CN101109705A - Reagent kit for detecting alcoholism and alcohol addiction susceptibility - Google Patents
Reagent kit for detecting alcoholism and alcohol addiction susceptibility Download PDFInfo
- Publication number
- CN101109705A CN101109705A CNA2006100292043A CN200610029204A CN101109705A CN 101109705 A CN101109705 A CN 101109705A CN A2006100292043 A CNA2006100292043 A CN A2006100292043A CN 200610029204 A CN200610029204 A CN 200610029204A CN 101109705 A CN101109705 A CN 101109705A
- Authority
- CN
- China
- Prior art keywords
- seq
- kit
- alcoholism
- primer
- probe
- 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
Abstract
The invention discloses a reagent box for detecting alcoholic intoxication and susceptibility to alcohol habituation. The reagent box comprises specificity primer pairs and specificity fluorescent probes for detecting position 161 SNP position in ALDH2 gene SEQ ID NO:1, and an ordinary assembly for fluorescent quantitative PCR detection. The reagent box predicts the alcoholic intoxication and susceptibility to alcohol habituation of an individual by detecting and analyzing the gene carried on position SNP on ALDH2 gene.
Description
Technical field
The present invention relates to the kit that a kind of disease susceptibility detects usefulness, especially a kind of kit that detects alcoholism and alcohol addiction susceptibility, (aldehyde dehydrogenase 2 family, individual neurological susceptibility to alcoholism and alcohol addiction is predicted in single nucleotide polymorphism ALDH2) (SNP) site by detecting the aldehyde dehydrogenase 2 gene.
Background technology
Drinking is one of human important life style.When drink is once gone into excessive alcohol or inebriant, the central nervous system that causes transfers the state of inhibition to by excitement, is called alcoholism or ethylism.
Alcoholism can produce inhibiting effect to central nervous system, causes lethargic sleep and stupor, and high concentration ethanol suppresses bulbar centre can cause breathing, circulatory function depletion.Ethanol generates a large amount of nicotinamide adenine dinucleotide reduced (NADH) at the liver cell intracellular metabolite in addition, make it to increase with the ratio (NADH/NAD) of oxidized form, even can be up to normal 2～3 times.Can take place in succession as lactic acid increase, ketoboidies accumulates the metabolic acidosis that causes; Hypoglycemia can appear after gluconeogenesis was obstructed.
Alcohol addiction can cause the back of drinking to produce light, excited floaty euphoria.After continuing to drink, produce tolerance, need to increase drinking amount and just can reach original effect.Can cause simultaneously the psychological dependence of alcohol and body are relied on: for the special pleasant sensation after obtaining to drink, serious hope is drunk, this is a psychological dependence, the body dependence being meant to drink repeatedly and making central nervous system that certain physiology, the biochemical variation taken place, so that need ethanol to be present in constantly in the body, to avoid taking place the special symptom that is referred to as abstinence syndrome.Feed reduces during long-term heavy drinking, can cause tangible nutritional deficiency.Ethanol has spread effect to mucous membrane and glandular secretion simultaneously, can cause esophagitis, gastritis, pancreatitis.Ethanol produces free radical in the metabolic process in vivo, can cause the peroxidating of cell membrane lipid, causes necrosis of liver cells, dysfunction of liver.
ALDH2 is aldehyde dehydrogenase 2 family (mitochondrial) (aldehyde dehydrogenase 2s, the mitochondria acetaldehyde dehydrogenase) encoding gene, major function is in liver the further dehydrogenation of alcohol metabolism product acetaldehyde to be become acetate, enter the tricarboxylic acid cycle of peripheral tissues, decompose generation CO
2And water.The defective of this gene or active decline can cause behind the heavy drinking acetaldehyde in the body can't be by timely metabolism, thereby cause a series of damages to liver, blood vessel, central nervous system.The deficiency of this gene comprises having distribution widely in China, Korea S, the Japan the East Asia crowd, has the easier usually generation alcoholism of individuality of this genoid type, and less generation alcohol addiction and the alcohol disease that therefore causes.SNP (single nucleotide polymorphism), promptly the single nucleotide polymorphism mark is the dna polymorphism that a class causes based on single nucleotide variation, is called third generation genetic marker by hereditary educational circles.Mainly be meant the dna sequence polymorphism that causes by the variation on the genome nucleotide level, comprise single base conversion, transversion, and the insertion/disappearance of single base etc.It is a class polymorphism mark that the most extensively exists in the genome, accounts for about 90%.The variation of these genome sequences can cause the difference of phenotype between individuality and Different Individual to disease, particularly the neurological susceptibility of complex disease and to the difference of environmental factor, drug response.
About Chinese population association analysis equal consistent showing, the ALDH2 defective genotype with drink discomfort, blush relevant after drinking.Study on large sample both at home and abroad shows that all rs671 SNP site on the ALDH2 gene (shown in the SEQ ID NO:1 in the sequence the 161st) genotype and ALDH2 gene generation defective have remarkable relation, rs671 SNP site is that to be positioned on the ten No. two chromosome a G/A on the ALDH2 gene polymorphic, causes that the 504th amino acid of albumen of ALDH2 gene code becomes Lys (K) by Arg (R).Low frequency allele type A encoded protein activity is lower, to the ability drop of acetaldehyde metabolism, causes the sense of discomfort of drinking, and blushes syndrome etc., causes alcoholism easily.In addition on the one hand, low frequency allele type A carrier is because the sense of discomfort of drinking makes this kind people be difficult for alcohol addiction, and the alcohol disease reduces on the contrary.
Therefore the polymorphism in the rs671 SNP site on the ALDH2 gene can be used for assessing individual neurological susceptibility to alcoholism and alcohol addiction, has passed through the authentication of biological gene detection technique application evaluation authentication center of country of the Chinese Academy of Sciences at present.
Summary of the invention
Polymorphism based on the rs671 SNP site on the ALDH2 gene can be used for assessing on the basis of individuality to the neurological susceptibility of alcoholism and alcohol addiction, the invention provides a kind of kit that detects alcoholism and alcohol addiction susceptibility.
This kit comprises: the Auele Specific Primer that detects the 161st SNP loci gene type among the ALDH2 genes of SEQ ID NO:1 is to reaching specificity fluorescent probe, Taq enzyme, dNTP mixed liquor, MgCl
2Solution, quantitative fluorescent PCR reaction buffer, deionized water.
Auele Specific Primer described in this kit designs being meant at the 161st SNP site among the SEQ ID NO:1, and it is right that the energy specific amplification goes out to comprise the primer of the dna fragmentation in the 161st SNP site among the SEQ ID NO:1.Design this class primer to being that those skilled in the art can be unlabored.Preferably, it is right to comprise the primer with sequence shown in SEQ ID NO:2 and 3 in the kit.Auele Specific Primer synthesizes the synthetic technology of available routine.Those skilled in the art will appreciate that primer of the present invention is not limited to this a pair of primer.
Specificity fluorescent probe described in this kit is meant at the 161st SNP site among the SEQ ID NO:1 and designs, can go out the probe of this SNP site alcoholism tumor susceptibility gene type by the fluorescent quantitative PCR technique specific detection.Designing this class probe is that those skilled in the art can be unlabored.Preferably, comprise Taqman probe in the kit with sequence shown in the SEQ ID NO:4.The specificity fluorescent probe can synthesize with the synthetic technology of routine.Those skilled in the art will appreciate that specificity fluorescent probe of the present invention is not limited to this probe, all probes that can be used for the 161st SNP loci gene type among the fluorescence quantitative PCR detection SEQ ID NO:1 all within the scope of the present invention.
The component and the content of kit of the present invention comprise:
1 μ l 10X quantitative fluorescent PCR reaction buffer,
0.1 μ l 25mM dNTP mixed liquor,
0.5 μ l 25mM MgCl
2Solution,
0.02 μ l (5units/ μ l) Taq archaeal dna polymerase,
20 μ M Auele Specific Primers are to each 0.225 μ l,
10 μ M specificity fluorescent probes, 0.25 μ l,
Deionized water 5.68 μ l.
This kit detects for a person-portion and uses, and the storage temperature of kit is-20 ℃.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The use of embodiment 1. detection kit
The extraction of step 1:DNA template
Genomic DNA with silica gel adsorption extracting mouth epithelial cells.
Step 2: quantitative fluorescent PCR reaction
Use can detect the fluorescent quantificationally PCR detecting kit of alcoholism and alcohol addiction susceptibility, wherein, contain following primer to and fluorescence probe:
It is 54 ℃ that adopted primer: 5 '-TTTGGTGGCTACAAGATGT-3 ' (SEQ ID NO:2) Tm value is arranged
Antisense primer: 5 '-CTCCGAGCCACCAGCA-3 ' (SEQ ID NO:3) Tm value is 54 ℃
Fluorescence probe: 5 '-GCTGCAGGCATACACTAAAGT-3 ' (SEQ ID NO:4) Tm value is 62 ℃
The quantitative fluorescent PCR reaction system is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed liquor, the 0.5 μ l 25mM MgCl of 20ng/ μ l
2The fluorescence probe 0.25 μ l of antisense primer 0.225 μ l, the 10 μ M that adopted primer 0.225 μ l, 20 μ M are arranged of solution, 0.02 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M, deionized water 5.68 μ l.
React on ABI9700 type pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on ABI7900 type quantitative real time PCR Instrument.
Step 3:SNP genotyping
The final sample fluorescence volume and the normal control that show on the quantitative real time PCR Instrument are compared, the final fluorescence signal of fluorescence probe is apparently higher than normal control, illustrate that the rs671 SNP locus gene on the ALDH2 gene of detected DNA carries the A type, being alcoholism susceptible type, also is not susceptible type of alcohol addiction simultaneously.
Embodiment 2. instructs people initiatively to prevent the service of alcoholism and alcohol addiction
Carried out this service in Shanghai Zhujian Biological Engineering Co., Ltd at present.
Step 1:DNA extracts
The detected person is served preceding consulting, sign Informed Consent Form, fill in the alcohol drinking patterns questionnaire by the detected person.Using the oral cavity sampling to wipe away according to the indication in the sampling box carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.
Step 2: Genotyping detects
Use kit provided by the invention, fluorescence quantitative PCR detection is carried out in the rs671 SNP site on the ALDH2 gene of detected person DNA, determine the genotype in this SNP site.
Step 3: instruct people initiatively to prevent alcoholism and alcohol addiction
By to the genotypic analysis of detected person SNP, provide the report of Genotyping examining report and detected person's individuation health guidance.The genetic test report describes the height of detected person's alcoholism and alcohol addiction susceptible degree in detail.The report of individuation health guidance in conjunction with its alcohol drinking patterns survey result, is assessed the relative risk of detected person's alcoholism and alcohol addiction inheritance susceptible based on detected person's Genotyping testing result.Make the personalized healthy action scheme that is directed to the detected person simultaneously, scheme is included in the recommendation on improvement on alcohol drinking patterns, the life style etc., and popularizes the health knowledge of prevention alcoholism and alcohol addiction for the detected person.
Sequence table
＜110〉Shanghai Zhujian Biological Engineering Co., Ltd
＜120〉a kind of kit that detects alcoholism and alcohol addiction susceptibility
<160>4
<210>1
<211>240
<212>DNA
＜213〉Genus Homo, and ethnic group (Homo sapiens, human)
<400>1
gggagtgtaa?cccataaccc?ccaagagtga?tttctgcaat?ctcgtttcaa?attacagggt 60
caactgctat?gatgtgtttg?gagcccagtc?accctttggt?ggctacaaga?tgtcggggag 120
tggccgggag?ttgggcgagt?acgggctgca?ggcatacact?gaagtgaaaa?ctgtgagtgt 180
gggacctgct?gggggctcag?ggcctgttgg?ggcttgaggg?tctgctggtg?gctcggagcc 240
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>2
tttggtggct?acaagatgt 19
<210>3
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>3
ctccgagcca?ccagca 16
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉probe
<400>4
gctgcaggca?tacactaaag?t 21
Claims (6)
1. kit that detects alcoholism and alcohol addiction susceptibility comprises: the Auele Specific Primer that detects the 161st SNP among the ALDH2 genes of SEQ ID NO:1 to and specificity fluorescent probe, Taq enzyme, dNTP mixed liquor, MgCl
2Solution, quantitative fluorescent PCR reaction buffer, deionized water.
2. kit according to claim 1, it is characterized in that: described Auele Specific Primer designs being meant at the 161st SNP site among the SEQ ID NO:1, and it is right that the energy specific amplification goes out to comprise the primer of the dna fragmentation in the 161st SNP site among the SEQ ID NO:1.
3. kit according to claim 1, it is characterized in that: described specificity fluorescent probe is meant at the 161st SNP site among the SEQ ID NO:1 and designs, can go out the probe of this SNP site alcoholism tumor susceptibility gene type by the fluorescent quantitative PCR technique specific detection.
4. kit according to claim 1 is characterized in that: contained Auele Specific Primer is right to being selected from the primer with sequence shown in SEQ ID NO:2 and 3.
5. kit according to claim 1 is characterized in that: contained specificity fluorescent probe is selected from the Taqman probe with sequence shown in the SEQ ID NO:4.
6. kit according to claim 1 is characterized in that: the component of kit and content comprise 1 μ l, 10 X quantitative fluorescent PCR reaction buffers, 0.1 μ l 25mM dNTP mixed liquor, 0.5 μ l 25mM MgCl
2Solution, 0.02 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M Auele Specific Primers is to each 0.225 μ l, 10 μ M specificity fluorescent probes, 0.25 μ l, deionized water 5.68 μ l.This kit detects for a person-portion and uses, and the storage temperature of kit is-20 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100292043A CN101109705A (en) | 2006-07-21 | 2006-07-21 | Reagent kit for detecting alcoholism and alcohol addiction susceptibility |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100292043A CN101109705A (en) | 2006-07-21 | 2006-07-21 | Reagent kit for detecting alcoholism and alcohol addiction susceptibility |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101109705A true CN101109705A (en) | 2008-01-23 |
Family
ID=39041869
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006100292043A Pending CN101109705A (en) | 2006-07-21 | 2006-07-21 | Reagent kit for detecting alcoholism and alcohol addiction susceptibility |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101109705A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102549171A (en) * | 2009-10-07 | 2012-07-04 | 学校法人武库川学院 | Genotype determination method |
| CN104880451A (en) * | 2015-06-26 | 2015-09-02 | 四川省科学城海天实业有限公司 | Fluorescent test paper used for testing alcohol in saliva and preparation method thereof |
| WO2022022663A1 (en) * | 2020-07-30 | 2022-02-03 | 苏州因顿医学检验实验室有限公司 | Method for constructing alcohol tolerance prediction model |
-
2006
- 2006-07-21 CN CNA2006100292043A patent/CN101109705A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102549171A (en) * | 2009-10-07 | 2012-07-04 | 学校法人武库川学院 | Genotype determination method |
| CN104880451A (en) * | 2015-06-26 | 2015-09-02 | 四川省科学城海天实业有限公司 | Fluorescent test paper used for testing alcohol in saliva and preparation method thereof |
| WO2022022663A1 (en) * | 2020-07-30 | 2022-02-03 | 苏州因顿医学检验实验室有限公司 | Method for constructing alcohol tolerance prediction model |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jiang et al. | A polymorphism in the promoter region of catalase is associated with blood pressure levels | |
| CN104212904B (en) | A kind of kit and using method thereof for fast detecting mankind CYP2C19 gene pleiomorphism | |
| KR101110396B1 (en) | Method of detecting variation and kit to be used therein | |
| CN101240324A (en) | Kit for estimating pre-pregnant nourishment metabolism inheritance capability | |
| CN101568646B (en) | Primer set for gene amplification, gene amplification reagent containing the same and use thereof | |
| CN114686597B (en) | SNP molecular marker for sex identification of silver dragon fish and application thereof | |
| CN108642152A (en) | The kit and method of Genotyping detection | |
| CN111118138A (en) | Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR | |
| CN101899519B (en) | Kit for detecting SNP locus of gene associated with individualized medication of warfarin and PCR amplification method thereof | |
| CN101548022B (en) | Probe for detection of mutation in abl gene and use thereof | |
| KR20120078739A (en) | Primer set for amplification of mthfr gene, mthfr gene amplification reagent comprising same, and use of same | |
| CN101622363B (en) | Probe for detecting mutation in jak2 gene and use thereof | |
| JP6531312B2 (en) | Method for detecting BCR-ABL inhibitor resistance related mutation and data acquisition method for predicting BCR-ABL inhibitor resistance using the same | |
| CN101109705A (en) | Reagent kit for detecting alcoholism and alcohol addiction susceptibility | |
| CN108796042B (en) | Nucleic acid composition for genetic anemia detection, detection kit and use method | |
| CN104293932A (en) | Method for detecting HLA-B * 5801 allele based on real-time fluorescence PCR | |
| CN101240326A (en) | Kit for detecting child lead poisioning inheritance susceptible risk | |
| CN111020019B (en) | Method for gene fusion detection based on nanopore technology | |
| Tang et al. | Isocitrate dehydrogenase mutation hot spots in acute lymphoblastic leukemia and oral cancer | |
| CN101240320A (en) | Kit for detecting cardiovascular disease incidence inheritance risk | |
| Baturina et al. | Pah and qdpr deficiency associated mutations In the novosibirsk region of the russian federation: correlation Of mutation type with disease manifestation and severity | |
| CN113549689B (en) | Kit and method for detecting PROS1 gene exon | |
| CN101608227A (en) | Kit for genetic detection of thyroid cancer | |
| CN102719524A (en) | Kit for warfarin personalized medication related gene SNP locus detection and PCR amplification method using same | |
| Poblete‐Gutiérrez et al. | The molecular basis of porphyria cutanea tarda in Chile: identification and functional characterization of mutations in the uroporphyrinogen decarboxylase gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |