CN108265113A - ALDH2 genetic polymorphism detection kits - Google Patents
ALDH2 genetic polymorphism detection kits Download PDFInfo
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Abstract
The invention discloses ALDH2 genetic polymorphism detection kits.Kit of the present invention has the following advantages that：(1) detection sensitivity is high, and lowest detection is limited to 7.8pg DNA；(2) it is easy to operate, without professional knowledge, it can realize and be detected by bed；(3) detection time is short, most short need 45 minutes；(4) detection specificity is high, using the fluorescence probe of specificity, has very high accuracy；(5) without nucleic acid extraction, low melting-point agarose (agarose) is added in, largely avoids PCR pollutions.The kit collocation ParaDNA gene magnifications detecting instrument of the present invention realizes detection (POCT) by bed, available for clinically instructing medicine of glonoin and prompting is drunk risk.
Description
Technical field
The invention belongs to biotechnologies, and in particular to ALDH2 genetic polymorphism detection kits.
Background technology
2 family (mitochondrial) of ALDH2 full name aldehyde dehydrogenase, i.e. mitochondria acetaldehyde
Dehydrogenase 2.ALDH2 has acetaldehyde dehydrogenase and esterase active simultaneously, participates in the metabolism of the drugs such as ethyl alcohol, nitroglycerin.
ALDH2*2 (rs671, NM_000690.3:c.1510G>A) be wild-type allele ALDH2*1 rs671 sites base
A is sported by G, is replaced so as to cause 504 glutamic acid of coded protein by lysine.Carry mutant allele
(ALDH2*2) individual ALDH2 enzymatic activitys decline, and heterozygous mutation individual enzymatic activity is only the 10% of wild type individual, is mutated
Homozygotic individual enzymatic activity lacks.Therefore, the individual alcohol metabolism ability for carrying saltant type ALDH2*2 allele declines, few
Amount, which is drunk i.e., there are the discomforts such as blush, palpitate quickly；The ability for being metabolized nitroglycerin declines, the effect that nitroglycerin resists myocardial ischemia
It should weaken.The carrying rate of ALDH2*2 mutant alleles is 30-50% in asian population.Carry ALDH2*2 mutant alleles
The patient with angina pectoris of gene should use other first aid medicines instead as far as possible, avoid nitroglycerin buccal invalid.
At present detection ALDH2*2 main method have Sanger sequencings, PCR-taqman fluorescence probe Ct values method, PCR maos
Thin electrophoresis fragments analysis method, gene chip hybridization method etc., but these methods are required to nucleic acid extraction, operating procedure is complicated, consumption
When it is longer, and these methods needs carried out in special gene magnification laboratory.
Invention content
First purpose of the present invention is to provide a kind of complete drawing for being used to detect ALDH2 gene rs671 loci polymorphisms
Object.
Provided by the present invention for detect ALDH2 gene rs671 loci polymorphisms primer set by primer 1, primer 2 and
Probe forms；
The primer 1 is following a1) or a2)：
A1) the single strand dna shown in sequence 1；
A2) by a1) single strand dna that limits carries out the missings of one or several bases, insertion and/or changes to obtain
And a1) limit single strand dna have identical function single strand dna；
The primer 2 is following b1) or b2)：
B1) the single strand dna shown in sequence 2；
B2) by b1) single strand dna that limits carries out the missings of one or several bases, insertion and/or changes to obtain
And b1) limit single strand dna have identical function single strand dna；
The probe is following c1) or c2)：
C1) the single strand dna shown in sequence 3；
C2) by c1) single strand dna that limits carries out the missings of one or several bases, insertion and/or changes to obtain
And c1) limit single strand dna have identical function single strand dna.
In above-mentioned primer set, the mole ratio of the primer 1, the primer 2 and the probe is 3:1:1.
In above-mentioned primer set,
The 6th of the probe and the 11st bit base are the base T of fluorophor modification.
In above-mentioned primer set,
The fluorophor is FAM.
In above-mentioned primer set, the 5 ' end of probe is protected with trimethoxy talan, 3 ' end phosphate groups
It is closed.
Second object of the present invention is to provide a kind of PCR examinations for being used to detect ALDH2 gene rs671 loci polymorphisms
Agent.
Include above-mentioned primer set provided by the present invention for the PCR reagent for detecting ALDH2 gene rs671 loci polymorphisms
And archaeal dna polymerase.
In above-mentioned PCR reagent,
The archaeal dna polymerase is Phire Hotstart II archaeal dna polymerases；
The PCR reagent is gathered by the primer 1, the primer 2, the probe, the Phire Hotstart II DNA
Synthase, agarose, dNTPs, buffer and water composition；
A concentration of 0.86 μM in the PCR reagent of the primer 1；
A concentration of 0.29 μM in the PCR reagent of the primer 2；
Concentration of the probe in the PCR reagent is 0.29 μM；
A concentration of 0.2U/ μ L of the Phire Hotstart II archaeal dna polymerases in the PCR reagent；
Mass fraction of the agarose in the PCR reagent is 0.2%.
Third object of the present invention is to provide a kind of kit for being used to detect ALDH2 gene rs671 loci polymorphisms.
Kit provided by the invention includes above-mentioned primer set or above-mentioned PCR reagent.
Fourth object of the present invention is to provide above-mentioned primer set or above-mentioned PCR reagent or the new use of mentioned reagent box
On the way.
The present invention provides above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box following 1) -8) in it is any
In application：
1) product of detection or auxiliary detection ALDH2 gene rs671 loci polymorphisms is prepared；
2) detect or assist detection ALDH2 gene rs671 loci polymorphisms；
3) it prepares detection or auxiliary detects the product of patient ALDH2 genotype to be measured；
4) it detects or assists to detect patient ALDH2 genotype to be measured；
5) it prepares detection or auxiliary detects the product whether patient to be measured carries ALDH2 mutation alleles；
6) it detects or assists to detect whether patient to be measured carries ALDH2 mutation alleles；
7) it prepares detection or auxiliary detects the product of patient's nitroglycerin metabolic capability and/or alcohol metabolism ability to be measured；
8) it detects or assists to detect patient's nitroglycerin metabolic capability and/or alcohol metabolism ability to be measured.
The 5th purpose of the present invention is to provide a kind of method detected or auxiliary detects patient ALDH2 genotype to be measured.
The method that detection provided by the invention or auxiliary detect patient ALDH2 genotype to be measured includes the following steps：
PCR amplification is carried out to the buccal swab sample of patient to be measured using above-mentioned primer set, obtains pcr amplification product
Tm values judge the genotype of sample to be tested according to the Tm values：
If the Tm values of clinical samples to be measured are 51.1-55.1, the genotype of patient to be measured is or candidate is AA genotype；
If the Tm values of clinical samples to be measured are 59.7-63.7, the genotype of patient to be measured is or candidate is GG genotype；
If the Tm values of clinical samples to be measured are 51.1-55.1 and 59.7-63.7, the genotype of patient to be measured is or candidate
For GA genotype；
The GG genotype is that rs671 (c.1510) site of two homologues of ALDH2 genes is the homozygosis of G
Type；
The AA genotype is that rs671 (c.1510) site of two homologues of ALDH2 genes is the homozygosis of A
Type；
The GA genotype is G and A for rs671 (c.1510) site of two homologues of ALDH2 genes
Heterozygous.
The 6th purpose of the present invention is to provide a kind of detection or auxiliary detects whether patient to be measured carries ALDH2 mutation etc.
The method of position gene.
Detection provided by the invention or auxiliary detect the method whether patient to be measured carries ALDH2 mutation alleles and include
Following steps：PCR amplification is carried out to the buccal swab sample of patient to be measured using above-mentioned primer set, obtains pcr amplification product
Tm values, judge whether patient to be measured carries ALDH2 mutation alleles according to the Tm values：
If the Tm values of clinical samples to be measured are 59.7-63.7, patient to be measured does not carry ALDH2 mutation alleles；
If the Tm values of clinical samples to be measured for it is following 1) or 2), patient to be measured carries ALDH2 mutation alleles：
1)51.1-55.1；
2) 51.1-55.1 and 59.7-63.7.
In the above method, the extension of time of the PCR amplification is 5 seconds.
In the above method, individual of the individual of ALDH2 mutation alleles for GA genotype or AA genotype is carried.
In above-mentioned primer set or PCR reagent or kit or method or application, the rs671 sites are ALDH2 genes
The 42421st, also as sequence 4 the 87th.
Archaeal dna polymerase Phire Hotstart II DNA polymerase, enzyme tool are added in the kit of the present invention
Have the advantages that start that speed is fast, Catalysis Rate is fast, high conversion rate, anti-PCR inhibitions are good, so that this kit is detecting
During buccal swab, without carrying out nucleic acid extraction.
The agarose of low melting point is added in the kit of the present invention, so kit is before use solid-state, when the agar
Sugar becomes liquid in high temperature pcr amplification reaction and melting curve analysis, does not influence the amplification and analysis of target gene, to be analyzed
Kit reverts to room temperature after process, and agarose becomes solid again, a large amount of target bases that can prevent amplification from generating in this way
Because segment forms Aerosol Pollution.
The advantages of kit of the present invention and effect are as follows：
(1) detection sensitivity is high, and lowest detection is limited to 7.8pg DNA；
(2) it is easy to operate, without professional knowledge, it can realize and be detected by bed；
(3) detection time is short, by the length control of pcr amplification product in 150bp or so, thus by prolonging in PCR cycle
Stretching the time foreshortens to 5s, and entire PCR amplification and analysis time is made only to need 21 minutes；
(4) detection specificity is high, using the fluorescence probe of specificity, has very high accuracy；
(5) adding in archaeal dna polymerase Phire Hotstart II DNA polymerase, speed is fast, is catalyzed speed with starting
The advantages that degree is fast, high conversion rate, good anti-PCR inhibitions, so that this kit is when detect buccal swab, without progress
Nucleic acid extraction.
(6) low melting-point agarose (agarose) is added in, largely avoids PCR pollutions.
The present invention provides the kit of a species specific detection ALDH2 gene pleiomorphisms, kit collocation
ParaDNA gene magnifications detecting instrument realizes detection (POCT) by bed, establishes a kind of other detection (POCT) ALDH2*2 gene
PCR- fluorescence probe melting curve methods.It is experimentally confirmed：The kit of the present invention has without nucleic acid extraction, quick, behaviour
Make the advantages that simple, specificity is high, high sensitivity, can not only realize effectively detection ALDH2 gene pleiomorphisms, but also can be used for
It clinically instructs medicine of glonoin and prompting is drunk risk.
Description of the drawings
Fig. 1 and Fig. 2 is the electrophoretic analysis figure of PCR product.
Fig. 3 is probe face principle.
Fig. 4 is the detection method of the kit of the present invention.
Fig. 5 is melting curve analysis result.ALDH2*1*1 carrier (blue, Tm=61.7 ± 0.8 DEG C), ALDH2*1*2
Carrier's (green, Tm=63.0 ± 0.8 DEG C and 54.5 ± 0.6 DEG C), ALDH2*2*2 carrier (red, Tm=54.5 ± 0.6
DEG C), the melting curve analysis result of negative control (black).
Fig. 6 is the melting curve result of minimum detection limit experiment.
Fig. 7 is the melting curve result of patient with angina pectoris.
Fig. 8 is the sequencing result of patient with angina pectoris.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
ALDH2*2 in following embodiments refers to that the base of the rs671 positions of people's ALDH2 genes sports A by G, so as to lead
504 glutamic acid of ALDH2 enzymes coded by cause are replaced by lysine, and ALDH2 enzymatic activitys decline.
ALDH2*1*1 carrier (wild type) in following embodiments is same for two through genotype identification ALDH2 genes
The individual that rs671 (c.1510) site of source chromosome does not mutate, the genotype of the individual is denoted as GG genotype；
ALDH2*1*2 carrier (heterozygous mutation) is in two homologues through genotype identification ALDH2 genes, and one homologous
The rs671 sites of chromosome sport A by G, and the rs671 sites of another item chromosome do not have the individual of mutation, by this
The genotype of individual is denoted as GA genotype；ALDH2*2*2 carrier (no mutant homozygote) is through genotype identification ALDH2 genes
The rs671 sites of two homologues are sported the individual of A by G, and by the individual, its genotype is denoted as AA genotype.Base
Because individual that type is GA genotype and AA genotype is the individual that carries ALDH2 mutation alleles.
Reagent and its parameter in following embodiments with it is as shown in table 1 at purchase.
At table 1, reagent and its parameter and purchase
Embodiment 1, ALDH2 genetic polymorphism detections kit and detection method
First, ALDH2 genetic polymorphism detections primer
1st, the design of primer
The present invention is according to the rs671 sites of target gene ALDH2 genes, and for the proliferation time for shortening PCR, by PCR
The length of amplified production is controlled in 150bp or so, and devising following three pairs of primer sequences, (three pairs of primer sequences are by Bioserch
Company synthesizes)：
ALDH2-F1：TTTGGAGCCCAGTCACCCTTTG；
ALDH2-R1：AGCCACCAGCAGACCCTCAA；
ALDH2-F2：TGTTTGGAGCCCAGTCACCCTT；
ALDH2-R2：AGCCACCAGCAGACCCTCAA；
ALDH2-F3：TGATGTGTTTGGAGCCCAGTCA；
ALDH2-R3：AGCCACCAGCAGACCCTCAA.
Table 2, rs671 site informations
2nd, PCR amplification
(1) Phire Hotstart II archaeal dna polymerases carry out PCR amplification
Respectively with throat swab liquid sample (ALDH2*1*1 carrier) and the throat swab liquid sample of nucleic acid extraction
The DNA solution (a concentration of 6.4ng/ μ L) of (ALDH2*1*1 carrier) is template, and three pairs of primers of step 1 design are respectively adopted
Use CFX96TouchTMFluorescence quantitative PCR instrument (BIO-RAD, 1855196) carries out PCR amplification, obtains PCR product.PCR amplification
System is as shown in table 3.PCR amplification program is as shown in table 6.
The PCR reaction systems of table 3, Phire enzymes
After PCR amplification, PCR product is subjected to electrophoretic analysis, shown in electrophoresis result Fig. 1.Electrophoresis result shows：
The template that ALDH2-F1/ALDH2-R1 primer pairs extract DNA and do not extract DNA has a small amount of amplified production generation；ALDH2-F2/
ALDH2-R2 primers are for extraction DNA and do not extract the template of DNA and can go out a nucleotide fragments with specific amplification, by this
Band recycling sequencing compares the band as the ALDH2 Gene Partial segments containing ALDH2 gene rs671 sites through BLAST；
The template that ALDH2-F3/ALDH2-R3 primer pairs extract DNA and do not extract DNA does not have apparent amplified production to generate, and has apparent
Primer dimer band.
Therefore selection ALDH2-F2/ALDH2-R2 primers can be directed to the throat swab sample specific amplification for not extracting DNA
ALDH2 genetic fragments.
(2) Taq archaeal dna polymerases carry out PCR amplification
Respectively with throat swab liquid sample (ALDH2*1*1 carrier) and the throat swab liquid sample of nucleic acid extraction
The DNA solution (a concentration of 6.4ng/ μ L) of (ALDH2*1*1 carrier) is template, using the ALDH2-F2/ of step 1 design
ALDH2-R2 primers use CFX96TouchTMFluorescence quantitative PCR instrument (BIO-RAD, 1855196) carries out PCR amplification, obtains PCR
Product.PCR amplification system is as shown in table 4.PCR amplification program is as shown in table 6.
The PCR reaction systems of table 4, Taq enzyme
After PCR amplification, PCR product is subjected to electrophoretic analysis, shown in electrophoresis result Fig. 2.Taq archaeal dna polymerases are directed to
DNA profiling after extraction nucleic acid can go out a nucleotide fragments with specific amplification, and for the throat swab sample for not extracting nucleic acid
This is without apparent amplified band.And Phire Hotstart II archaeal dna polymerases are directed to extraction DNA and do not extract the mould of DNA
Plate can go out a nucleotide fragments with specific amplification, by the band recycle be sequenced, through BLAST compare the band be containing
The ALDH2 Gene Partial segments in ALDH2 gene rs671 sites.
The above results show：Phire Hotstart II archaeal dna polymerases can expand the throat swab without nucleic acid extraction
Sample, and testing result is accurate.
3rd, ALDH2 genetic polymorphism detections kit
The present invention ALDH2 genetic polymorphism detections kit by step 1 ALDH2-F2/ALDH2-R2 primers, visit
Needle and PCR reaction systems composition.
1st, primer
Primer sequence is as follows：
Preceding primer sequence is：TGTTTGGAGCCCAGTCACCCTT (sequence 1)；
Primer sequence is afterwards：AGCCACCAGCAGACCCTCAA (sequence 2).
PCR product sequence is as shown in sequence 4, the 87th rs671 site for ALDH2 genes in sequence 4.
2nd, probe
The fluorescence probe of following specificity is devised according to the base sequence of ALDH2 gene rs671 location proximates：5’-
TMS-TTCACTTCAGTGTATGCC (sequence 3)-phosphate-3 ' (F=fluoroscein dT).Wherein, the 6th in sequence 3
Position and the base T of the 11st are the base T of FAM fluorescent markers, and the probe 5 ' end is protected with trimethoxy talan (TMS)
Shield, 3 ' ends are closed with phosphate group phosphate.Wherein, shown in TMS such as formulas (I), the base T such as formulas (II) of FAM fluorescent markers
It is shown.The present invention probe sequence synthesized by Bioserch companies of the U.S., operation principle be its under single-chain state, fluorescent base
Group can quench, after probe hybridizes with target gene segment and (hybridizes with 76-93 of PCR product), fluorescent base
Group restores, therefore can carry out the Tm values obtained after liquation according to fluorescence probe and target gene segment, judges target base
Because whether the rs671 sites of segment mutate (Fig. 3).
3rd, PCR reaction systems
The PCR reaction systems of the present invention is 35 μ L, each constituent in system and its concentration such as table 5 in system
It is shown.
The PCR reaction systems of table 5, kit
The archaeal dna polymerase Phire Hotstart II DNA used in the PCR reaction systems of kit of the present invention
Polymerase has many advantages, such as to start that speed is fast, Catalysis Rate is fast, high conversion rate, anti-PCR inhibitions are good, so that this
Kit is when detecting buccal swab, without carrying out nucleic acid extraction.
The agarose used in the PCR reaction systems of kit of the present invention, fusing point is low, so kit is solid before use
State becomes liquid when the agarose is in high temperature pcr amplification reaction and melting curve analysis, do not influence target gene amplification and
It analyzes, kit reverts to room temperature after process to be analyzed, and agarose becomes solid again, can prevent what amplification from generating in this way
A large amount of target gene segments form Aerosol Pollution.
3rd, the application method of ALDH2 genetic polymorphism detections kit
1st, the sampling of sample device is moved
Using Medical oral cavity swab in patient to be measured (ALDH2*1*1 carrier, ALDH2*1*2 carrier and ALDH2*2*2
Carrier) oral cavity two cheek on the inside of scrape a small amount of mouth desquamated cells, the end that sample device is then moved using special four-head is rubbed
At the cotton for wiping buccal swab, the sample on swab is transferred to the end of sampler.With ddH2O is negative control.
2nd, intercalation reaction plate
The end of sampler is inserted respectively into four holes of reaction plate, 35 μ L PCR are contained in each hole of reaction plate
Reaction system, and confirm sealing, the reaction plate sealed.
3rd, PCR amplification
By the reaction plate of sealing be put into ParaDNA instruments (LGC Ltd., ParaDNA instruments contain 4 it is independent
PCR react and detection unit Para-010) in carry out PCR amplification.PCR amplification program is as shown in table 6.
Table 6, PCR amplification program
4th, melting curve analysis
After amplification program, ParaDNA instruments carry out melting curve analysis, and analysis program is 35 DEG C -80 DEG C, heating speed
Rate is 0.1 DEG C/s.
The results are shown in Figure 5 for melting curve analysis.In Fig. 5, the melting curve of ALDH2*1*1 carrier is blue, Tm
Be worth is 61.7 ± 0.8 DEG C；The melting curve of ALDH2*1*2 carrier is green, Tm values for 63.0 ± 0.8 DEG C and 54.5 ±
0.6℃；The melting curve of ALDH2*2*2 carrier is red, and Tm values are 54.5 ± 0.6 DEG C；The melting curve of negative control
For black.
It therefore can be according to the genotype of the Tm values analysis sample to be tested in the melting curve figure of sample to be tested：
If the Tm values of sample to be tested are 51.1-55.1, the genotype of sample to be tested is or candidate is AA genotype；
If the Tm values of sample to be tested are 59.7-63.7, the genotype of sample to be tested is or candidate is GG genotype；
If the Tm values of sample to be tested are 51.1-55.1 and 59.7-63.7, the genotype of sample to be tested is or candidate is GA
Genotype.
Can also whether ALDH2 mutation etc. be carried according to the Tm values analysis sample to be tested in the melting curve figure of sample to be tested
Position gene：
If the Tm values of sample to be tested are 59.7-63.7, sample to be tested does not carry ALDH2 mutation alleles；
If the Tm values of sample to be tested for it is following 1) or 2), sample to be tested carries ALDH2 mutation alleles.
1)51.1-55.1；
2) 51.1-55.1 and 59.7-63.7.
The sensitivity technique of embodiment 2, ALDH2 genetic polymorphism detection kits
The DNA of the Pharyngeal swab samples of ALDH2*1*2 carrier is extracted, obtained DNA extracting solutions are passed through
After Nanodrop1000 (Thermo Scientific, ND-1000) is quantitative successively diluted concentration be 250pg/ μ L, 125pg/ μ L,
62.5pg/ μ L, 31.25pg/ μ L, 15.625pg/ μ L, 7.8pg/ μ L, 3.9pg/ μ L, 1.95pg/ μ L DNA solution.Then divide
The above-mentioned DNA solution that 2 μ L are not drawn with liquid-transfering gun detects various concentration respectively according to the method in the step of embodiment 1 three
ALDH2 gene pleiomorphisms in DNA solution.
The results are shown in Figure 5.The lowest detection DNA content of the ALDH2 genetic polymorphism detection kits of the present invention reaches
7.8pg/μL。
The application of embodiment 3, ALDH2 genetic polymorphism detection kits
Take 6 patient with angina pectoris (from Beijing Fuwai Hospital, all patient with angina pectoris are determined through clinical diagnosis, and
Through informed consent) mouth swab sample, the mouth swab sample equivalent of each patient is divided into two parts, and portion uses the present invention's
ALDH2 genetic polymorphism detections kit detects the genotype of clinical samples to be measured according to the method in 1 step 3 of embodiment, separately
Portion is sent to the sequencing of Sanger sequencing companies.
Testing result is as shown in table 7.The results are shown in Figure 7 for melting curve, and sequencing result is as shown in Figure 8.From figure and table
In it can be seen that：The testing result of kit of the present invention is consistent with PCR sequencing PCR testing result, illustrates that the kit of the present invention can be with
The genotype of clinical samples to be measured is effectively detected, can be used for detecting ALDH2 gene pleiomorphisms.
Table 7, testing result
The performance verification of embodiment 4, ALDH2 genetic polymorphism detection kits
The mouth swab sample of 100 Chinese is randomly selected, each mouth swab sample equivalent is divided into two parts, and portion makes
The base of sample to be tested is detected according to the method in 1 step 3 of embodiment with the ALDH2 genetic polymorphism detections kit of the present invention
Because of type, another is sent to the sequencing of Sanger sequencing companies.
It is as shown in table 8 to detect comparison result.The result shows that：The testing result of kit of the present invention and PCR sequencing PCR testing result
Unanimously, the accuracy for illustrating the kit of the present invention is 100%, and specificity is 100%, can effectively detect sample to be tested
ALDH2 gene pleiomorphisms.Experimental result shows to have in Chinese population simultaneously 43% to carry ALDH2*2 allele, metabolism
Nitroglycerin and alcohol ability are general.
Table 8, detection comparison result
Sequence table
<110>Binjiang Hua Kang（Beijing）Bio tech ltd
<120>ALDH2 genetic polymorphism detection kits
<160>4
<210>1
<211>20bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>1
gtggctacaa gatgtcgggg 20
<210>2
<211>20bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>2
accctcaagc cccaacaggc 20
<210>3
<211>19bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>3
ccgtatgtga ctacacttt 19
<210>4
<211>159bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>4
tgtttggagc ccagtcaccc tttggtggct acaagatgtc ggggagtggc cgggagttgg 60
gcgagtacgg gctgcaggca tacactgaag tgaaaactgt gagtgtggga cctgctgggg 120
gctcagggcc tgttggggct tgagggtctg ctggtggct 159
Claims (10)
1. it is a kind of for detecting the primer set of ALDH2 gene rs671 loci polymorphisms, by primer 1, primer 2 and probe groups
Into；
The primer 1 is following a1) or a2)：
A1) the single strand dna shown in sequence 1；
A2) by a1) single strand dna that limits carry out the missings of one or several bases, insertion and/or change it is obtaining, with
A1) single strand dna limited has the single strand dna of identical function；
The primer 2 is following b1) or b2)：
B1) the single strand dna shown in sequence 2；
B2) by b1) single strand dna that limits carry out the missings of one or several bases, insertion and/or change it is obtaining, with
B1) single strand dna limited has the single strand dna of identical function；
The probe is following c1) or c2)：
C1) the single strand dna shown in sequence 3；
C2) by c1) single strand dna that limits carry out the missings of one or several bases, insertion and/or change it is obtaining, with
C1) single strand dna limited has the single strand dna of identical function.
2. the primer set according to claim 1, it is characterised in that：The primer 1, the primer 2 and the probe
Mole ratio is 3:1:1.
3. primer set according to claim 1 or 2, it is characterised in that：
The 6th of the probe and the 11st bit base are the base T of fluorophor modification, and the fluorophor is specially FAM.
4. it is a kind of for detecting the PCR reagent of ALDH2 gene rs671 loci polymorphisms, including institute any in claim 1-3
The primer set and archaeal dna polymerase stated.
5. PCR reagent according to claim 4, it is characterised in that：
The archaeal dna polymerase is Phire Hotstart II archaeal dna polymerases；
The PCR reagent is polymerize by the primer 1, the primer 2, the probe, the Phire Hotstart II DNA
Enzyme, agarose, dNTPs, buffer and water composition；
A concentration of 0.86 μM in the PCR reagent of the primer 1；
A concentration of 0.29 μM in the PCR reagent of the primer 2；
Concentration of the probe in the PCR reagent is 0.29 μM；
A concentration of 0.2U/ μ L of the Phire Hotstart II archaeal dna polymerases in the PCR reagent；
Mass fraction of the agarose in the PCR reagent is 0.2%.
6. it is a kind of for detecting the kit of ALDH2 gene rs671 loci polymorphisms, including institute any in claim 1-3
The PCR reagent described in primer set or claim 4 or 5 stated.
7. any primer set or the PCR reagent described in claim 4 or 5 or claim 6 institute in claim 1-3
The kit stated is following 1) -8) in it is any in application：
1) product of detection or auxiliary detection ALDH2 gene rs671 loci polymorphisms is prepared；
2) detect or assist detection ALDH2 gene rs671 loci polymorphisms；
3) it prepares detection or auxiliary detects the product of patient ALDH2 genotype to be measured；
4) it detects or assists to detect patient ALDH2 genotype to be measured；
5) it prepares detection or auxiliary detects the product whether patient to be measured carries ALDH2 mutation alleles；
6) it detects or assists to detect whether patient to be measured carries ALDH2 mutation alleles；
7) it prepares detection or auxiliary detects the product of patient's nitroglycerin metabolic capability and/or alcohol metabolism ability to be measured；
8) it detects or assists to detect patient's nitroglycerin metabolic capability and/or alcohol metabolism ability to be measured.
8. a kind of method detected or auxiliary detects patient ALDH2 genotype to be measured, includes the following steps：
PCR amplification is carried out to the buccal swab sample of patient to be measured using the primer set any in claim 1-3, is obtained
To the Tm values of pcr amplification product, the genotype of sample to be tested is judged according to the Tm values：
If the Tm values of clinical samples to be measured are 51.1-55.1, the genotype of patient to be measured is or candidate is AA genotype；
If the Tm values of clinical samples to be measured are 59.7-63.7, the genotype of patient to be measured is or candidate is GG genotype；
If the Tm values of clinical samples to be measured are 51.1-55.1 and 59.7-63.7, the genotype of patient to be measured is or candidate is GA
Genotype.
9. a kind of method detected or whether auxiliary detection patient to be measured carries ALDH2 mutation alleles, includes the following steps：
PCR amplification is carried out to the buccal swab sample of patient to be measured using the primer set any in claim 1-3, is obtained
The Tm values of pcr amplification product judge whether patient to be measured carries ALDH2 mutation alleles according to the Tm values：
If the Tm values of clinical samples to be measured are 59.7-63.7, patient to be measured does not carry ALDH2 mutation alleles；
If the Tm values of clinical samples to be measured for it is following 1) or 2), patient to be measured carries ALDH2 mutation alleles：
1)51.1-55.1；
2) 51.1-55.1 and 59.7-63.7.
10. method according to claim 8 or claim 9, it is characterised in that：The extension of time of the PCR amplification is 5 seconds.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611251299.3A CN108265113A (en) | 2016-12-29 | 2016-12-29 | ALDH2 genetic polymorphism detection kits |
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| CN201611251299.3A CN108265113A (en) | 2016-12-29 | 2016-12-29 | ALDH2 genetic polymorphism detection kits |
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| CN108588196A (en) * | 2018-04-23 | 2018-09-28 | 北京中能通达科技发展中心（有限合伙） | A method of prevent PCR from forming Aerosol Pollution |
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| CN110527711A (en) * | 2019-06-12 | 2019-12-03 | 江苏莱尔生物医药科技有限公司 | A kind of rapid PCR amplification kit and its application method |
| CN110982885A (en) * | 2019-12-20 | 2020-04-10 | 郑州安图生物工程股份有限公司 | Gene polymorphism detection primer, probe and kit |
| CN113337595A (en) * | 2021-05-31 | 2021-09-03 | 山东第一医科大学附属皮肤病医院(山东省皮肤病性病防治研究所、山东省皮肤病医院) | Application of single nucleotide polymorphism rs671 in screening leprosy patients |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588196A (en) * | 2018-04-23 | 2018-09-28 | 北京中能通达科技发展中心（有限合伙） | A method of prevent PCR from forming Aerosol Pollution |
| CN108588196B (en) * | 2018-04-23 | 2021-12-14 | 北京中能通达科技发展中心（有限合伙） | Method for preventing aerosol pollution formed by PCR |
| CN109371127A (en) * | 2018-10-22 | 2019-02-22 | 江苏美因康生物科技有限公司 | The kit and method of a kind of while quick detection UGT1A1*6 type and UGT1A1*28 type gene pleiomorphism |
| CN110527711A (en) * | 2019-06-12 | 2019-12-03 | 江苏莱尔生物医药科技有限公司 | A kind of rapid PCR amplification kit and its application method |
| CN110982885A (en) * | 2019-12-20 | 2020-04-10 | 郑州安图生物工程股份有限公司 | Gene polymorphism detection primer, probe and kit |
| CN113337595A (en) * | 2021-05-31 | 2021-09-03 | 山东第一医科大学附属皮肤病医院(山东省皮肤病性病防治研究所、山东省皮肤病医院) | Application of single nucleotide polymorphism rs671 in screening leprosy patients |
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