CN105671153A - CYP2C9*3 detection parting kit based on probe AllGlo and parting method of CYP2C9*3 detection parting kit - Google Patents
CYP2C9*3 detection parting kit based on probe AllGlo and parting method of CYP2C9*3 detection parting kit Download PDFInfo
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- CN105671153A CN105671153A CN201610083701.5A CN201610083701A CN105671153A CN 105671153 A CN105671153 A CN 105671153A CN 201610083701 A CN201610083701 A CN 201610083701A CN 105671153 A CN105671153 A CN 105671153A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a CYP2C9*3 detection parting kit based on a probe AllGlo and a parting method of the CYP2C9*3 detection parting kit and relates to single nucleotide polymorphism (SNP). The kit comprises a real-time fluorescence quantification PCR reagent, positive control and negative control. CYP2C9*3 is an SNP locus in a CYP2C9 gene of human chromosome 10 provided by NCBI. The parting method comprises the steps that firstly, DNA in an EDTA anti-freezing whole blood sample is extracted through a conventional method; secondly, the real-time fluorescence quantification PCR reagent provided by the CYP2C9*3 detection parting kit based on the probe AllGlo is used for conducting real-time fluorescence quantification PCR amplification on the DNA; thirdly, parting is conducted on the CYP2C9*3 locus of the human gene CYP2C9 according to detected fluorescence signals. On the premise of keeping high specificity and sensitivity of the probe AllGlo, the detecting price is lower than that of a direct sequencing method, the process is simpler, and consumed time is shorter.
Description
Technical field
The present invention relates to single nucleotide polymorphism (SNP), especially relate to a kind of CYP2C9*3 detection and genotyping test kit based on AllGlo probe and classifying method thereof.
Background technology
Single nucleotide polymorphism (singlenucleotidepolymorphism, SNP), the DNA sequence polymorphism being primarily referred to as in genomic level caused by the variation of single core thuja acid. It is modal one in the heritable variation of the mankind. SNPs is widely present in human genome, on average just has 1 in every 500～1000 base pairs, estimates that its sum is even more up to 3,000,000. As third generation genetic marker, SNP is closely related with the many phenotypic differences of human body, drug sensitivity and disease susceptibility. The SNP a large amount of existence in genome so that it is become a strong instrument, have played very important effect in disease location and clone, the design of medicine and test and biological basic research.
Individual Diagnosis treatment is the general orientation of future medicine development. The target spot of individuation is positioned certain gene or even single core thuja acid, it has been found that the genetic marker of disease, it will help carry out disease prevention according to the different genetic backgrounds of each patient, diagnose and treat. The SNP of gene is the important genetic base forming interindividual variation, correlation analysis by SNP with disease and Drug therapy, make doctor not only by detected SNP prediction, determine the gene relevant with disease, also can will hold the individual patients reaction characteristics for certain medicine in advance simultaneously, and selecting therapeutic effect best according to this feature, dangerous minimum drugs on patients precisely treats for untoward reaction etc.
SNP plays important role in the prevention of disease, diagnosis, treatment and individuality medicine develop, and therefore carries out SNP detection and genotyping quickly and accurately and is significant.
At present, the method for SNP typing mainly has direct Sequencing technology law, restriction fragment length polymorphism technology (RFLP), Tagman fluorescence probe method, amplification refractory mutation system (ARMS), high-resolution fusion curve analytic process (HRM) etc.Wherein, direct sequencing is at present directly perceived, the SNP classifying method that accuracy is the highest comparatively speaking, but its step is many and disperse, and relatively costly, workload is big, and the cycle is long, expensive, is not suitable for the detection of large sample multidigit point; Restriction fragment length polymorphism technology (RFLP) is as a kind of DNA marker technology the earliest, instrument requirements is low, it is only necessary to a PCR instrument and electrophoresis apparatus can be tested, but its experiment complex operation, the detection cycle is long, is not suitable for large sample detection; Tagman fluorescence probe method accuracy is better, but its design synthesis program is complicated, and is not suitable for the typing of a small amount of sample in many sites, the site that especially frequency is on the low side; Amplification refractory mutation system (ARMS method) is quick, easy, but can not operate by stopped pipe, and Polymorphism Analysis is difficult to high flux, automatization; High-resolution fusion curve analytic process (HRM) has the advantages such as simple, quick, but the method specificity is not enough, and apparatus selection is few.
AllGlo probe, as the fluorescent probe of a new generation, is possessing on the basis of TaqMan-MGB probe advantage, is greatly improved signal to noise ratio, reduces background fluorescence signal. At present, AllGlo probe has been successfully applied to detection herpesvirus, hepatitis B virogene sudden change (Feng, Z.L.Yu, X.Y.Lu, Z.M.Geng, D.Y.Zhang, L.Chen, S.J.RapiddetectionofthehepatitisBvirusYMDDmutantusingAll GloTNProbes [J] .ClinChimActa, 2011,412 (11-12): 1018-1021), invasive aspergillosis (Wu, D.S.Shen, J.Z.Zhou, X.Q.Shen, S.F.Wu, X.M.TheestablishmentandevaluationofdiagnosticaccuracyofA llGlo (TM) probe-basedtechniquesforinvasiveaspergillosis [J] .ZhonghuaNeiKeZaZhi, 2010,49 (2): 142-145).
Warfarin is a kind of anticoagulant of wide clinical application, can be effectively used for the thrombus prevention of prosthetic valve replacement and Atrial Fibrillation. The treatment window narrower due to it and the individual treatment dose difference being widely present, therefore maintenance one safely and effectively warfarin treatment dosage is the key of clinical success treatment. CYP2C9 is a kind of enzyme that can inactivate warfarin, research finds that CYP2C9*3 may result in CYP2C9 and reduces the enzymatic activity of 80%, thus may result in interindividual warfarin dose therapeutically effective difference (TakanashiK, TainakaH, KobayashiK, YasumoriT, HosakawaM, ChibaK.CYP2C9Ile359andLeu359variants:enzymekineticstudyw ithsevensubstrates.Pharmacogenetics.2000; 10 (2): 95 104). Many researchs clearly prove, carrying the patient in CYP2C9*3 allelic mutation site only needs the warfarin of low dosage just can maintain stable therapeutic effect, therefore before medication, patient is carried out the detection and genotyping of CYP2C9*3, suitable therapeutic dose can be provided for patient, avoid dosage not enough or excessively medication, provide important reference frame for clinical treatment medication. Under the background that Individual Diagnosis is fast-developing with treatment, CYP2C9*3 and being closely connected of warfarin individual dose difference have become the SNP site of great medical science potentiality, are therefore badly in need of a kind of more rapid efficient SNP classifying method to meet the development of accurate medical science.
Summary of the invention
An object of the present invention is in that for the drawbacks described above that the test kit used in existing detection SNP method and detection method exist, it is provided that based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe.
The two of the purpose of the present invention are in that to provide the detection and genotyping method of the CYP2C9*3 based on AllGlo probe.
Described based on AllGlo probe CYP2C9*3 detection and genotyping test kit, including real-time fluorescence quantitative PCR reagent, positive control and negative control;
SNP site in described CYP2C9*3 is American National Biotechnology Information center (NCBI) No. 10 chromosome CYP2C9 genes of offer people.
Described real-time fluorescence quantitative PCR reagent includes following component: 10 × Taq buffer, 10mmol MgCl2, the TaqHotstartDNA polymerase of 5u/ μ L, 10mmoldNTPs mixed liquor, 50 × LowROX, 100mL nuclease free water, the specific forward primer of 10 μm of ol/LCYP2C9*3, the specific reverse primer of 10 μm of ol/LCYP2C9*3 and AllGlo probe; Described 10 × Taq buffer includes 100mmolTris-HCl and 500mmolKCl.
The nucleotides sequence of the specific forward primer of described CYP2C9*3 is classified as:
SEQIDNO.1:5 ＇-AGCCACATGCCCTACACAGAT-3 ＇;
The nucleotides sequence of the specific reverse primer of described CYP2C9*3 is classified as:
SEQIDNO.2:5 ＇-GAGAAACAAACTTACCTTGGGAATG-3 ＇.
Described AllGlo probe is the specific probe of CYP2C9*3, and its nucleotides sequence is classified as:
SEQIDNO.3:CYP2C9*3-A:MAR-CCAGAGATACATTGAC-MAR;
SEQIDNO.4:CYP2C9*3-C:JUP-CCAGAGATACCTTGAC-JUP.
Described positive control includes: isozygoty reference substance 1, the positive of the positive is isozygotied reference substance 2, positive heterozygous control product; The described positive reference substance 1 that isozygotys is the DNA sample of AA for CYP2C9*3 typing, and the described positive reference substance 2 that isozygotys is the DNA sample of CC for CYP2C9*3 typing, and described positive heterozygous control product are CYP2C9*3 typing is the DNA sample of AC.
Described negative control is the nuclease free water of 1mL.
Described based on AllGlo probe CYP2C9*3 detection and genotyping method, its step is as follows:
1) conventional method is adopted to extract the DNA in EDTA anticoagulated whole blood sample;
2) adopt the described real-time fluorescence quantitative PCR reagent provided based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe that DNA is carried out real-time fluorescence quantitative PCR amplification;
3) according to the fluorescence signal detected, people CYP2C9 gene C YP2C9*3 site is carried out typing.
Described AllGlo probe can adopt the fluorescent quantitation probe of A1leLogicBiosciencesCorporation company of the U.S., it has general T aqman, Taqman-MGB and all advantages of molecular beacon (molecularbeacon) probe, overcome the maximum drawback of these several probes at present, it has broken the restriction of reporter group one end, traditional Taqman one end quenching group, make use of the special fluorescent dye of the several frequently seen wavelength that A1leLogicBiosciencesCorporation company of the U.S. develops, it is marked at above oligonucleotide reporter group and essence each other to go out group, and above containing the special chemical group that can improve Tm value (annealing temperature), improve probe specificity, hybrid specificities is greatly improved, after hybridization hydrolysis, the dyestuff of two ends labelling all becomes again reporter group, improve fluorescence increment.
Described AllGlo probe has the advantage that and 1. improves Tm value (up to more than 10 DEG C), and probe is shorter can reach 15～16 bases, it is possible to be suitable for the sequential design probe that A, T comparision contents is high; 2. increasing the selection of multiple fluorescence quantitative, because not only every kind of dyestuff is reporter group but also be quenching group, the Taqman probe that breaks traditions selects difficulty because of wavelength reason labelling, is not limited by quenching group wavelength;3. being greatly improved signal to noise ratio, without background signal, space length is near, better quenching effects; 4. less costly, price just corresponds to the half of Taqman-MGB probe, has MGB probe and is had superiority.
Present invention employs AllGlo probe technique, devise specific primer and corresponding AllGlo probe, probe is 2 kinds of special fluorophors (MAR, JUP) of labelling respectively, and this technology reaction condition is optimized, establish a kind of AllGlo probe SNP detection and genotyping method, have a extensive future.
Beneficial effects of the present invention is as follows:
The invention provides a kind of SNP detection and genotyping test kit based on AllGlo probe and detection method, its specificity and sensitivity high, can quick and precisely carry out SNP typing, have the advantage that compared with existing common technique
1., compared with direct sequencing, AllGlo probe is under keeping the premise of high specific and sensitivity, and detection price is more less expensive than direct sequencing, and process is simpler and easy consuming time shorter.
2., compared with taqman-MGB probe, AllGlo probe adopts identical fluorophor, reporter group and quenching group each other, and the fluorescence signal of release is higher on the one hand, and background signal is lower on the other hand; And synthesis program is simple, price just corresponds to the half of Taqman-MGB.
3. compared with restriction fragment length polymorphism technology (RFLP), SNP classifying method based on AllGlo probe will be greatly shortened in the response time, flux is bigger, and sensitivity and specificity will apparently higher than the SNP classifying method based on restriction enzyme site.
4. compared with amplification refractory mutation system (ARMS), higher based on AllGlo probe SNP classifying method detection sensitivity, it is possible to achieve " stopped pipe operation ", effectively prevent outside contamination, there is good specificity and accuracy.
5. compared with high-resolution fusion curve analytic process (HRM), higher based on AllGlo probe SNP classifying method specificity, and multiple instrument can apply AllGlo probe and carry out SNP detection and genotyping, and available instrumentation selects many, not needing particular technology human users, range of application is broader.
6. the present invention establishes the detection and genotyping method carrying out CYP2C9*3 based on AllGlo probe, and applicable crowd carries out the SNP detection and genotyping of individuation, has promoted SNP in the development of clinical drug research and personalized medicine.
Detailed description of the invention
Embodiment 1
The present invention includes following component based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe: real-time fluorescence quantitative PCR reagent, positive control and negative control.
1. real-time fluorescence quantitative PCR reagent includes following component: 10 × Taq buffer (10 × Taq buffer includes 100mmolTris-HCl and 500mmolKCl), 10mmol MgCl2, the TaqHotstartDNA polymerase of 5u/ μ L, 10mmoldNTPs mixed liquor, 50 × LowROX, 100mL nuclease free water, the specific forward primer of 10 μm of ol/LCYP2C9*3, the specific reverse primer of 10 μm of ol/LCYP2C9*3 and AllGlo probe.
2. positive control includes: isozygoty reference substance 1, the positive of the positive is isozygotied reference substance 2, positive heterozygous control product. The described positive reference substance 1 that isozygotys is the DNA sample of AA for CYP2C9*3 typing, and the described positive reference substance 2 that isozygotys is the DNA sample of CC for CYP2C9*3 typing, and described positive heterozygous control product are CYP2C9*3 typing is the DNA sample of AC.
3. negative control is the nuclease free water of 1mL.
Embodiment 2
The detection and genotyping of peripheral blood CYP2C9*3, comprises the following steps:
1. collect EDTA anticoagulation cirumferential blood:
Extract fresh blood specimen 2mL be loaded on EDTA anticoagulant tube subpackage multitube, often pipe subpackage 400～500 μ L, take 200 μ L for DNA extraction, all the other whole blood samples be placed in-80 DEG C frozen.
2. extract DNA:
Adopt poba gene group DNA extraction kit (DP348) of the production of Tian Gen bio tech ltd, sample (whole blood) DNA extraction is carried out according to operating instruction, 200 μ LEDTA anticoagulated whole bloods operate to specifications and carry out, finally with the elution buffer TB dissolving DNA of 50 μ L, concentration and purity is measured respectively on nucleic acid spectrophotometric instrument NanoDrop2000, take purity A260/A280 ratio between 1.7～1.9, the DNA extracted taking concentration 10～50ng/ μ L detects for SNP typing, and remaining DNA is all frozen in-80 DEG C.
3. real-time fluorescence quantitative PCR amplification:
Each component of quantitative fluorescent PCR is placed in room-temperature dissolution by 3.1, and mixing is placed on ice.
3.2 preparation PCR reaction mixture such as table 1.
Table 1
3.3 in 8 unions subpackage prepare PCR reaction mixture, often pipe adds the DNA of 1 μ L, fully mixes, and whole process carries out on ice, it is to avoid the generation of bubble. One negative control of each Setup Experiments, arranges a positive and isozygotys reference substance 1, and a positive is isozygotied reference substance 2 and positive heterozygous control product;
The multiple instruments such as 3.4 detections carry out on real-time fluorescence quantitative PCR instrument, can use ABI7300,7500 (AppliedBiosystems companies of the U.S.);
3.5 arrange quantitative fluorescent PCR reaction condition such as table 2.
Table 2
4. interpretation of result and process: application ABI instrument carries software and carries out interpretation of result. According to comparison amplification, typing produces three kinds of genotype:
The first genotype: AA, isozygotys reference substance 1 on the same axis with the positive;
The second genotype: AC, with positive heterozygous control product on the same axis;
The third genotype: CC, isozygotys reference substance 2 on the same axis with the positive.
Embodiment 3. tissue samples SNP detection and genotyping
1. tissue samples processes:
Tissue (spleen tissue consumption should lack 10mg) should smash process for cell suspension, then the centrifugal 1min of 10000rpm (～11200 × g), using up supernatant, add 200 μ L buffer GA (sky root DNA extraction kit DP304), concussion is to thoroughly suspending;
2. tissue samples DNA extraction and enrichment:
Adopt the DNA extraction kit (DP304) of the production of Tian Gen bio tech ltd, the DNA extraction of tissue samples is carried out according to operating instruction, finally with the elution buffer TE dissolving DNA of 50 μ L, on nucleic acid spectrophotometric instrument NanoDrop2000, measure concentration and purity respectively;
3. the SNP detection and genotyping of tissue samples.
Subsequent step is with reference to step 3～4 of embodiment 2.
Can range of application:
CYP2C9*3 detection and genotyping test kit and detection method thereof based on AllGlo probe can be applicable to human tissue sample and the detection and genotyping of hemocyte CYP2C9*3, and the ultimate principle of the present invention, principal character and advantages of the present invention have more than been shown and described.
Claims (8)
1. based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe, it is characterised in that include real-time fluorescence quantitative PCR reagent, positive control and negative control;
Described CYP2C9*3 is the SNP site in No. 10 chromosome CYP2C9 genes of the provided people in American National Biotechnology Information center.
2. as claimed in claim 1 based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe, it is characterised in that described real-time fluorescence quantitative PCR reagent includes following component: 10 × Taq buffer, 10mmol MgCl2, the TaqHotstartDNA polymerase of 5u/ μ L, 10mmoldNTPs mixed liquor, 50 × LowROX, 100mL nuclease free water, the specific forward primer of 10 μm of ol/LCYP2C9*3, the specific reverse primer of 10 μm of ol/LCYP2C9*3 and AllGlo probe;Described 10 × Taq buffer includes 100mmolTris-HCl and 500mmolKCl.
3. as claimed in claim 1 based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe, it is characterised in that the nucleotides sequence of the specific forward primer of described CYP2C9*3 is classified as:
SEQIDNO.1:5 ＇-AGCCACATGCCCTACACAGAT-3 ＇;
The nucleotides sequence of the specific reverse primer of described CYP2C9*3 is classified as:
SEQIDNO.2:5 ＇-GAGAAACAAACTTACCTTGGGAATG-3 ＇.
4. as claimed in claim 1 based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe, it is characterised in that described AllGlo probe is the specific probe of CYP2C9*3, and its nucleotides sequence is classified as:
SEQIDNO.3:CYP2C9*3-A:MAR-CCAGAGATACATTGAC-MAR;
SEQIDNO.4:CYP2C9*3-C:JUP-CCAGAGATACCTTGAC-JUP.
5. as claimed in claim 1 based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe, it is characterised in that described positive control includes: isozygoty reference substance 1, the positive of the positive is isozygotied reference substance 2, positive heterozygous control product; The described positive reference substance 1 that isozygotys is the DNA sample of AA for CYP2C9*3 typing, and the described positive reference substance 2 that isozygotys is the DNA sample of CC for CYP2C9*3 typing, and described positive heterozygous control product are CYP2C9*3 typing is the DNA sample of AC.
6. as claimed in claim 1 based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe, it is characterised in that described negative control is the nuclease free water of 1mL.
7. based on AllGlo probe CYP2C9*3 detection and genotyping method, it is characterised in that its step is as follows:
1) conventional method is adopted to extract the DNA in EDTA anticoagulated whole blood sample;
2) adopt the real-time fluorescence quantitative PCR reagent provided such as CYP2C9*3 detection and genotyping test kit based on AllGlo probe as described in arbitrary in claim 1～6 that DNA is carried out real-time fluorescence quantitative PCR amplification;
3) according to the fluorescence signal detected, people CYP2C9 gene C YP2C9*3 site is carried out typing.
8. as claimed in claim 1 based on the CYP2C9*3 detection and genotyping test kit of AllGlo probe, it is characterised in that described AllGlo probe adopts the fluorescent quantitation probe of A1leLogicBiosciencesCorporation company of the U.S..
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