CN110819708A - Method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR - Google Patents
Method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR Download PDFInfo
- Publication number
- CN110819708A CN110819708A CN201911299216.1A CN201911299216A CN110819708A CN 110819708 A CN110819708 A CN 110819708A CN 201911299216 A CN201911299216 A CN 201911299216A CN 110819708 A CN110819708 A CN 110819708A
- Authority
- CN
- China
- Prior art keywords
- fluorescent
- probe
- primer
- quantitative pcr
- gene polymorphism
- 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.)
- Pending
Links
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 27
- 101150038502 ALDH2 gene Proteins 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 65
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 108010009513 Mitochondrial Aldehyde Dehydrogenase Proteins 0.000 claims description 23
- 239000007850 fluorescent dye Substances 0.000 claims description 19
- 230000003321 amplification Effects 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000007400 DNA extraction Methods 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 7
- NZCHHEFOTMKOJX-UHFFFAOYSA-K [6-[[3-carboxy-4-(3-oxido-6-oxoxanthen-9-yl)phenyl]carbamothioylamino]hexoxy-oxidophosphoryl] [5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound O1C(COP([O-])(=O)OP([O-])(=O)OCCCCCCNC(=S)NC=2C=C(C(=CC=2)C2=C3C=CC(=O)C=C3OC3=CC([O-])=CC=C32)C(O)=O)C(O)C(O)C1N1C=CC(=O)NC1=O NZCHHEFOTMKOJX-UHFFFAOYSA-K 0.000 claims description 7
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- 238000010791 quenching Methods 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 claims description 4
- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 3
- JZODKRWQWUWGCD-UHFFFAOYSA-N 2,5-di-tert-butylbenzene-1,4-diol Chemical compound CC(C)(C)C1=CC(O)=C(C(C)(C)C)C=C1O JZODKRWQWUWGCD-UHFFFAOYSA-N 0.000 claims description 2
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- QOFZZTBWWJNFCA-UHFFFAOYSA-N texas red-X Chemical compound [O-]S(=O)(=O)C1=CC(S(=O)(=O)NCCCCCC(=O)O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 QOFZZTBWWJNFCA-UHFFFAOYSA-N 0.000 claims description 2
- 102000009645 Mitochondrial Aldehyde Dehydrogenase Human genes 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 231100000956 nontoxicity Toxicity 0.000 abstract description 3
- 102100033816 Aldehyde dehydrogenase, mitochondrial Human genes 0.000 description 22
- SNIOPGDIGTZGOP-UHFFFAOYSA-N Nitroglycerin Chemical compound [O-][N+](=O)OCC(O[N+]([O-])=O)CO[N+]([O-])=O SNIOPGDIGTZGOP-UHFFFAOYSA-N 0.000 description 12
- 239000000006 Nitroglycerin Substances 0.000 description 12
- 229960003711 glyceryl trinitrate Drugs 0.000 description 12
- 230000008569 process Effects 0.000 description 7
- 239000003814 drug Substances 0.000 description 6
- 108700028369 Alleles Proteins 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 206010002383 Angina Pectoris Diseases 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 125000006853 reporter group Chemical group 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 101150028074 2 gene Proteins 0.000 description 2
- 101710185492 Acetaldehyde dehydrogenase 2 Proteins 0.000 description 2
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102200133167 rs671 Human genes 0.000 description 2
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 1
- 241000534000 Berula erecta Species 0.000 description 1
- 201000000057 Coronary Stenosis Diseases 0.000 description 1
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102000002247 NADPH Dehydrogenase Human genes 0.000 description 1
- 108010014870 NADPH Dehydrogenase Proteins 0.000 description 1
- 108010045510 NADPH-Ferrihemoprotein Reductase Proteins 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 206010047141 Vasodilatation Diseases 0.000 description 1
- 102000005773 Xanthine dehydrogenase Human genes 0.000 description 1
- 108010091383 Xanthine dehydrogenase Proteins 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 230000036782 biological activation Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000024883 vasodilation Effects 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
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
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- 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 provides a primer probe combination for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR, wherein the nucleotide sequence is shown in a sequence table; meanwhile, a method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR is provided. The invention adopts a real-time fluorescent quantitative PCR Taqman-MGB probe method to detect ALDH2 gene polymorphism, and adopts a wild type and mutant type double-probe detection method, thereby not only improving specificity, but also reducing cost; the Taqman-MGB probe is superior to a common probe in the aspects of accuracy, repeatability, specificity, sensitivity and the like of an experimental result. Therefore, the method for the ALDH2 gene polymorphism established by the invention has the advantages of accurate result, high specificity and sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency and the like.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer probe combination and a detection method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR.
Background
Nitroglycerin (NTG) is a basic drug of nitrate vasodilatation drugs, is widely applied to the treatment of various diseases and crisis such as chronic cardiac insufficiency, ventricular myocardial infarction, angina pectoris and hypertension clinically, and is one of the classical treatment drugs for treating acute attack of angina pectoris. However, clinical observations have shown that nitroglycerin does not alleviate angina attacks in all patients diagnosed with coronary stenosis, and the in vivo biotransformation mechanism of nitroglycerin has not yet been fully elucidated. Several enzymes including glutathione S-transferase, cytochrome P450 reductase, cytochrome P450, old yellow enzyme, xanthine oxidoreductase, etc. have been proposed, but since Chen et al 2002 reported that mitochondrial acetaldehyde dehydrogenase 2(mtALDH2) plays an important role in nitroglycerin biological activation and that inhibition of ALDH2 may be related to nitroglycerin resistance, researchers have come to appreciate that ALDH2 may play a crucial role in nitroglycerin exerting therapeutic effects.
Mitochondrial acetaldehyde dehydrogenase 2(ALDH2) is a key enzyme involved in metabolism of drugs such as ethanol, nitroglycerin and the like, and the polymorphism of ALDH2 x 2(Glu504Lys, rs671) can cause that 504 th glutamic acid of the encoded protein is replaced by lysine, thereby affecting the activity of ALDH2 enzyme. The study showed that the enzymatic activity of ALDH2 of individuals carrying the mutant allele (ALDH2 x 2) was reduced, that of heterozygous individuals was only 10% of that of wild-type individuals, and that of mutant homozygous individuals was absent. Through data statistics, the carrying rate of ALDH2 x 2 allele in Asian population is 30-50%, the alcohol metabolism ability of individual carrying ALDH2 x 2 allele is reduced, and discomfort such as flushing, heart beat acceleration and the like can occur when a little alcohol is drunk; the ability to metabolize nitroglycerin is reduced, and the effect of nitroglycerin on myocardial ischemia is weakened, so that other emergency drugs should be used as far as possible for angina pectoris patients carrying ALDH2 x 2 allele, and the nitroglycerin is prevented from being taken orally inefficiently. Metabolic enzyme activity: ALDH2 × 1/' 1(GG) > ALDH2 × 1/' 2(GA) > ALDH2 × 2/' 2 (AA).
At present, there are many methods for detecting polymorphism of ALDH2 × 2 gene, such as restriction length polymorphism analysis (RFLP), direct sequencing, blot hybridization, Taqman technique, allele-specific Amplification (ARMS), and the like. Among them, the most commonly used sequencing method can directly detect the position and type of a mutation site, but the method has the disadvantages of complicated operation steps, long detection period and easy pollution of an amplification product.
Disclosure of Invention
In order to solve the defects, the technical purpose of the invention is to provide a primer probe combination and a detection method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR, wherein the method uses a detection method of primer and double-probe combination, and has the advantages of accurate result, high specificity and sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency and the like.
In order to achieve the purpose, the invention is realized by the following technical scheme: a primer probe combination for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR, wherein the nucleotide sequence of the primer probe is as follows: the upstream primer is shown by SEQ ID NO.1, the downstream primer is shown by SEQ ID NO.2, the fluorescent probe 1 is shown by SEQ ID NO.3, and the fluorescent probe 2 is shown by SEQ ID NO. 4.
The primer probe combination is specially designed for detecting ALDH2 x 2 locus of ALDH2 gene, and can amplify specific gene fragment of ALDH2 x 2 locus of ALDH2 gene with high efficiency, high specificity and accuracy. In the primer probe combination, the fluorescent probe is marked with a fluorescent group at a 5 'terminal region and is marked with a quenching group at a 3' terminal region, and the fluorescent groups of the fluorescent probe 1 and the fluorescent probe 2 are different. Wherein the fluorescent group is selected from any one of FAM, TET, VIC, ROX, Texas Red-X, Cy3 and Cy5, and is more preferably one of FAM, TET, VIC and ROX; the quenching group is selected from at least one of TAMRA, BHQ, DABCYL and NFQ-MGB, preferably at least one of TAMRA, BHQ and NFQ-MGB. According to a most preferred embodiment of the invention, the selection of the fluorescent probe is: the 5 ' end of the fluorescent probe 1 is marked with a fluorescent group of FAM, the 5 ' end of the fluorescent probe 2 is marked with a VIC fluorescent group, and the 3 ' ends of all the fluorescent probes are marked with quenching groups of NFQ-MGB.
The invention adopts a real-time fluorescent quantitative PCR Taqman-MGB probe method to detect ALDH2 gene polymorphism, and adopts a wild type and mutant type double-probe detection method, thereby not only improving specificity, but also reducing cost; the Taqman-MGB probe is superior to a common probe in the aspects of accuracy, repeatability, specificity, sensitivity and the like of an experimental result. Therefore, the method for the ALDH2 gene polymorphism established by the invention has the advantages of accurate result, high specificity, high sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency and the like.
Multiple tests prove that the primer probe combination provided by the invention has better specificity and amplification accuracy, can be applied to preparation of a kit for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR, and can quickly and accurately obtain the result of ALDH2 gene polymorphism after sample collection tests by preparing a finished product kit.
The kit mainly comprises the primer probe combination provided by the invention, wherein the final concentration of the primer is preferably as follows: the final concentration of the upstream primer and the final concentration of the downstream primer are both 0.2-1.0 mu M; the final concentration of the fluorescent probes is 0.05-0.5. mu.M. Other PCR reagents may be selected according to conventional techniques, for example, a preferred embodiment further comprises DNA polymerase, buffer, uracil DNA glycosylase, dNTPs and ultrapure water.
When the finished product kit is manufactured, a reagent for extracting the DNA of a sample or a professional DNA extraction kit can be selectively configured according to actual needs; the DNA of the sample to be detected can be obtained more conveniently and quickly, and the convenience and the rapidity of the detection finished product kit are enhanced. The sample to be tested can be any sample of blood, cells, tissues or buccal swabs containing genomic DNA.
On the basis of the primer probe provided by the invention, the invention further provides a detection method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR, and the primer probe is adopted for fluorescent quantitative PCR detection.
According to a preferred embodiment, the detection method comprises the following specific steps:
(1) extracting genome DNA from a sample to be detected as an amplification template;
(2) preparing a fluorescent quantitative PCR reaction system containing the primer probe combination and the amplification template, wherein the final concentration of the upstream primer and the final concentration of the downstream primer in the reaction system are both 0.2-1.0 mu M; the final concentration of the fluorescent probes is 0.05-0.5 mu M;
the PCR reaction system comprises: 2 xTaqMan PCR reaction mixture (DNA polymerase, buffer solution, uracil DNA glycosylase, dNTPs (containing dUTP)), upstream and downstream primers for amplifying ALDH2 x 2, wild type, mutant type specific fluorescent probes (fluorescent probe 1 and fluorescent probe 2), ultrapure water and DNA template, wherein the volume of the reaction system is 10-50 μ l;
(3) fluorescence PCR amplification reaction, conditions: pre-denaturation at 94-98 deg.C for 5-15 min; denaturation at 94-98 deg.C for 10-60 s; annealing at 60-70 deg.C for 60-90 s; extending for 15-300s at 68-72 ℃; 30-50 cycles;
(4) after amplification was complete, genotypes were read automatically from the scatter plot, each point representing a sample, and each data set representing one genotype: wild type (. sup.1/. sup.1 GG), heterozygous mutant (. sup.1/. sup.2 GA), homozygous mutant (. sup.2/. sup.2 AA), blank control (NTC).
The invention is suitable for any common fluorescent quantitative PCR instrument on the market, and particularly comprises an ABI fluorescent quantitative PCR instrument (ABI7300, ABI7500, ABI ViiA7 and ABI Stepo plus (Stepo)); roche (Roche) fluorescent quantitative PCR instrument LightCycler 480; bio-rad (Berle) fluorescent quantitative PCR instruments CFX96TM and the like. Different fluorescent probes can be selected according to different PCR instruments for PCR amplification.
The detection method has the following advantages and effects:
(1) the two probes of the mutant type and the wild type are simultaneously added into a single tube, so that the operation is simple, the cost is reduced, the result analysis is clearer, the detection efficiency is improved, the detection of a large batch of samples can be carried out, and the clinical operation is facilitated.
(2) The Taqman-MGB specific probe is used for detecting the gene polymorphism, so that the specificity is strong, and the result accuracy is high. The detection result is completely consistent with the sequencing result.
(3) The whole detection process is carried out in the same PCR tube, cover opening analysis is not needed, and aerosol pollution is not easily caused; electrophoresis analysis is not needed, and toxic and harmful reagents are not contacted.
(4) The entire detection process from sample reception, DNA extraction and PCR amplification can be completed within 4 hours. And a large number of samples can be detected simultaneously, so that the detection efficiency is improved, and the time cost is saved.
The detection method can quickly, accurately and efficiently detect the polymorphism of ALDH2 x 2, minimizes the occurrence probability of false positive and false negative, and provides technical support for giving out the initial dosage of drugs such as nitroglycerin and the like according to the gene polymorphism result of ALDH2 x 2.
Drawings
Fig. 1 is a scatter plot of three genotypes (. about.1/. about.1 GG,. about.2/. about.2 AA,. about.1/. about.2 GA) of ALDH 2. about.2 site and blank control (NTC).
Detailed Description
To further illustrate the present invention, reference is made to the following examples. Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.
Example 1
A method for detecting ALDH2 gene polymorphism and preparing a DNA sample to be detected comprises the following steps: the genomic DNA was extracted with a blood/cell/tissue genomic DNA extraction kit (DP304), and the concentration and purity of the DNA were determined using NP80-touch (IMPLEN, Germany), followed by preservation of the genomic DNA.
Example 2
1. Primer and probe design
Respectively designing specific primers and specific fluorescent probes according to base sequences of ALDH2 x 2 gene sites, designing the primers at two ends of the SNP sites by the specific primers through Primer Premier 5.0, and analyzing and selecting the most suitable primers through software; the specific probe is located in the region between a pair of primers, wherein the Tm should be between 65-70 ℃ and usually 5-10 ℃ higher than the primers. Wherein the 5 'end of the target probe is marked by a fluorescence reporter group (FAM/VIC), and the 3' end of the target probe is marked by a fluorescence quenching group (NFQ-MGB); in actual operation, the fluorescent reporter group can be replaced according to actual conditions, as long as the inconsistency between the wild type fluorescent reporter group and the mutant type fluorescent reporter group is ensured. In this example, probe 1(ALDH2 × 2P1) detected the wild type, and probe 2(ALDH2 × 2P2) detected the mutant type.
The primer probe sequences of the invention are shown in Table 1:
TABLE 1 PCR primer Probe sequences
2. System preparation and PCR amplification
Amplifying an ALDH2 gene target fragment by fluorescent quantitative PCR reaction, mixing primers and probes according to a ratio of 1: 1 respectively, and amplifying the primers and probes according to the reaction program shown in the table 2 and the table 3; blank control is set in the experimental process, and ultrapure water is used as a template.
TABLE 2 PCR amplification reaction System
| Reagent composition | Volume of |
| 2×TaqMan PCR Mix | 10μl |
| Probe Mix | 1μl |
| Primer Mix | 1.4μl |
| DNA | 1μl |
| Water (W) | 6.6μl |
TABLE 3 fluorescent PCR reaction conditions
Note: indicating the position of received fluorescence signal
In example 2, ABI7500 is selected for amplification, but the invention is not limited to ABI7500, and the invention is applicable to any fluorescent quantitative PCR instrument, and different fluorescent probes can be selected for detection according to different PCR instruments.
3. Result judgment
After amplification was complete, genotypes were automatically read from the scatter plot (as shown in FIG. 1), each point representing a sample, and each data set representing one genotype: wild type (. sup.1/. sup.1 GG), heterozygous mutant (. sup.1/. sup.2 GA), homozygous mutant (. sup.2/. sup.2 AA), blank control (NTC).
Compared with the gold standard sequencing method, the detection result of the invention has the result coincidence rate of 100 percent, and the detection method has good specificity.
Example 3: configuration of the kit
According to the above experimental results, the present example provides a preferred kit for detecting the polymorphism of ALDH2 gene by fluorogenic quantitative PCR, wherein the kit comprises the following reagents:
2 × TaqMan PCR Mix: DNA polymerase, buffer, uracil DNA glycosylase, dNTPs (containing dUTP);
probe Mix: probe 1 (10. mu.M), probe 2 (10. mu.M), mixed in equal proportions;
primer Mix: a primer pair, an F upstream primer (10 mu M), an R downstream primer (10 mu M) are mixed in equal proportion;
a sample DNA extraction reagent;
sample collection receptacles (e.g., containing oral swabs and swab receptacles/tubes);
ultrapure water.
The reagent is reasonably placed in the kit, the instructions (optionally reconfigured with a PCR test tube or a pore plate or a pipette) of the kit are put into the kit, the instructions comprise the step of collecting the sample to be detected, the collected sample is put into a storage box/tube, the step of DNA extraction is carried out, and finally the PCR amplification process is carried out according to the detection method.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Sequence listing
<110> Beijing and Hei medical diagnostic technology GmbH
<120> method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR
<130>ss191-216
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>18
<212>DNA
<213> Artificial sequence ()
<400>1
agttgggcga gtacgggc 18
<210>2
<211>18
<212>DNA
<213> Artificial sequence ()
<400>2
gcagaccctc aagcccca 18
<210>3
<211>23
<212>DNA
<213> Artificial sequence ()
<400>3
aggcatacac tgaagtgaaa act 23
<210>4
<211>23
<212>DNA
<213> Artificial sequence ()
<400>4
aggcatacac taaagtgaaa act 23
Claims (10)
1. A primer probe combination for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR, wherein the nucleotide sequence of the primer probe is as follows: the upstream primer is shown by SEQ ID NO.1, the downstream primer is shown by SEQ ID NO.2, the fluorescent probe 1 is shown by SEQ ID NO.3, and the fluorescent probe 2 is shown by SEQ ID NO. 4.
2. The primer probe combination of claim 1, wherein the ALDH2 gene polymorphism comprises the ALDH2 x 2 site of ALDH2 gene.
3. The primer-probe combination of claim 1 or 2, wherein the fluorescent probe is labeled with a fluorophore at the 5 '-terminal region and a quencher at the 3' -terminal region, and the fluorophores of fluorescent probe 1 and fluorescent probe 2 are different.
4. The primer probe combination of claim 3, wherein the fluorescent group is selected from FAM, TET, VIC, ROX, Texas Red-X, Cy3, Cy 5; the quenching group is selected from TAMRA, BHQ, DABCYL, NFQ-MGB.
5. The primer-probe combination of claim 3 or 4, wherein the 5 ' end of the fluorescent probe 1 is labeled with a fluorescent group of FAM, the 5 ' end of the fluorescent probe 2 is labeled with a VIC fluorescent group, and the 3 ' ends of all the fluorescent probes are labeled with a quencher group of NFQ-MGB.
6. Use of the primer probe combination of any one of claims 1 to 5 for preparing a kit for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR.
7. A kit for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR, which comprises the primer probe combination of any one of claims 1-5.
8. The kit of claim 7, further comprising a DNA polymerase, a buffer, a uracil DNA glycosylase, dNTPs, and ultrapure water.
9. The kit according to claim 8, further comprising a test sample DNA extraction reagent or DNA extraction kit.
10. A detection method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR, which is characterized in that the primer probe combination of any one of claims 1-5 is used for carrying out fluorescent quantitative PCR detection on a sample to be detected, and comprises the following steps:
(1) extracting genome DNA from a sample to be detected as an amplification template;
(2) preparing a fluorescent quantitative PCR reaction system containing the primer probe combination and the amplification template, wherein the final concentration of the upstream primer and the final concentration of the downstream primer are both 0.2-1.0 mu M; the final concentration of the fluorescent probes is 0.05-0.5 mu M;
(3) fluorescence PCR amplification reaction, conditions: pre-denaturation at 94-98 deg.C for 5-15 min; denaturation at 94-98 deg.C for 10-60 s; annealing at 60-70 deg.C for 60-90 s; extending for 15-300s at 68-72 ℃; 30-50 cycles;
(4) after amplification was complete, genotypes were read automatically from the scatter plot, each point representing a sample, and each data set representing one genotype: wild type: 1/' 1 GG; hybrid mutant type: 1/' 2 GA; homozygous mutant type: 2/' 2 AA; blank control: the NTC.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911299216.1A CN110819708A (en) | 2019-12-16 | 2019-12-16 | Method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911299216.1A CN110819708A (en) | 2019-12-16 | 2019-12-16 | Method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110819708A true CN110819708A (en) | 2020-02-21 |
Family
ID=69545992
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911299216.1A Pending CN110819708A (en) | 2019-12-16 | 2019-12-16 | Method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110819708A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116024318A (en) * | 2023-02-23 | 2023-04-28 | 常州国药医学检验实验室有限公司 | Kit for detecting human ALDH2 gene polymorphism based on fluorescent quantitative PCR |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120276533A1 (en) * | 2011-04-28 | 2012-11-01 | Arkray, Inc. | Method for Simultaneously Detecting Polymorphisms of Acetaldehyde Dehydrogenase 2 and Alcohol Dehydrogenase 2 |
| CN105132545A (en) * | 2015-08-25 | 2015-12-09 | 安徽同科生物科技有限公司 | Primers and probes for detecting ALDH2, and detection method |
| CN105441439A (en) * | 2015-12-01 | 2016-03-30 | 武汉海吉力生物科技有限公司 | Primers, probe and kit for detecting ALDH2 (aldehyde dehydrogenase gene2) gene polymorphism |
| CN106834519A (en) * | 2017-03-27 | 2017-06-13 | 重庆迪安医学检验中心有限公司 | A kind of primer of TaqMan MGB sonde methods detection mankind's ALDH2 gene pleiomorphisms and its application |
| CN106947807A (en) * | 2017-03-01 | 2017-07-14 | 上海荻硕贝肯医学检验所有限公司 | Primer, probe, kit and method for detecting mankind's ALDH2 gene pleiomorphisms |
| CN108441553A (en) * | 2018-03-13 | 2018-08-24 | 普迈德（北京）科技有限公司 | It is a kind of it is accurate detection ALDH2 gene pleiomorphisms kit and its application |
| CN109055367A (en) * | 2018-09-06 | 2018-12-21 | 武汉康录生物技术股份有限公司 | A kind of mankind ALDH2 genetic polymorphism detection kit and its preparation method and application |
| CN109055560A (en) * | 2018-09-30 | 2018-12-21 | 广州艾基生物技术有限公司 | The detection kit and method of ALDH2 gene RS671 polymorphism |
-
2019
- 2019-12-16 CN CN201911299216.1A patent/CN110819708A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120276533A1 (en) * | 2011-04-28 | 2012-11-01 | Arkray, Inc. | Method for Simultaneously Detecting Polymorphisms of Acetaldehyde Dehydrogenase 2 and Alcohol Dehydrogenase 2 |
| CN105132545A (en) * | 2015-08-25 | 2015-12-09 | 安徽同科生物科技有限公司 | Primers and probes for detecting ALDH2, and detection method |
| CN105441439A (en) * | 2015-12-01 | 2016-03-30 | 武汉海吉力生物科技有限公司 | Primers, probe and kit for detecting ALDH2 (aldehyde dehydrogenase gene2) gene polymorphism |
| CN106947807A (en) * | 2017-03-01 | 2017-07-14 | 上海荻硕贝肯医学检验所有限公司 | Primer, probe, kit and method for detecting mankind's ALDH2 gene pleiomorphisms |
| CN106834519A (en) * | 2017-03-27 | 2017-06-13 | 重庆迪安医学检验中心有限公司 | A kind of primer of TaqMan MGB sonde methods detection mankind's ALDH2 gene pleiomorphisms and its application |
| CN108441553A (en) * | 2018-03-13 | 2018-08-24 | 普迈德（北京）科技有限公司 | It is a kind of it is accurate detection ALDH2 gene pleiomorphisms kit and its application |
| CN109055367A (en) * | 2018-09-06 | 2018-12-21 | 武汉康录生物技术股份有限公司 | A kind of mankind ALDH2 genetic polymorphism detection kit and its preparation method and application |
| CN109055560A (en) * | 2018-09-30 | 2018-12-21 | 广州艾基生物技术有限公司 | The detection kit and method of ALDH2 gene RS671 polymorphism |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116024318A (en) * | 2023-02-23 | 2023-04-28 | 常州国药医学检验实验室有限公司 | Kit for detecting human ALDH2 gene polymorphism based on fluorescent quantitative PCR |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8153377B2 (en) | Method for detecting and quantifying rare mutations/polymorphisms | |
| US8999644B2 (en) | Method for detecting the presence of a DNA minor contributor in a DNA mixture | |
| CN112538528A (en) | Primer group and kit for detecting ALDH2 gene polymorphism | |
| CN111118138A (en) | Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR | |
| CN110564861B (en) | Fluorescent marker composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof | |
| CN117730163A (en) | Dilated cardiomyopathy gene detection marker and application thereof | |
| Tranah et al. | Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies | |
| CN108048565A (en) | A kind of primer for detecting ApoE gene pleiomorphisms and its detection method and application | |
| CN110846408A (en) | Primer combination for detecting TTN gene mutation and application thereof | |
| CN110863040A (en) | Method for detecting CYP3A5 gene polymorphism by fluorescent quantitative PCR | |
| CN105624315A (en) | Primers and reagent kit for detecting polymorphism of ALDH2 gene c.1510 locus | |
| CN110819709A (en) | Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) | |
| CN110878351A (en) | Method for detecting MTHFR gene polymorphism by fluorescent quantitative PCR | |
| CN110819708A (en) | Method for detecting ALDH2 gene polymorphism by fluorescent quantitative PCR | |
| CN111621553A (en) | Reagent for detecting NPC1L1 mutant genotyping and application thereof | |
| CN112538524A (en) | Detection product for detecting gene SNP related to deafness | |
| CN110819701A (en) | Method for detecting CYP2D6 gene polymorphism by fluorescent quantitative PCR | |
| Shi et al. | Application of kinetic polymerase chain reaction and molecular beacon assays to pooled analyses and high‐throughput genotyping for candidate genes | |
| CN111411148B (en) | One-tube ALDH2 genotyping kit and detection method thereof | |
| CN117448448B (en) | IL-1B gene detection primer probe combination, kit and application thereof | |
| CN113444791B (en) | ARMS-PCR kit for assisting in screening familial papillary thyroid carcinoma | |
| CN109082460B (en) | Non-competitive probe design method, detection method and application applied to SNP typing | |
| CN117385010A (en) | qPCR method for detecting gene polymorphism | |
| CN117187380A (en) | One-tube APOE genotyping detection kit and detection method thereof | |
| CN117448446A (en) | FCGR2A gene detection primer probe combination, kit and application thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200221 |