CN105624315A - Primers and reagent kit for detecting polymorphism of ALDH2 gene c.1510 locus - Google Patents
Primers and reagent kit for detecting polymorphism of ALDH2 gene c.1510 locus Download PDFInfo
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Abstract
The invention discloses primers and a reagent kit for detecting the polymorphism of the ALDH2 gene c.1510 locus. The specific primers, a specific probe and the specific reagent kit which are designed for the ALDH2 gene locus have the advantages of being high in sensitivity, good in specificity, high in response speed and low in cost, and are suitable for large-scale clinical application; rapid, effective and accurate typing qualitative detection of ALDH2 can be achieved, and a reference can be provided for timely nitroglycerin treatment of stenocardia, drinking guidance and occurrence of related caner.
Description
Technical field
The present invention relates to a kind of ALDH2 gene primer that c.1510 loci polymorphism detects and test kit.
Background technology
Stenocardia causes coronary insufficiency to produce owing to coronary atherosclerosis is narrow, clinical upper use sublingual administration pannonit is treated, pannonit is by release nitrogen protoxide (NO), NO is identical with endothelium relaxation, activate guanylate cyclase, the cyclic guanosine monophosphate (CGMP) in unstriated muscle and its hetero-organization is increased, cause myosin light chain to be scaled acidifying, regulate smooth muscle contraction state, cause vasodilation thus allevating angina pectoris. General sublingual administration pannonit took effect in 15 minutes, need to be hospitalized for treatment in time if can not alleviate. Experiment finds aldehyde dehydrogenase 2 (aldehydedehydrogenase2, ALDH2) it is the key factor of pannonit onset, the metabolism of pannonit needs the enzyme of ALDH2 genes encoding, ALDH2 gene has genetic polymorphism, and wherein close with asian population is that the c.1510 position Nucleotide of ALDH2 gene exists G/A polymorphism. Its heritable variation type makes pannonit metabolic rate greatly reduce, and the enzymic activity of wild-type is 10 times of mutated enzyme activity. Thus nitrogen protoxide cannot be produced, it is difficult to play drug effect or drug effect reduction. In addition, one of key enzyme in ALDH2 or alcohol metabolism, after drinking, alcohol is respectively by ethanol dehydrogenase (ADH) with acetaldehyde dehydrogenase (ALDH) is metabolized to carbonic acid gas and water excretes. ALDH2 genic mutation type can make alcohol metabolism be obstructed, and causes the accumulation of acetaldehyde in body. Acetaldehyde, in the accumulation of blood of human body, can cause the peroxidation of cell membrane lipid, occurs the incidences such as alcoholism, alcohol dependence, hepatopathy, the esophageal carcinoma, laryngocarcinoma to improve.
Therefore, the gene pleiomorphism of the SNP site G on ALDH2 gene > A is great on treatment stenocardia impact, by the detection of G > ASNP point gene type on ALDH2 gene is come auxiliary clinical diagnosis, for clinician selects medicine to provide reference. The people simultaneously excessive drinking being addicted to drink has certain directive significance, to reduce the occurrence risk of disease.
At present, the technique means of detection gene locus polymorphism has a lot, but great majority all stop at laboratory level, in the routine testing of the medical institutions that can't really apply, below introduces the detection technique existed at present:
(1) single-strand conformation polymorphism (PCR-SSCP)
Point mutation or SNP is utilized to be impacted by short DNA single chain conformation, thus the difference causing electrophoretic velocity detects. Feature is the examination that can carry out specific region sudden change, but needs to run PAGE glue, comparatively loaded down with trivial details, and has certain loss.
(2) high resolving power melt curve analysis analyzes (HRM)
This is nearly new technology grown up for 2 years, and principle is easy, it is not necessary to reaction conditions and system are optimized especially, it is easy to realizes, can carry out the examination suddenlyd change, but need the support of particular instrument and reagent. And catastrophe point in fragment can only be detected, can not determine the particular location of detection catastrophe point, it is easy to cause false positive results.
(3) Taqman probe method (quantitative PCR)
Applicable known SNP site, the detection that site quantity is few, flux is high. But probe synthesis expense is expensive, unknown SNP site can not be found simultaneously, and easily cause false positive results.
(4) denaturing high-performance chromatography (DHPLC)
For the abrupt climatic change of specific site, it is necessary to grope various condition, less stable, influence factor is too many, so being not easy to carry out.
(5) digestion with restriction enzyme method
Select suitable restriction endonuclease that PCR primer is carried out enzyme according to sudden change or SNP site to cut, then electroresis appraisal. The method is reliable and stable, but it is noted that digesting efficiency is complete, and not every sudden change or SNP site have enzyme to select, it is easy to pollute.
(6) chip technology
Having high-throughput, miniatureization, automatization, the feature such as anti-pollution compared with traditional instrument detection method, being only applicable to full-length genome SNP scans, and is not suitable for the SNP site detection of single gene, and precision is low, expensive.
What the ALDH2 genetic polymorphism detection test kit of the current proud Science and Technology Ltd. in domestic Shanghai hundred adopted is method for gene chip access authentication.
(7) Allele-specific diagnostic PCR primer (ARMS)
Utilize the principle that 3 ' end end bit base of PCR primer could effectively must increase with its template DNA complementation, design ApoE gene amplimer, under strict conditions, only when primer 3 ' base and template are matched, pcr amplification band could occur, thus detect out sudden change. Utilize Taqman probe to realize the detection of sudden change in sample DNA on real-time fluorescence quantitative PCR platform, there is extremely high specificity and susceptibility.
(8) direct sequencing
The gold standard of snp analysis, due to the detection of gene sequencing method is the complete sequence in district of easily suddenling change, and therefore can detect unknown mutation site. But order-checking experimental implementation is comparatively complicated, experimental period is longer, electrophoresis easily causes laboratory pollution.
Summary of the invention
The object of the present invention is exactly to solve the problem, it is provided that a kind of ALDH2 gene primer that c.1510 loci polymorphism detects and test kit.
In order to realize above-mentioned purpose, the present invention adopts following technical scheme:
The ALDH2 gene primer that c.1510 loci polymorphism detects, comprises ALDH2-A-F, and nucleotide sequence is as shown in SEQIDNO:1, and ALDH2-G-F, nucleotide sequence is as shown in SEQIDNO:2, and ALDH2-R, nucleotide sequence is as shown in SEQIDNO:3.
The ALDH2 gene test kit that c.1510 loci polymorphism detects, comprises above-mentioned three kind primer.
Preferred: mentioned reagent box also comprises one group of interior label primer, the nucleotide sequence of HER2-F is as shown in SEQIDNO:5, and the nucleotide sequence of HER2-R is as shown in SEQIDNO:6, and the nucleotide sequence of HER2-FP is as shown in SEQIDNO:7.
Preferred: mentioned reagent box also comprises, 5 ' end of described fluorescent PCR probe (ALDH2-FP, nucleotide sequence is as shown in SEQIDNO:4) has fluorescent reporter group, and 3 ' end has quenching of fluorescence group.
Preferred: described fluorescent reporter group and quenching of fluorescence group are arranged in pairs or groups such as following table:
More preferably: 5 ' end of described fluorescent PCR probe has fluorescent reporter group 6-FAM or HEX, and 3 ' end has quenching of fluorescence group B HQ1.
Preferred: test kit also comprises Mg2+, archaeal dna polymerase, the quantitative fluorescent PCR reaction buffer of dNTPs, ALDH2 positive quality control product, negative quality control product, interior mark quality control product.
Preferred: described positive quality control product behaviour ALDH2 (A/G) type plasmid, negative quality control product is purified water, interior mark quality control product HER2 plasmid.
Preferred: PCR reaction buffer comprises: ALDH2PCR reaction solution 12.5ul, ALDH2 (A) primed probe mixed solution 6.5ul, ALDH2 (G) primed probe mixed solution 6.5ul, sample to be tested DNA2ul, purified water 4.0ul, is made into 25ul reaction system altogether; Wherein the consisting of of ALDH2 (A) primed probe mixed solution: ALDH2-A-F1.5ul (10P), ALDH2-FP1.5ul (0.25P), ALDH2-R1.5ul (10P), HER2-F0.5ul (10P), HER2-R0.5ul (10P), HER2-FP1.0ul (0.25P), consisting of of ALDH2 (G) primed probe mixed solution: ALDH2-G-F1.5ul, ALDH2-FP1.5ul, ALDH2-R1.5ul, HER2-F0.5ul, HER2-R0.5ul, HER2-FP1.0ul.
Preferred: PCR reaction conditions is such as following table:
Inventive principle: minute
The present invention chooses c.1510 site (G/A) the SNP region of ALDH2, and design specificity ARMS primer, then utilizes the SNP type that whole blood DNA carries out ALDH2 not detect.
Allele specific amplification (Allelesspecificamplification, ASA)-hinder abruptly-changing system (Amplificationrefractorymutationsystem also known as amplification, ARMS), utilize the principle that 3 ' end end bit base of PCR primer could effectively must increase with its template DNA complementation, design ApoE gene amplimer, under strict conditions, only when primer 3 ' base and template are matched, pcr amplification band could occur, thus detect out sudden change. This test kit have employed Taqman fluorescence probe, and this probe is with a fluorescence radiation group and a fluorescent quenching group, and complete probe is under specific light source excites, and the fluorescence that luminophore produces is quenched group and all absorbs, sample unstressed configuration. In PCR process, Taq enzyme is while extended DNA chain, the specificity fluorescent probe degraded be combined with template by 5 ' �� 3 ' exonuclease activity of self, makes fluorescent reporter group be separated with quenching group, and the fluorescent reporter group after separation excites lower generation fluorescence at specific light source. Monitor the change of whole PCR process fluorescent signal.
Meanwhile, this product marks system of quality control in adopting, for monitoring the restraining factors that reaction system may exist. Interior mark template and target gene are without homology, and what interior mark probe was selected is another detection passage not conflicted with target gene probe.
Fluorescence probe method is when pcr amplification at the fluorescent probe adding a specificity adding one pair of primer simultaneously, and this probe is an oligonucleotide, and two ends mark a report fluorophor and a cancellation fluorophor respectively. When probe is complete, the fluorescent signal that reporter group is launched is quenched group and absorbs; During pcr amplification the 5 '-3 of Taq enzyme ' probe enzyme cut degraded by 5 prime excision enzyme activity, report fluorophor is separated with cancellation fluorophor, thus fluorescence monitoring system can receive fluorescent signal, namely often increase a DNA, a fluorescence molecule is just had to be formed, it is achieved that the accumulation of fluorescent signal is formed completely synchronous with PCR primer. Different shaped does not need to be divided in two different PCR pipe, if detection sample is heterozygous, then all has amplified fragments in two PCR pipe, and CT value difference is less than 5. If detection sample is homozygote, then corresponding PCR pipe has amplified fragments, and another PCR pipe does not increase, and carries out somatotype with this. This detection ALDH2 genetic method principle is clear, simple to operate, and result interpretation clear, easy, the cycle of operation is short, and cost is lower, and specificity and accuracy all can reach more than 97%. Can be used for the daily quick detection of medical institutions.
The useful effect of the present invention:
The primer special for ALDH2 gene locus of inventive design, probe (ALDH2-FP, nucleotide sequence is as shown in SEQIDNO:4, nucleotides sequence is classified as 5-AGTGAAAACTGTGAGTGTGGGACCTGCT-3) and test kit, there is susceptibility height, specificity is good, reaction is quick and cost is low advantage, be suitable for clinical on a large scale development; Quick, effectively and accurately somatotype qualitative detection to ALDH2 can be realized, for the generation offer reference of the timely treatment of stenocardia, guidance of drinking, associated cancer treated by pannonit, solve existing primer and can only separate two types, or the problem of specificity and susceptibility difference.
The ALDH2 gene tester having obtained patent in existing market is gene chips and PCR-gel electrophoresis. Gene chip is that the DNA extracted is carried out pcr amplification, afterwards product is put into hybridization instrument and hybridizes, and finally takes out chip by biochip identification reading instrument analytical results. Gene chip great advantage is high-throughput. But also there is many deficiencies simultaneously. Comprise that cost costliness, complicated operation, detection sensitivity be lower and poor repeatability etc. And PCR-gel electrophoresis processes identical early stage, being presented by gel electrophoresis by the product increased afterwards, the accuracy of the method result is lower and has certain danger, is not suitable for the operation of great amount of samples simultaneously. And this test kit adopts PCR-fluorescence probe method, combining both advantages, the operating time is short, result interpretation simple and avoids people is the error caused, and is applicable to widely using of the mechanisms such as hospital.
Three primers of the present invention are according to ARMS-PCR design of primers principle, Taq DNA polymerase is utilized to lack 3 ' to the 5 ' 5 prime excision enzyme activity held, specific base is placed in primer 3 ' hold, 3 ' end base respectively with sudden change and wild template base complementrity, thus the template of sudden change and wild template region are separated; Simultaneously owing to Taqman probe has sequence-specific, only it is attached to complementary region, the specificity fluorescent probe that Taq DNA polymerase is degraded be combined with template by 5 ' �� 3 ' exonuclease activity of self, and the copy number of fluorescent signal and amplification has relation one to one, therefore high specificity is highly sensitive. Final realization is to the accurate somatotype of sample, and the primer of inventive design is all higher than other primer specificity and susceptibility.
The interior label primer of inventive design chooses one section of sequence conservative in human genome DNA, and interior mark probe 5 ' end is marked with reporter group HEX, and 3 ' end is marked with not luminous fluorescent quenching group BHQ1. When probe is complete time, the fluorescent energy that reporter group is launched is quenched group and absorbs, and instrument can't detect signal. Along with the carrying out of PCR, Taq enzyme runs into, in DNA extension process, the probe being combined with template, and probe will be cut off by its 5 ' �� 3 ' exonuclease activity, and reporter group, away from fluorescent quenching group, produces fluorescent signal. Therefore the strength of signal detected just represents the copy number of template DNA.
The interior mark system of inventive design is for monitoring whether the reaction of this quantitative fluorescent PCR exists suppression; In addition, it is poor that quantitative real time PCR Instrument may exist between the hole higher than allowed band, causes amplification efficiency difference between different pipe; In addition people adds the appearance that sample mistake also may cause false negative result, and the interior mark system of the present invention eliminates above-mentioned hidden danger, ensure that the accuracy of detected result.
The PCR reaction buffer that the present invention selects and reaction conditions, with the primer of inventive design with the use of ensure that under the prerequisite that sample is carried out accurate somatotype, achieve the polymorphism detecting sample fast, easily, as long as 1.0 to 1.5 hours can obtain reaction result, and cost is low, it is suitable for clinical on a large scale development. Realize to ALDH2 gene c.1510 loci polymorphism detect fast, effectively and accurately, thus can ensure timely be clinical application provide reference. Three kinds of types that primer of the present invention, test kit can effectively isolate ALDH2 gene c.1510 site are other.
Accompanying drawing explanation
Fig. 1 is the curve that embodiment 1 positive quality control product employing quantitative real time PCR Instrument detection sample to be tested obtains;
Fig. 2 is the curve that embodiment 1 negative quality control product employing quantitative real time PCR Instrument detection sample to be tested obtains;
Fig. 3 is the curve that the homozygous employing quantitative real time PCR Instrument detection sample to be tested of embodiment 1AA obtains;
Fig. 4 is the curve that embodiment 1AG heterozygous employing quantitative real time PCR Instrument detection sample to be tested obtains;
Fig. 5 is the curve that the homozygous employing quantitative real time PCR Instrument detection sample to be tested of embodiment 1GG obtains.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the invention will be further described.
Amplification region of the present invention selects ALDH2 specific sequence as primer amplification target spot.
The principle of design of primer
1) length of PCR primer is at 80��150bp;
2) the G/C content of primer sequence is 40��60%;
3) avoid primer self or and primer between form 4 or more than 4 pairing continuously, avoid primer self formation ring-type hairpin structure;
4) primer Tm 55��65 DEG C, the Tm difference between primer avoids exceeding 2 DEG C;
5) primer sequence optimum length 18-24bp;
6) 3 ' end of primer is avoided using base A, and 3 ' end of primer avoids 3 or more than 3 consecutive identical bases occur;
The screening principle of primer
In the present invention, the screening principle of primer three kinds of other samples of type to be carried out accurate somatotype.
Wherein, the nucleotide sequence of ALDH2-A-F is as shown in SEQIDNO:1, or is substituted by the nucleotide sequence shown in SEQIDNO:1, lacks, adds the nucleotide sequence formed;
The nucleotide sequence of ALDH2-G-F is as shown in SEQIDNO:2, or is substituted by the nucleotide sequence shown in SEQIDNO:2, lacks, adds the nucleotide sequence formed;
The nucleotide sequence of ALDH2-R is as shown in SEQIDNO:3, or is substituted by the nucleotide sequence shown in SEQIDNO:3, lacks, adds the nucleotide sequence formed.
The nucleotide sequence of ALDH2-FP is as shown in SEQIDNO:4, or is substituted by the nucleotide sequence shown in SEQIDNO:4, lacks, adds the nucleotide sequence formed;
The nucleotide sequence of HER2-F is as shown in SEQIDNO:5, or is substituted by the nucleotide sequence shown in SEQIDNO:7, lacks, adds the nucleotide sequence formed;
The nucleotide sequence of HER2-R is as shown in SEQIDNO:6, or is substituted by the nucleotide sequence shown in SEQIDNO:8, lacks, adds the nucleotide sequence formed.
The nucleotide sequence of HER2-FP is as shown in SEQIDNO:7, or is substituted by the nucleotide sequence shown in SEQIDNO:8, lacks, adds the nucleotide sequence formed.
Wherein, the nucleotides sequence of SEQIDNO:1 is classified as 5-TCAGGGCTGCAGGCATACACTA-3
The nucleotides sequence of SEQIDNO:2 is classified as 5-TTTGGCTGCAGGCATACACAG-3
The nucleotides sequence of SEQIDNO:3 is classified as 5-CCACCAGCAGACCCTCAA-3
The nucleotides sequence of SEQIDNO:4 is classified as 5-AGTGAAAACTGTGAGTGTGGGACCTGCT-3
The nucleotides sequence of SEQIDNO:5 is classified as 5-CCACACTCACAGTTTTCACTTC-3
The nucleotides sequence of SEQIDNO:6 is classified as 5-CCTTTGGTGGCTACAAGATG-3
The nucleotides sequence of SEQIDNO:7 is classified as 5-CCACTCCCCGACATCTTGTAGCCACC-3
Preferably, the primer of above-described somatotype detection ALDH2 gene and probe, fluorescent reporter group is 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, FAM), chlordene-6-methyl fluorescein (Hexachloro-6-methylfluorescein, HEX), VIC fluorescence dye, four chloro-6-Fluoresceincarboxylic acid (tetrachloro-6-carboxyfluorescein, TET), carboxy-X-rhodamine (Carboxy-x-rhodamine, ROX), 6-carboxyl tetramethylrhodamine (6-carboxytetramethylrhodamine, TAMRA), sulphonyl rhodamine (Sulforhodamine101, TexasRed), 6-carboxyl-4 ', 5 '-two chloro-2 ', 7 '-dimethoxy fluorescein succinimidyl ester (6-Carboxy-4 ', 5 '-dichloro-2 ', 7 '-dimethoxyfluorescein, JOE), flower cyanines 3 (cyanine3, Cy3), flower cyanines 3.5 (cyanine3.5, Cy3.5), flower cyanines 5 (cyanine5, Cy5) and flower cyanines 5.5 (cyanine5.5, Cy5.5) at least one in, fluorescent quenching group is at least one in 6-carboxyl tetramethylrhodamine, 4-(4-dimethylamino phenylazo-) phenylformic acid, black hole quencher 1, black hole quencher 2 and black hole quencher 3. above fluorescent reporter group and fluorescent quenching group are existing commercial goods.
Wherein black hole quencher 1 (BlackHoleQuencher1, BHQ1), black hole quencher 2 (BlackHoleQuencher2, BHQ2), black hole quencher 3 (BlackHoleQuencher3, BHQ3), 4-(4-dimethylamino phenylazo-) phenylformic acid (4-(4 '-dimethylaminophenylazo) benzoicacid, DABCYL) it is the product of biological search technique company (BiosearchTechnologies, Inc).
Preferably, when fluorescent quenching group is selected from 4-(4-dimethylamino phenylazo-) phenylformic acid, fluorescent reporter group is selected from 6-Fluoresceincarboxylic acid, four chloro-6-Fluoresceincarboxylic acids, 6-carboxyl-4 ', 5 '-two chloro-2 ', at least one in 7 '-dimethoxy fluorescein succinimidyl ester, chlordene-6-methyl fluorescein, Hua Jing 3.
When fluorescent quenching group is selected from 6-carboxyl tetramethylrhodamine, fluorescent reporter group is selected from 6-Fluoresceincarboxylic acid, four chloro-6-Fluoresceincarboxylic acids, 6-carboxyl-4 ', 5 '-two chloro-2 ', at least one in 7 '-dimethoxy fluorescein succinimidyl ester or chlordene-6-methyl fluorescein.
When fluorescent quenching group is selected from black hole quencher 1, fluorescent reporter group is selected from 6-Fluoresceincarboxylic acid, four chloro-6-Fluoresceincarboxylic acids, 6-carboxyl-4 ', 5 '-two chloro-2 ', at least one in 7 '-dimethoxy fluorescein succinimidyl ester, chlordene-6-methyl fluorescein or flower cyanines 3.
When fluorescent quenching group is selected from black hole quencher 2, at least one that fluorescent reporter group is selected from 6-carboxyl tetramethylrhodamine, Hua Jing 3, carboxy-X-rhodamine or sulphonyl rhodamine.
When fluorescent quenching group is selected from black hole quencher 3, the one that fluorescent reporter group is selected from Hua Jing 5 or flower cyanines 5.5.
The collocation of fluorescent reporter group and fluorescent quenching group sees table:
| Fluorescent quenching group | Fluorescent reporter group |
| DABCYL | At least one in 6-FAM, TET, JOE, HEX, Cy3 |
| TAMRA | At least one in 6-FAM, TET, JOE, HEX |
| BHQ1 | At least one in 6-FAM, TET, JOE, HEX, Cy3 |
| BHQ2 | At least one in TAMRA, Cy3, ROX, Texas Red |
| BHQ3 | Cy5 or Cy5.5 |
Preferably, the primer of above-mentioned somatotype detection ALDH2 gene and probe, the nucleotide sequence 5 ' end of described Taqman fluorescence probe is marked with fluorescent reporter group 6-FAM, HEX, and 3 ' end is marked with fluorescent quenching group B HQ1.
Process sample to be tested, carries out PCR;
This test kit does not comprise nucleic acid extraction composition, sample and positive quality control product process adopt the nucleic acid extraction kit of commercialization, recommendation TIANampBloodDNAKit (CatNO.DP318), specifically operates see this test kit specification sheets, but it is noted that following item.
Extracting DNA to need to measure concentration with ultraviolet spectrophotometer, sample pollutes without albumen or RNA, OD260/OD280=1.8��2.0, OD260/OD230>=2.0; DNA content requires: recommend DNA applied sample amount 50��250ng; The DNA suggestion extracted detects immediately, otherwise please in-20 �� 5 DEG C of preservations.
Result is analyzed
Primer synthesis and purity detecting: display purifying rank is ULTRAPAGE level in the synthesis report that a) combination mechanism is provided, it is provided that ULTRAPAGE electrophoresis result be shown as single band; Mass spectral results figure is shown as single peak, or assorted peak intensity is not higher than the 1/10 of main peak intensity; B) ratio of the solution O D260/OD280 of 10 ��Ms is between 1.60��2.05.
Sample to be tested processes: the present invention is detected sample behaviour whole blood sample, and whole blood DNA extracts and existing whole blood DNA can be adopted to extract test kit. It is sure not after the collecting whole blood of periphery to place too for a long time in room temperature, preserves under 4 DEG C of conditions and be no more than one month, preserve under-20 �� 5 DEG C of conditions and be no more than 1 year.
Pcr amplification:
Wherein the reaction system of PCR reaction comprises:
ALDH2PCR reaction solution (purchased from Bai Ye trade (Shanghai) company limited) 12.5ul, ALDH2 (A) primed probe mixed solution 6.5ul, ALDH2 (G) primed probe mixed solution 6.5ul, sample to be tested DNA2ul, purified water 4.0ul, is made into 25ul reaction system altogether; Wherein the consisting of of ALDH2 (A) primed probe mixed solution: ALDH2-A-F1.5ul (10P), ALDH2-FP1.5ul (0.25P), ALDH2-R1.5ul (10P), HER2-F0.5ul (10P), HER2-R0.5ul (10P), HER2-FP1.0ul (0.25P). Consisting of of ALDH2 (G) primed probe mixed solution: ALDH2-G-F1.5ul, ALDH2-FP1.5ul, ALDH2-R1.5ul, HER2-F0.5ul, HER2-R0.5ul, HER2-FP1.0ul.
PCR reaction conditions is:
The present invention also relates to a kind of ALDH2 gene locus polymorphic detection test kit clock, and described test kit comprises:
| Sequence number | Product forms | Main component | Specification and loading amount |
| 1 | ALDH2PCR reaction solution P | dNTP��Mg2+, Taq enzyme etc. | 600 �� L �� 1 pipes |
| 2 | ALDH2 (A) primed probe mixed solution P | Primer, probe etc. | 156 �� L �� 1 pipes |
| 3 | ALDH2 (G) primed probe mixed solution P | Primer, probe etc. | 156 �� L �� 1 pipes |
| 4 | ALDH2 (A/G) positive quality control product P | ALDH2 (AG type) plasmid | 210 �� L �� 1 pipes |
| 5 | Mark quality control product P in ALDH2 | HER2 plasmid | 100 �� L �� 1 pipes |
| 6 | The negative quality control product P of ALDH2 | Purified water | 1200 �� L �� 1 pipes |
Embodiment 1
1. sample process
1.1 sample process and nucleic acid extraction
This test kit does not comprise nucleic acid extraction composition, sample (1,2, No. 3) process adopts the nucleic acid extraction kit of commercialization, recommendation TIANampBloodDNAKit (CatNO.DP318), specifically operates see this test kit specification sheets, but it is noted that following item.
Extracting DNA to need to measure concentration with ultraviolet spectrophotometer, sample pollutes without albumen or RNA, OD260/OD280=1.8��2.0, OD260/OD230>=2.0; DNA content requires: recommend DNA applied sample amount 50��250ng; The DNA suggestion extracted detects immediately, otherwise please in-20 �� 5 DEG C of preservations.
1.2 prepare
Experiment takes out each component of test kit before starting, and also sustained oscillation of fully thawing mixes even in 15 seconds, centrifugal 15 seconds of 2000rpm.
Prepare the PCR instrument example reaction pipe of respective numbers, except sample, also should comprise 1 positive quality control product and 1 negative quality control product reaction tubes.
2. reagent configuration
2.1 determine stoichiometric number N, N=sample number (n) to be checked+negative and positive quality control product number (2)+1. Calculate the amount of each reagent being added in reaction mixture, it be calculated as follows:
Table 1 system configurations
2.2 get 1.5ml centrifuge tube (sterilizing) configures reaction system, and reagent all adds rear vortex oscillation 10 seconds, centrifugal 15 seconds of 2000rpm.
Then above-mentioned mixed solution 23 �� L/ pipe point is filled in PCR reaction tubes (aseptic and RNase-Free) by 2.3.
3. add sample
By processed sample, people ALDH2 (A/G) positive quality control product P, negative quality control product P, interior mark quality control product add in PCR reaction tubes by table 3 specified amount respectively. Cover tightly pipe lid (avoiding bubble to produce), centrifugal 15 seconds of 2000rpm, the liquid on tube wall is all got rid of to, at the bottom of pipe, then carrying out pcr amplification reaction immediately.
Table 2 adds sample
Sample putting position recommended by table 3
| Title | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| People ALDH2 (A) | �� | �� | �� | �� | �� | �� | STD | NTC |
| People ALDH2 (G) | �� | �� | �� | �� | �� | �� | STD | NTC |
Note: 1.-6. represent sample; STD representative ALDH2 (A/G) positive quality control product P, NTC represent negative quality control product.
4. quantitative real time PCR Instrument amplification program is arranged
Table 4 response procedures
Target gene: FAM passage; Interior mark: HEX passage
5 result analysis condition settings
After reaction terminates, manual setting baseline to 2000 is carried out according to fluorescence curve, the starting point (Start) of manual setting baseline (Non-adaptivebaseline) is generally set as 5��8, and terminating point (Stop) is generally set as 12��15. After setting, it is possible to obtain the Ct value of each sample from " Ct (dRn) " of " Textreport " window.
6, result interpretation
1). in each sample, mark passage (HEX) Ct value answers��35, and one of them Ct < 35 in target gene passage two amplified reactions, (without amplification curve, CT value is by 40 calculating for target gene passage), meet after this condition by following table interpretation:
2) if. mark passage (HEX) Ct value in sample > 35, it is proposed that again extract sample, then detect;
Table 5 result interpretation
3) if. mark channel C T value��35 in sample, and target gene channel C t >=35, then it is invalid to detect.
Detected result is as Figure 1-5.
The present invention, on the basis of conventional ARMS primer, holding the 2nd base place or the 3rd base place to introduce a base mismatch apart from primer 3 ', enhances the specificity of primer, inhibits non-specific amplification.
The position introducing mutating alkali yl in ARMS primer is most important to the specificity of primer, and the amplification of the normal template of primer pair can not be checked in some mutational sites; Or owing to restraining effect causes excessively by force mutagenesis template also can not increase. Therefore, to guarantee that the ARMS primer of screening only can increase mutagenesis template in reaction system, and the normal template that do not increase.
The present embodiment devises for detecting ALDH2 gene c.1510 2 forward primers of loci polymorphism and 1 shared reverse primer through optimal screening. Its sequence is respectively as shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3.
Comparative example 1:
The specificity of ARMS primer is particularly important to the accuracy of detected result, for improving accuracy, need to introduce mispairing at 3 ' end of ARMS primer. If not introducing mispairing, then the phenomenon of somatotype mistake easily occurs.
Share reverse primer and probe sequence constant, the forward primer being used for detecting ALDH2 gene c.1510 site is not artificially introduced mispairing, its primer sequence is as follows: 5-TTTGGCTGCAGGCATACACTG-3.
Below do not introduce mispairing forward primer, share reverse primer and probe to ALDH2 gene c.1510 site AA, AG, GG tri-kinds of type samples detect, has there is amplification curve in result AA type pattern detection hole. The above results illustrates: reduce without the artificial primer specificity introducing base mismatch, it is easy to cause somatotype mistake.
Comparative example 2:
In the introducing process in site, it is necessary to rationally arrange according to sequence signature. If it is improper to introduce position, in extension increasing sequence, G/C content is too high, it is easy to forms secondary structure or produces non-specific amplification, thus affects the accuracy of detected result.
Other primers and component all remain unchanged, the forward primer being used for detecting ALDH2 gene c.1510 site is changed into: 5-TTTGGCTGCAGGCATACTCAG-3, to ALDH2 gene c.1510 site AA, AG, GG tri-kinds of type samples detect, result AA type pattern detection hole has occurred amplification curve equally, namely create non-specific amplification, sample accurately can not be detected.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of the technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (9)
1. the ALDH2 gene primer that c.1510 loci polymorphism detects, is characterized in that: comprise ALDH2-A-F, and nucleotide sequence is as shown in SEQIDNO:1, ALDH2-G-F, nucleotide sequence is as shown in SEQIDNO:2, and ALDH2-FP, nucleotide sequence is as shown in SEQIDNO:3.
2. the ALDH2 gene test kit that c.1510 loci polymorphism detects, is characterized in that: comprise three primers according to claim 1.
3. test kit as shown in claim 2, is characterized in that: also comprise one group of interior label primer, HER2-F, its nucleotide sequence as shown in SEQIDNO:5, HER2-R, its nucleotide sequence is as shown in SEQIDNO:6, HER2-FP, its nucleotide sequence is as shown in SEQIDNO:7.
4. test kit as shown in claim 2, is characterized in that: also comprise fluorescent PCR probe ALDH2-R, and nucleotide sequence is such as SEQIDNO:4, and 5 ' end of shown described fluorescent PCR probe has fluorescent reporter group, and 3 ' end has quenching of fluorescence group.
5. test kit as shown in claim 4, is characterized in that: described fluorescent reporter group and quenching of fluorescence group are arranged in pairs or groups such as following table:
6. test kit as shown in claim 2, is characterized in that: test kit also comprises Mg2+, archaeal dna polymerase, the quantitative fluorescent PCR reaction buffer of dNTPs, ALDH2 positive quality control product, negative quality control product, interior mark quality control product.
7. test kit as shown in claim 6, is characterized in that: described positive quality control product behaviour ALDH2 (A/G) type plasmid, and negative quality control product is purified water, interior mark quality control product HER2 plasmid.
8. test kit as shown in claim 6, it is characterized in that: described PCR reaction buffer comprises: ALDH2PCR reaction solution 12.5ul, ALDH2 (A) primed probe mixed solution 6.5ul, ALDH2 (G) primed probe mixed solution 6.5ul, purified water 4.0ul, is made into 23ul reaction system altogether; Wherein the consisting of of ALDH2 (A) primed probe mixed solution: ALDH2-A-F1.5ul (10P), ALDH2-FP1.5ul (0.25P), ALDH2-R1.5ul (10P), HER2-F0.5ul (10P), HER2-R0.5ul (10P), HER2-FP1.0ul (0.25P), consisting of of ALDH2 (G) primed probe mixed solution: ALDH2-G-F1.5ul, ALDH2-FP1.5ul, ALDH2-R1.5ul, HER2-F0.5ul, HER2-R0.5ul, HER2-FP1.0ul.
9. test kit as shown in claim 8, is characterized in that: PCR reaction conditions is such as following table:
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