CN105671143A - Kit for detecting human COX-1 gene polymorphism and application thereof - Google Patents
Kit for detecting human COX-1 gene polymorphism and application thereof Download PDFInfo
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- CN105671143A CN105671143A CN201510581647.2A CN201510581647A CN105671143A CN 105671143 A CN105671143 A CN 105671143A CN 201510581647 A CN201510581647 A CN 201510581647A CN 105671143 A CN105671143 A CN 105671143A
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- 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
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- 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
- C12Q1/6851—Quantitative amplification
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Abstract
The invention relates to a kit for detecting human COX-1 gene polymorphism and an application thereof. The kit includes the following substances: a specific primer and a specific fluorescent probe for detecting an rs1330344 SNP locus on COX-1 gene, a Taq DNA polymerase, a dNTP mixed liquid, a MgCl2 solution, a fluorescent quantitative PCR reaction buffer liquid and deionized water. The kit overcomes the defects in various gene mutation detection methods, is high in detection accuracy, is simple and convenient, is short in detection time and is simple in result analysis. The kit is suitable for clinical laboratories and can perform qualitative detection to COX-1 gene polymorphic sites, wherein a PCR fluorescent amplification reaction is carried out according to the SNP locus. A result can be determined just on the basis of whether two different fluorescent curves are positive or not, so that the kit is free of manual error, is low in false positive and false negative rate, and is improved in efficiency and reduced in detection cost.
Description
Technical field
The present invention relates to the technique of gene detection in biomedical sector clinical detection technique, in particular to a kind of test kit and the application thereof that detect mankind's COX-1 gene pleiomorphism.
Background technology
Epoxide hydrolase (cyclo-oxygen-ase, COX) is that prostaglandin(PG) (PGs) synthesizes necessary enzyme, is also that PGs synthesizes the key rate-limiting enzyme in initial step. COX has two kinds of isozyme COX-1 and COX-2. COX-1 is that structure-type is called as key element enzyme or house keeper's enzyme; mainly it is present in the tissues such as blood vessel, stomach, kidney; home and abroad scholar generally thinks that the PG that it produces participates in body normal physiological processes and protection function, as maintained gastrointestinal mucosa integrity, regulate platelet function and renal hemodynamic. Human body COX-1 gene is positioned on karyomit(e) 9q32～9q33.3, long about 22kb, comprises 11 exons, encodes 576 amino acid altogether. COX-1 gene mononucleotide polymorphism phenomenon (such as No. rs1330344), the change of base replacement and promotor connecting portion can be caused, the function of remarkably influenced intron or exon, change the conformation of COX-1 albumen so that it is to the susceptibility extremely inequality one of the resistance effect that drugs with function (acetylsalicylic acid etc.) produces.
Before the present invention makes, the method carrying out detection in Gene Mutation at present has tens of kinds. Gold standard method is gene sequencing method. Gene sequencing method is current most widely used general, one method more accurately, but the personnel that the method needs expensive plant and instrument and specialty operate, and waste time and energy, are difficult to clinically promote. Gene chips has the advantage of fast high-flux, but operation is upper complicated, and step is many, and cost height is also not easily popularized. The method detecting transgenation with Restrictive fragment length polymorphism (RFLP) has advantage with low cost, but process is loaded down with trivial details, and time length also is not suitable for popularizing.
Summary of the invention
The object of the present invention just is to overcome above-mentioned defect, develops a kind of test kit and the application thereof that detect mankind's COX-1 gene pleiomorphism.
The technical scheme of the present invention is:
A kind of test kit detecting mankind's COX-1 gene pleiomorphism, the material comprising box body and be loaded in box body, its main technology is characterised in that: the material in described box body comprises Auele Specific Primer and specificity fluorescent probe, Taq DNA polymerase, dNTP mixed solution, the MgCl of the rs1330344 SNP site on detection COX-1 gene2Solution, quantitative fluorescent PCR reaction buffer and deionized water;
Sense primer and the sequence of antisense primer in described Auele Specific Primer be:
Sense primer: 5 '-CTTGTTTAAGGTCACGCTATGG-3 ',
Antisense primer: 5 '-GTGCCTCCTGCTGACTTCC-3 ';
Described specificity fluorescent probe comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-AGGCTCTTCCCATGAGT-MGB-3 '
Saltant type probe: 5 '-HEX-AGGCTCTTCCCATAAGT-MGB-3 '.
In described box body, the content of each material is: concentration is the Auele Specific Primer totally 0.45 μ l of 10 μMs, wherein sense primer and each 0.225 μ l of antisense primer; Concentration is the specificity fluorescent probe totally 0.2 μ l of 5 μMs, wherein wild-type probe and each 0.1 μ l of saltant type probe; Concentration is the Taq DNA polymerase 0.02 μ l of 5 units/μ l; Concentration is the dNTP mixed solution 0.1 μ l of 20mM; Concentration is the MgCl2 solution 0.5 μ l of 25mM; 5X quantitative fluorescent PCR reaction buffer 2 μ l; Deionized water 4.73 μ l.
In described Auele Specific Primer, the Tm value of sense primer is 57 DEG C, and the Tm value of antisense primer is 57.1 DEG C; In described specificity fluorescent probe, the Tm value of wild-type probe is 69 DEG C, and the Tm value of saltant type probe is 69 DEG C.
The storage temperature of described test kit is-20 DEG C.
Another technical scheme of the present invention is:
Detecting an application for the test kit of mankind's COX-1 gene pleiomorphism, its thing is levied and is: described test kit is for detecting the genotype of rs1330344 SNP site on COX-1 gene.
Compared with prior art, advantage is detection accuracy height, it is simple and convenient to detect, detection time is short, the simple advantage of result analysis in the present invention, is applicable to clinical labororatory and uses:
(1) test kit of the present invention is utilized COX-1 gene polymorphism sites can be carried out qualitative detection, PCR amplified fluorescence reaction is carried out according to SNP site, only whether need to play line according to two kinds of different fluorescence curves just can carry out result judgement, personal errors can not be produced, false positive and false negative rate are low, it is to increase degradation in efficiency testing cost.
(2) without the need to DNA extraction, only need cracking complete blood cell, the suspension after lysis is added reaction system and can complete amplification, remove the step of DNA extraction and cost from and avoid aerosol to pollute, it is to increase detection efficiency and accuracy, shortening detection time.
(3) fluorescent detection probe that test kit of the present invention uses is Taqman probe, and this probe expense is cheap; The omnidistance stopped pipe operation of reaction that the present invention simultaneously is all, PCR does not need to carry out PCR primer purifying after reacting, electrophoresis, enzyme are cut, hybridization etc., while saving testing cost, substantially reduce sense cycle, improve the efficiency of detection, reduce PCR primer in addition and pollute the risk causing false positive.
Accompanying drawing explanation
Rs1330344 SNP site homozygous wildtype amplification curve schematic diagram on Fig. 1 COX-1 gene.
Rs1330344 SNP site heterozygous amplification curve schematic diagram on Fig. 2 COX-1 gene.
Rs1330344 SNP site homozygous mutation type amplification curve schematic diagram on Fig. 3 COX-1 gene.
Embodiment
Doing embodiments of the invention to illustrate in detail below, the present embodiment is implemented under premised on technical solution of the present invention, gives detailed enforcement mode and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Detect a test kit for mankind's COX-1 gene pleiomorphism, the specific amplification primer 1 that test kit comprises rs1330344 SNP site on following COX-1 gene to and 2 specificity fluorescent probes.
On COX-1 gene, the sequence of the sense primer in the Auele Specific Primer of rs1330344 SNP site detection and antisense primer is:
Sense primer: 5 '-CTTGTTTAAGGTCACGCTATGG-3 ',
Antisense primer: 5 '-GTGCCTCCTGCTGACTTCC-3 ';
Described specificity fluorescent probe comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-AGGCTCTTCCCATGAGT-MGB-3 '
Saltant type probe: 5 '-HEX-AGGCTCTTCCCATAAGT-MGB-3 '.
Described a kind of test kit detecting mankind's COX-1 gene locus polymorphism, also comprises Taq DNA polymerase, dNTP mixed solution, MgCl2Solution, quantitative fluorescent PCR reaction buffer and deionized water.
Described wild-type specificity fluorescent probe is Taqman probe, and the fluorophor that this probe 5 ' is held is FAM, and the 3 ' quenching group held is MGB.
Described saltant type specificity fluorescent probe is Taqman probe, and the fluorophor that this probe 5 ' is held is HEX, and the 3 ' quenching group held is MGB.
Test kit forms
The configuration composition of test kit coordinates configuration, it may also be useful to time only needs to add sample and can complete experimental configuration, and this kind of configuration is more convenient, is preferred disposition.
Complex reaction liquid composition forms:
| Composition | Volume |
| The Auele Specific Primer of 10 μMs | 0.45μl |
| Sense primer and antisense primer | 0.225μl |
| The specificity fluorescent probe of 5 μMs | 0.2μl |
| The Taq archaeal dna polymerase of 5 units/μ l | 0.02μl |
| The dNTP mixed solution of 20mM | 0.1μl |
| The MgCl2 of 25mM | 0.5μl |
| 5X quantitative fluorescent PCR reaction buffer | 2μl |
| Deionized water | 4.73μl |
Use the operation steps of test kit
Step 1: the preparation of complete blood cell lysate
Get person under inspection's peripheric venous blood 300 μ l, add 700 μ l cell pyrolysis liquids, put upside down mixed even 5 times, centrifugal 1 minute of 12000rpm, remove supernatant, and centrifuge tube is upside down on clean thieving paper and stops 2 minutes, it is ensured that be deposited in pipe, adding 300 μ l distilled waters, vortex concussion is mixed into lysis suspension.
Step 2: quantitative fluorescent PCR reacts
Quantitative fluorescent PCR reaction system cumulative volume is 10 μ l, comprising: the Auele Specific Primer totally 0.45 μ l that lysis suspension 2 μ l that concentration is 20ng/ μ l, concentration are 10 μMs, wherein sense primer and each 0.225 μ l of antisense primer; Concentration is the specificity fluorescent probe totally 0.2 μ l of 5 μMs, wherein wild-type probe and each 0.1 μ l of saltant type probe; Concentration is the Taq DNA polymerase 0.02 μ l of 5 units/μ l; Concentration is the dNTP mixed solution 0.1 μ l of 20mM; Concentration is the MgCl2 solution 0.5 μ l of 25mM; 5X quantitative fluorescent PCR reaction buffer 2 μ l; Deionized water 4.73 μ l.
ABI7500 type quantitative real time PCR Instrument reacts, reaction conditions is: 2min at 50 DEG C, 10min at 95 DEG C, then carries out 40～55 PCR cycle (15s at 92 DEG C, 1min at 57 DEG C), reaction reads fluorescence volume at ABI7900HT type quantitative real time PCR Instrument after terminating.
Step 3: gene type assay and determine genotype
ABI7500 type quantitative real time PCR Instrument is adopted to carry software SDS (SequenceDetectionSystems) V2.0, according to the FAM fluorescence in detection reaction pipe and HEX fluorescence PGR curve, judging the somatotype of rs1330344 SNP site on COX-1 gene, the concrete foundation of somatotype sees the following form.
Rs1330344 SNP site somatotype foundation on COX-1 gene
| PCR reaction tubes curve amplification | FAM fluorescence | HEX fluorescence |
| Homozygous wildtype | Positive (playing line) | Negative (not playing line) |
| Heterozygous | Positive (playing line) | Positive (playing line) |
| Homozygous mutation type | Negative (not playing line) | Positive (playing line) |
As shown in Figure 1, fluorescent quantitative PCR curve only FAM fluorescence play line, HEX fluorescence does not play line, is rs1330344 SNP site homozygous wildtype on COX-1 gene.
As shown in Figure 2, fluorescent quantitative PCR curve FAM and HEX2 bar fluorescence all play line, are rs1330344 SNP site heterozygous on COX-1 gene.
As shown in Figure 3, fluorescent quantitative PCR curve only HEX fluorescence play line, FAM fluorescence does not play line, is rs1330344 SNP site homozygous mutation type on COX-1 gene.
Obviously, above-described embodiment is only the example done for clearly illustrating, and not to the restriction of the mode of enforcement. For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description. Here without the need to also cannot all enforcement modes be given exhaustive. And the apparent change therefore amplified or variation are still within the protection domain of the invention.
Claims (5)
1. one kind is detected the test kit of mankind's COX-1 gene pleiomorphism, the material comprising box body and be loaded in box body, it is characterised in that: the material in described box body comprises Auele Specific Primer and specificity fluorescent probe, Taq DNA polymerase, dNTP mixed solution, the MgCl of the rs1330344 SNP site on detection COX-1 gene2Solution, quantitative fluorescent PCR reaction buffer and deionized water;
Sense primer and the sequence of antisense primer in described Auele Specific Primer be:
Sense primer: 5 '-CTTGTTTAAGGTCACGCTATGG-3 ',
Antisense primer: 5 '-GTGCCTCCTGCTGACTTCC-3 ';
Described specificity fluorescent probe comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-AGGCTCTTCCCATGAGT-MGB-3 '
Saltant type probe: 5 '-HEX-AGGCTCTTCCCATAAGT-MGB-3 '.
2. a kind of test kit detecting mankind's COX-1 gene pleiomorphism as claimed in claim 1, it is characterized in that: in described box body, the content of each material is: concentration is the Auele Specific Primer totally 0.45 μ l of 10 μMs, wherein sense primer and each 0.225 μ l of antisense primer; Concentration is the specificity fluorescent probe totally 0.2 μ l of 5 μMs, wherein wild-type probe and each 0.1 μ l of saltant type probe; Concentration is the Taq DNA polymerase 0.02 μ l of 5 units/μ l; Concentration is the dNTP mixed solution 0.1 μ l of 20mM; Concentration is the MgCl2 solution 0.5 μ l of 25mM; 5X quantitative fluorescent PCR reaction buffer 2 μ l; Deionized water 4.73 μ l.
3. a kind of test kit detecting mankind's COX-1 gene pleiomorphism as claimed in claim 1, it is characterised in that: in described Auele Specific Primer, the Tm value of sense primer is 57 DEG C, and the Tm value of antisense primer is 57.1 DEG C; In described specificity fluorescent probe, the Tm value of wild-type probe is 69 DEG C, and the Tm value of saltant type probe is 69 DEG C.
4. a kind of test kit detecting mankind's COX-1 gene pleiomorphism as claimed in claim 1, it is characterised in that: the storage temperature of described test kit is-20 DEG C.
5. detecting an application for the test kit of mankind's COX-1 gene pleiomorphism, its thing is levied and is: described test kit is for detecting the genotype of rs1330344 SNP site on COX-1 gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510581647.2A CN105671143A (en) | 2015-09-10 | 2015-09-10 | Kit for detecting human COX-1 gene polymorphism and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510581647.2A CN105671143A (en) | 2015-09-10 | 2015-09-10 | Kit for detecting human COX-1 gene polymorphism and application thereof |
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| CN105671143A true CN105671143A (en) | 2016-06-15 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277413A (en) * | 2010-06-08 | 2011-12-14 | 广州益善生物技术有限公司 | Specific primer and liquid-phase chip for SNP detection of COX-1 genes |
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- 2015-09-10 CN CN201510581647.2A patent/CN105671143A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277413A (en) * | 2010-06-08 | 2011-12-14 | 广州益善生物技术有限公司 | Specific primer and liquid-phase chip for SNP detection of COX-1 genes |
Non-Patent Citations (2)
| Title |
|---|
| MARI´A ASUNCIO´N GARCI´A-GONZA´ LEZ ET AL.: "rognostic Role of Host Cyclooxygenase and Cytokine Genotypes in a Caucasian Cohort of Patients with Gastric Adenocarcinoma", 《PLOS ONE》 * |
| WWW.LIFETECHNOLOGIES.COM: "TaqMan® SNP Genotyping Assays", 《WWW.LIFETECHNOLOGIES.COM》 * |
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