CN105671142A - Kit for detecting human PEAR1 gene polymorphism and application thereof - Google Patents

Kit for detecting human PEAR1 gene polymorphism and application thereof Download PDF

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Publication number
CN105671142A
CN105671142A CN201510581594.4A CN201510581594A CN105671142A CN 105671142 A CN105671142 A CN 105671142A CN 201510581594 A CN201510581594 A CN 201510581594A CN 105671142 A CN105671142 A CN 105671142A
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probe
primer
pear1
pear1 gene
type probe
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徐运
邵渊
张希根
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Ren Tian Bio Tech Ltd Nanjing
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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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

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Abstract

The invention relates to a kit for detecting human PEAR1 gene polymorphism and an application thereof. The kit includes the following substances: a specific primer and a specific fluorescent probe for detecting an rs2768759 locus on PEAR1 gene, a specific primer and a specific fluorescent probe for detecting an rs12041331 locus on the PEAR1 gene, a Taq DNA polymerase, 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 the two polymorphic sites, the rs2768759 and the rs12041331, on the PEAR1 gene, 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 without manual error, so that the kit is improved in efficiency and reduced in detection cost. The kit is free of DNA extraction, wherein a suspension liquid after cell pyrolysis is added to the reaction system to complete amplification.

Description

A kind of test kit and application thereof detecting mankind's PEAR1 gene pleiomorphism
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 PEAR1 gene pleiomorphism.
Background technology
Platelet-endothelial assembles acceptor 1 (PEAR1), and a kind of new discovery transmembrane protein, take part in induced platelet contact activation process. After collagen acts on thrombocyte, cruel ammonia acid and phosphorylate serine residues in PEAR1 kytoplasm, thus the thrombocyte contacted with each other is formed and more claims stable thrombus. PEAR1 can regulate CRP-XL and adenosine diphosphate (ADP) (adenosinediphosphate simultaneously, ADP) path is formed, path is relevant with rs12041331 two SNP site to No. rs2768759 of PEAR1 gene, and its gene pleiomorphism all may affect the antiplatelet effects of medicament for resisting platelet aggregation (such as acetylsalicylic acid etc.). By the detection to PEAR1 gene pleiomorphism, it is possible to better understand patient to the result for the treatment of of medicament for resisting platelet aggregation.
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 PEAR1 gene pleiomorphism.
The technical scheme of the present invention is:
A kind of test kit detecting mankind's PEAR1 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 the Auele Specific Primer in rs2768759 site on detection PEAR1 gene and specificity fluorescent probe, the Auele Specific Primer of rs12041331 SNP site and specificity fluorescent probe, Taq DNA polymerase, dNTP mixed solution, MgCl2Solution, quantitative fluorescent PCR reaction buffer and deionized water;
The sense primer in rs2768759 site-specific primer and the sequence of antisense primer on described PEAR1 gene be:
Sense primer: 5 '-CCACCCTCAGCCCTATCACT-3 ',
Antisense primer: 5 '-AGATTGAATCATGGGAGTGGG-3 ';
Rs12041331 locus specificity fluorescent probe on described PEAR1 gene comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-CACCTGTCCAGTTCC-MGB-3 '
Saltant type probe: 5 '-HEX-CACCTGTCCCGTTCC-MGB-3 ';
The sense primer in rs12041331 site-specific primer and the sequence of antisense primer on described PEAR1 gene be:
Sense primer: 5 '-GGTGAGGGGTTATCCTATGCTAC-3 ',
Antisense primer: 5 '-GATTAGAGTTCCTGGTGGACAAGA-3 ';
Rs2768759 locus specificity fluorescent probe on described PEAR1 gene comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-TCTCACTTCCATCACC-MGB-3 '
Saltant type probe: 5 '-HEX-TCTCACTTCCGTCACC-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 detection rs2768759 site-specific primer, the Tm value of sense primer is 58.4 DEG C, and the Tm value of antisense primer is 58.3 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 68 DEG C; In described detection rs12041331 site-specific primer, the Tm value of sense primer is 58.7 DEG C, and the Tm value of antisense primer is 59.1 DEG C; In described specificity fluorescent probe, the Tm value of wild-type probe is 66 DEG C, and the Tm value of saltant type probe is 66 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 PEAR1 gene pleiomorphism, its thing is levied and is: described test kit is for detecting on PEAR1 gene the genotype of No. rs2768759 and rs12041331 SNP site.
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 No. rs2768759, PEAR1 gene and No. rs12041331 two pleomorphism 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, PGR 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
Rs2768759 SNP site homozygous wildtype amplification curve schematic diagram on Fig. 1 PEAR1 gene.
Rs2768759 SNP site heterozygous amplification curve schematic diagram on Fig. 2 PEAR1 gene.
Rs2768759 SNP site homozygous mutation type amplification curve schematic diagram on Fig. 3 PEAR1 gene.
Rs12041331 SNP site homozygous wildtype amplification curve schematic diagram on Fig. 4 PEAR1 gene.
Rs12041331 SNP site heterozygous amplification curve schematic diagram on Fig. 5 PEAR1 gene.
Rs12041331 SNP site homozygous mutation type amplification curve schematic diagram on Fig. 6 PEAR1 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 PEAR1 gene pleiomorphism, the specific amplification primer 1 that test kit comprises No. rs2768759, following PEAR1 gene and No. rs12041331 two pleomorphism sites to and 2 specificity fluorescent probes.
Sense primer and the sequence of antisense primer in the rs2768759 site-specific primer detected on PEAR1 gene be:
Sense primer: 5 '-CCACCCTCAGCCCTATCACT-3 ',
Antisense primer: 5 '-AGATTGAATCATGGGAGTGGG-3 ';
Rs12041331 locus specificity fluorescent probe for detecting on PEAR1 gene comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-CACCTGTCCAGTTCC-MGB-3 '
Saltant type probe: 5 '-HEX-CACCTGTCCCGTTCC-MGB-3 '.
Sense primer and the sequence of antisense primer in the rs12041331 site-specific primer detected on PEAR1 gene be:
Sense primer: 5 '-GGTGAGGGGTTATCCTATGCTAC-3 ',
Antisense primer: 5 '-GATTAGAGTTCCTGGTGGACAAGA-3 ';
Rs2768759 locus specificity fluorescent probe for detecting on PEAR1 gene comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-TCTCACTTCCATCACC-MGB-3 '
Saltant type probe: 5 '-HEX-TCTCACTTCCGTCACC-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 fluorescent PCR curve, judging on PEAR1 gene the somatotype of No. rs2768759 and rs12041331 two gene locuss, the concrete foundation of somatotype sees the following form.
No. rs2768759 and rs12041331 two SNP site somatotype foundations on PEAR1 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 rs2768759 SNP site homozygous wildtype on PEAR1 gene.
As shown in Figure 2, fluorescent quantitative PCR curve FAM and HEX2 bar fluorescence all play line, are rs2768759 SNP site heterozygous on PEAR1 gene.
As shown in Figure 3, fluorescent quantitative PCR curve only HEX fluorescence play line, FAM fluorescence does not play line, is rs2768759 SNP site homozygous mutation type on PEAR1 gene.
As shown in Figure 4, fluorescent quantitative PCR curve only FAM fluorescence play line, HEX fluorescence does not play line, is rs12041331 SNP site homozygous wildtype on PEAR1 gene.
As shown in Figure 5, fluorescent quantitative PCR curve FAM and HEX2 bar fluorescence all play line, are rs12041331 SNP site heterozygous on PEAR1 gene.
As shown in Figure 6, fluorescent quantitative PCR curve only HEX fluorescence play line, FAM fluorescence does not play line, is rs12041331 SNP site homozygous mutation type on PEAR1 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 PEAR1 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 No. rs2768759 on detection PEAR1 gene and rs12041331 two SNP site2Solution, quantitative fluorescent PCR reaction buffer and deionized water;
The sense primer in rs2768759 site-specific primer and the sequence of antisense primer on described PEAR1 gene be:
Sense primer: 5 '-CCACCCTCAGCCCTATCACT-3 ',
Antisense primer: 5 '-AGATTGAATCATGGGAGTGGG-3 ';
Rs12041331 locus specificity fluorescent probe on described PEAR1 gene comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-CACCTGTCCAGTTCC-MGB-3 '
Saltant type probe: 5 '-HEX-CACCTGTCCCGTTCC-MGB-3 ';
The sense primer in rs12041331 site-specific primer and the sequence of antisense primer on described PEAR1 gene be:
Sense primer: 5 '-GGTGAGGGGTTATCCTATGCTAC-3 ',
Antisense primer: 5 '-GATTAGAGTTCCTGGTGGACAAGA-3 ';
Rs2768759 locus specificity fluorescent probe on described PEAR1 gene comprises wild-type probe and mutagenicity probe, and their sequence is:
Wild-type probe: 5 '-FAM-TCTCACTTCCATCACC-MGB-3 '
Saltant type probe: 5 '-HEX-TCTCACTTCCGTCACC-MGB-3 '.
2. a kind of test kit detecting mankind's PEAR1 gene pleiomorphism as claimed in claim 1, detect No. rs2768759 and rs12041331 two SNP site respectively, 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; The quantitative PGR reaction buffer 2 μ l of 5X fluorescence; Deionized water 4.73 μ l.
3. a kind of test kit detecting mankind's PEAR1 gene pleiomorphism as claimed in claim 1, it is characterised in that: in described detection rs2768759 site-specific primer, the Tm value of sense primer is 58.4 DEG C, and the Tm value of antisense primer is 58.3 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 68 DEG C; In described detection rs12041331 site-specific primer, the Tm value of sense primer is 58.7 DEG C, and the Tm value of antisense primer is 59.1 DEG C; In described specificity fluorescent probe, the Tm value of wild-type probe is 66 DEG C, and the Tm value of saltant type probe is 66 DEG C.
4. a kind of test kit detecting mankind's PEAR1 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 PEAR1 gene pleiomorphism, its thing is levied and is: described test kit is for detecting on PEAR1 gene the genotype of No. rs2768759 and rs12041331 two SNP site.
CN201510581594.4A 2015-09-10 2015-09-10 Kit for detecting human PEAR1 gene polymorphism and application thereof Pending CN105671142A (en)

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Cited By (6)

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CN108949964A (en) * 2018-08-21 2018-12-07 潍坊德诺泰克生物科技有限公司 For detecting the primed probe group and its application of rs12041331
CN109207583A (en) * 2018-10-22 2019-01-15 南通中科基因医学检验所有限公司 Aspirin personalized medicine gene detecting kit and detection method
CN109306377A (en) * 2018-05-21 2019-02-05 上海迈浦生物科技有限公司 SNaPShot serotype specific primer and its detection method
CN111154842A (en) * 2020-02-20 2020-05-15 圣湘生物科技股份有限公司 Composition, kit and method for detecting polymorphism of aspirin resistance gene of human
CN111893173A (en) * 2020-07-22 2020-11-06 福州艾迪康医学检验所有限公司 Primer, method and kit for detecting PEAR1 SNP locus
WO2023001304A1 (en) * 2021-07-22 2023-01-26 上海循曜生物科技有限公司 Drug for preventing, alleviating or treating mucosal adhesion and use thereof

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109306377A (en) * 2018-05-21 2019-02-05 上海迈浦生物科技有限公司 SNaPShot serotype specific primer and its detection method
CN108949964A (en) * 2018-08-21 2018-12-07 潍坊德诺泰克生物科技有限公司 For detecting the primed probe group and its application of rs12041331
CN109207583A (en) * 2018-10-22 2019-01-15 南通中科基因医学检验所有限公司 Aspirin personalized medicine gene detecting kit and detection method
CN111154842A (en) * 2020-02-20 2020-05-15 圣湘生物科技股份有限公司 Composition, kit and method for detecting polymorphism of aspirin resistance gene of human
CN111154842B (en) * 2020-02-20 2023-04-14 圣湘生物科技股份有限公司 Composition, kit and method for detecting polymorphism of aspirin resistance gene of human
CN111893173A (en) * 2020-07-22 2020-11-06 福州艾迪康医学检验所有限公司 Primer, method and kit for detecting PEAR1 SNP locus
WO2023001304A1 (en) * 2021-07-22 2023-01-26 上海循曜生物科技有限公司 Drug for preventing, alleviating or treating mucosal adhesion and use thereof

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Application publication date: 20160615