits and products; the kit biomaterial, unless otherwise specified, is commercially available. In the examples, the PCR apparatus was purchased from Eppendorf, germany, model Mastercycler nexus; ABI3500XL genetic Analyzer was purchased from ABI corporation of America; U-Taq DNA polymerase, reaction mix, SIZ-500 are the products of this company.
Example 1
The 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 disease (MTHFS, HLA-DQA1, PLA2R 1), type II diabetes (KCNJ 11, IGF2BP2, CDKAL1, MTHFR, CDKN 2A/2B), cerebral hemorrhage (MTHFR, APOE); wherein MTHFR can detect kidney disease and cerebral hemorrhage simultaneously. MTHFS (rs 6495446), HLA-DQA1 (rs 2187668), PLA2R1 (rs 4664308), KCNJ11 (rs 5219), IGF2BP2 (rs 4402960), CDKAL1 (rs 4712523), MTHFR (rs 1801133), CDKN2A/2B (rs 10811661), APOE (rs 7412, 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, wherein the specific downstream primer introduces mismatched bases at the 1 st, the 2 nd or the 3 rd position of the 3' tail end.
The primer and the sequence thereof are as follows:
specific primers for the detection site MTHFS (rs 6495446) are 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 the detection site HLA-DQA1 (rs 2187668) 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 detection site PLA2R1 (rs 4664308) 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 detection site KCNJ11 (rs 5219) 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 the detection site IGF2BP2 (rs 4402960) 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 (rs 4712523) 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 the detection site CDKN2A/2B (rs 10811661) were 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 detection of the site APOE (rs 7412) 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 detection of site APOE (rs 429358) 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 (rs 1801133) 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；
wherein the underlined base is the introduced mismatched base.
The primer is marked by fluorescent dyes FAM and HEX.
The kit comprises a molecular weight internal standard and is labeled with a fluorescent dye, SIZ.
The kit comprises a reaction mixture, hot start Taq enzyme and sdH 2 O; wherein the reaction mixture comprises: mgCl 2 7.5mM，Tris-HCl buffer 150mM，KCl 100mM，dNTPs 8.0mM，BSA 1.8mg/mL。
Example 2
1. And (3) collecting a detection material: the test material is a volunteer oral swab, 2 parts in total.
2. Template DNA extraction: template DNA was extracted from oral swab samples of 2 volunteers using the Chelex-100 method.
3. And (3) PCR amplification:
(1) The used multiplex amplification detection primers are:
(2) SNP locus multiplex primer configuration table:
(3) The PCR amplification system is as follows:
PCR amplification Using the above-described composite amplification primers
10. Mu.L System:
(4) The PCR amplification procedure was:
denaturation at 95℃for 5min, denaturation at 94℃for 15s, annealing at 58℃for 55s, extension at 70℃for 35s, 29 cycles, final incubation at 60℃for 5min, and storage at 10 ℃.
4. Capillary electrophoresis detection:
taking SIZ-500 as an internal standard of DNA molecular weight, mixing 1 mu L of the PCR amplification product and 12 mu L of formamide internal standard SIZ-5001 mu L, adding into a 96-well plate, denaturing for 3min at 95 ℃, immediately carrying out ice bath for 3min, centrifuging, and placing into an ABI 3130xl genetic analyzer for electrophoresis detection, wherein the genotyping detection results of loci of 2 volunteers are shown in figures 2 and 3.
5. Detection results and suggestions:
(1) Gene detection results in volunteers 353191:
(2) Gene detection results in volunteers 409957:
sequence listing
<110> De Mey Biotechnology Co., ltd
<120> composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tgcctaaagt taccattcct ca 22
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
attagctaga attaagagac tac 23
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atttgctaga ataaagagac tgc 23
<210> 4
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
acatgcccat tttatttgat tactt 25
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ttaccacatg gacctcactt 20
<210> 6
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ttaccacatg gtcctcat 18
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ttgaccaaga aggtaagagc at 22
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
caggtagaac aagacctttc ttat 24
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
caggtagaac aagacctttc ttac 24
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
tcgaggaata cgtgctgaca 20
<210> 11
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gcacggaacc tgggctc 17
<210> 12
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gcacggtacc tgggctt 17
<210> 13
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
tctgctttga ccattcctta tct 23
<210> 14
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
taaggtagga tggacagaag attg 24
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
taaggaagga tggacagtag attt 24
<210> 16
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
atgggtaaag agtccaggtt aga 23
<210> 17
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
ctccttctgt tgcaccca 18
<210> 18
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ctccttctgt agcacccg 18
<210> 19
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
aggaggagcc agaagacaga t 21
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
cacctccagc tttagatttc cc 22
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
cacctccagc tttagtattc tc 22
<210> 22
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
ccaggcgctc gcggat 16
<210> 23
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ccgatgacct gcagaagc 18
<210> 24
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
ccgttgacct gcagaagt 18
<210> 25
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
gcagctcctc ggtgctct 18
<210> 26
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ggacatggag gacgtgc 17
<210> 27
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
ggacatggag gacgtgt 17
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
tcatccctat tggcaggtta 20
<210> 29
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gcgagatgat gaaatcgg 18
<210> 30
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gcgagatgat gaaatcga 18
Claims (5)
1. The composite amplification kit for simultaneously detecting three chronic disease susceptibility genes is characterized by comprising primers for amplifying SNP loci of related loci of kidney disease, type II diabetes and cerebral hemorrhage; each locus and SNP locus thereof are: the gene locus and SNP locus of kidney disease are MTHFS rs6495446, HLA-DQA1 rs2187668 and PLA2R1 rs4664308; the loci and SNP loci of type II diabetes are KCNJ11 rs5219, IGF2BP2 rs4402960, CDKAL1 rs4712523, MTHFR rs1801133 and CDKN2A/2B rs10811661; the loci and SNP loci of cerebral hemorrhage are MTHFR rs1801133, APOE rs7412 and rs429358; wherein, rs represents the number of the locus in the dbSNP database;
the primer comprises a common upstream primer and a specific downstream primer aiming at each SNP locus, wherein the specific downstream primer introduces mismatched bases at the 1 st position, the 2 nd position or the 3 rd position of the 3' tail end;
the primer and the sequence thereof are as follows:
specific primers for detection site rs6495446 are 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 detection 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 detection 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 detection 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 detection 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 detection site 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 detection 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；
specific primers for the detection site 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 detection site 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 detection 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；
wherein the underlined base is the introduced mismatched base.
2. The composite amplification kit for simultaneously detecting three chronic disease susceptibility genes according to claim 1, wherein when the detection site is rs6495446 wild type site C, the common upstream primer SEQ ID No.1 and SEQ ID No.3 are combined by using a primer; when the detection site is rs6495446 mutant site T, the common upstream primer SEQ ID NO.1 and SEQ ID NO.2 are used as primer combinations; when the detection site is rs2187668 wild type site G, the common upstream primer SEQ ID NO.4 and SEQ ID NO.5 are used as primer combinations; when the detection site is rs2187668 mutant site A, the common upstream primer SEQ ID NO.4 and SEQ ID NO.6 are used as primer combinations; when the detection site is rs4664308 wild type site A, the common upstream primer SEQ ID NO.7 and SEQ ID NO.8 are used as primer combinations; when the detection site is rs4664308 mutant site G, the common upstream primer SEQ ID NO.7 and SEQ ID NO.9 are used as primer combinations; when the detection site is rs5219 wild type site T, the common upstream primer SEQ ID NO.10 and SEQ ID NO.12 are used as primer combinations; when the detection site is rs5219 mutant site C, the common upstream primer SEQ ID NO.10 and SEQ ID NO.11 are used as primer combinations; when the detection site is rs4402960 wild type site G, the common upstream primer SEQ ID NO.13 and SEQ ID NO.14 are used as primer combinations; when the detection site is rs4402960 mutant site T, the common upstream primer SEQ ID NO.13 and SEQ ID NO.15 are used as primer combinations; when the detection site is rs4712523 wild type site A, the common upstream primer SEQ ID NO.16 and SEQ ID NO.17 are used as primer combinations; when the detection site is rs4712523 mutant site G, the common upstream primer SEQ ID NO.16 and SEQ ID NO.18 are used as primer combinations; when the detection site is rs10811661 mutant site C, the common upstream primer SEQ ID NO.19 and SEQ ID NO.20 are used as primer combinations; when the detection site is rs10811661 wild type site T, the common upstream primer SEQ ID NO.19 and SEQ ID NO.21 are used as primer combinations; when the detection site is rs7412 wild type site C, the common upstream primer SEQ ID NO.22 and SEQ ID NO.23 are used as primer combinations; when the detection site is rs7412 mutant site T, the common upstream primer SEQ ID NO.22 and SEQ ID NO.24 are used as primer combinations; when the detection site is rs429358 mutant site C, the common upstream primer SEQ ID NO.25 and SEQ ID NO.26 are used as primer combinations; when the detection site is rs429358 wild type site T, the common upstream primer SEQ ID NO.25 and SEQ ID NO.27 are used as primer combinations; when the wild type site G of the site rs1801133 is detected, the upstream primers SEQ ID NO.28 and SEQ ID NO.29 are used as the primer combination, and when the mutant type site A of the site rs1801133 is detected, the upstream primers SEQ ID NO.28 and SEQ ID NO.30 are used as the primer combination.
3. The multiplex amplification kit for simultaneously detecting three chronic disease susceptibility genes according to claim 1, wherein the primers are labeled with fluorescent dyes FAM and HEX.
4. The multiplex amplification kit for simultaneously detecting three chronic disease susceptibility genes according to claim 1, wherein the kit contains a molecular weight internal standard and is labeled by a fluorescent dye SIZ.
5. The composite amplification kit for simultaneously detecting three chronic disease susceptibility genes according to claim 1, wherein the kit comprises a reaction mixture, a hot start Taq enzyme, sdH 2 O; wherein the reaction mixture comprises: mgCl 2 7.5mM，Tris-HCl buffer 150mM，KCl 100mM，dNTPs 8.0mM，BSA 1.8mg/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111403363.6A CN113846158B (en) | 2021-11-24 | 2021-11-24 | Composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111403363.6A CN113846158B (en) | 2021-11-24 | 2021-11-24 | Composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113846158A CN113846158A (en) | 2021-12-28 |
| CN113846158B true CN113846158B (en) | 2024-01-30 |
Family
ID=78982124
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111403363.6A Active CN113846158B (en) | 2021-11-24 | 2021-11-24 | Composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113846158B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018346A (en) * | 2018-01-15 | 2018-05-11 | 无锡中德美联生物技术有限公司 | Composite amplification reagent kit and its application of female skin and hair quality SNP genetic polymorphisms site are detected at the same time |
| CN112852956A (en) * | 2021-03-23 | 2021-05-28 | 上海康黎诊断技术有限公司 | Kit for guiding medication of human hyperlipidemia |
| CN112941182A (en) * | 2021-03-11 | 2021-06-11 | 南京先声医学检验有限公司 | Gene detection method for medicine for treating rheumatic immune diseases based on nucleic acid mass spectrometry and application of gene detection method |
| CN113403380A (en) * | 2021-06-11 | 2021-09-17 | 中国科学院北京基因组研究所(国家生物信息中心) | Complex disease related SNP site primer composition and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2227780A4 (en) * | 2008-03-19 | 2011-08-03 | Existence Genetics Llc | Genetic analysis |
-
2021
- 2021-11-24 CN CN202111403363.6A patent/CN113846158B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018346A (en) * | 2018-01-15 | 2018-05-11 | 无锡中德美联生物技术有限公司 | Composite amplification reagent kit and its application of female skin and hair quality SNP genetic polymorphisms site are detected at the same time |
| CN112941182A (en) * | 2021-03-11 | 2021-06-11 | 南京先声医学检验有限公司 | Gene detection method for medicine for treating rheumatic immune diseases based on nucleic acid mass spectrometry and application of gene detection method |
| CN112852956A (en) * | 2021-03-23 | 2021-05-28 | 上海康黎诊断技术有限公司 | Kit for guiding medication of human hyperlipidemia |
| CN113403380A (en) * | 2021-06-11 | 2021-09-17 | 中国科学院北京基因组研究所(国家生物信息中心) | Complex disease related SNP site primer composition and application |
Non-Patent Citations (4)
| Title |
|---|
| 2型糖尿病的全基因组关联研究进展;陆凤;钱云;沈洪兵;;中华疾病控制杂志(第11期);表2 * |
| 基因芯片在筛查儿童过敏性紫癜差异基因中的研究;李娟;李群;刘洪;齐海锋;黄玉辉;郑卫民;傅睿;;江西医药(第10期);第995页左栏倒数第2段 * |
| 特发性膜性肾病相关基因单核苷酸多态性研究进展;程桂雪;刘建华;秦晓松;;中国免疫学杂志(第09期);第1405页右栏第2段 * |
| 陈超等.新技术与精准医学.上海交通大学出版社,2017,第36页第1段、第37页第2段、及38页表4-1. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113846158A (en) | 2021-12-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108486231B (en) | Primer probe composition for detecting polymorphism of human CYP2C19 gene, kit and application | |
| JP2001514013A (en) | Method for analyzing LTC4 synthase polymorphism and use for diagnosis | |
| WO2010001969A1 (en) | Method for amplification of target nucleic acid sequence, method for detection of mutation by using the method, and reagents for use in the methods | |
| CN105008551B (en) | Simple detection method for RNA modification and method for detecting type 2 diabetes using same | |
| KR101546058B1 (en) | SNP markers for metabolic syndrome and use thereof | |
| WO2011062258A1 (en) | Primer set for amplification of mthfr gene, mthfr gene amplification reagent comprising same, and use of same | |
| KR20090012222A (en) | Primer set for gene amplification, gene amplification reagent containing the same and use thereof | |
| KR101220806B1 (en) | Identification of group of hypertension-susceptibility genes | |
| US20150057317A1 (en) | Gabr-a2 diagnostic | |
| CN115141884B (en) | Novel ATP7B mutant gene and diagnostic reagent thereof | |
| CN111118138A (en) | Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR | |
| CN110846408A (en) | Primer combination for detecting TTN gene mutation and application thereof | |
| KR101649179B1 (en) | Target sequence amplification method, polymorphism detection method, and reagents for use in the methods | |
| CN113846158B (en) | Composite amplification kit for simultaneously detecting three chronic disease susceptibility genes and application thereof | |
| KR101646189B1 (en) | Marker for diagnosing intrinsic atopic dermatitis and use thereof | |
| JP2015156853A (en) | Method and kit for detecting alcohol metabolism gene | |
| JP4491276B2 (en) | Method and kit for detecting the presence of a single nucleotide polymorphism in a target DNA sequence | |
| Shimizu et al. | Genotyping of wild-type cytochrome P450 2A6 and whole-gene deletion using human blood samples and a multiplex real-time polymerase chain reaction method with dual-labeled probes | |
| KR101992952B1 (en) | Composition, kit for predicting the risk of developing cardiovascular disease related to Cholesterol efflux capacity, and method using the same | |
| KR101046344B1 (en) | Stroke Diagnosis Method Using SNP2 and Polymorphs as Biomarkers | |
| KR101468580B1 (en) | Single nucleotide polymorphism of Human multidrug and toxin extrusion 1 gene and use thereof | |
| KR101071081B1 (en) | Polynucleotides comprising single nucleotide polymorphism derived from DEFA4 gene, microarrays and diagnostic kits comprising the same, and detection methods using the same | |
| KR101075392B1 (en) | Polynucleotides comprising single nucleotide polymorphism derived from FGA gene, microarrays and diagnostic kits comprising the same, and detection methods using the same | |
| KR101501083B1 (en) | Marker composition for diagnosing diabetes and method for diagnosing or determining the risk of developing diabetes using the same | |
| KR100790226B1 (en) | Method for determining the susceptibility to toxic hepatitis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240311 Address after: 214177 88 Huicheng Road, Huishan Economic Development Zone, Wuxi City, Jiangsu Province Patentee after: Jiangsu Anke Huajie Biotechnology Co.,Ltd. Country or region after: China Address before: No.18-1, Wenhui Road, Huishan District, Wuxi City, Jiangsu Province Patentee before: AGCU SCIENTECH Inc. Country or region before: China |
|
| TR01 | Transfer of patent right |