CN107119107A - A kind of method and kit for detecting mankind's CYP2C19 gene pleiomorphisms - Google Patents
A kind of method and kit for detecting mankind's CYP2C19 gene pleiomorphisms Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The present invention provides a kind of method of new detection mankind's CYP2C19 gene pleiomorphisms, comprises the following steps：(1) ARMS primers are designed, 3 ' ends of the ARMS primers are mutational site, and by any bit missense mutation in the nucleotides of mutational site upstream the 1st 3；(2) after the design allele ARMS primers for each SNP, one section is introduced with human genome sequencing without homologous artificial sequence oligodeoxynucleotide at 5 ' ends of two specific ARMS primers respectively；(3) according to the general artificial sequence of the introducing, the Taqman probes that design one is matched with universal sequence intermediate segment sequence complete complementary, the specific fragment of specific recognition ARMS primers amplification；The detection method and kit of the present invention can greatly increase the ability that allele-specific primers distinguishes not iso-allele；Ensure to greatly save cost while testing result accuracy, improve efficiency.
Description
Technical field
The present invention relates to a kind of gene pleiomorphism detecting method, especially a kind of detection mankind's CYP2C19 gene pleiomorphisms
Method and kit.
Background technology
1.CYP2C19 gene brief introductions
Cytochrome P 450 Enzyme (Cytochome P450, CYP450) is one group of structurally and functionally related superfamily base
It is one group of enzyme containing heme because of the isodynamic enzyme family of coding.It is primarily involved in medicine I associated metabolic, 80% medicine generation
Thank associated therewith.Wherein CYP2 is maximum family, there is 15 subfamilies (CYP2A-2Q), about 20% clinical metabolic medicine
Through its metabolism, CYP2C19 is one of most important drug metabolic enzyme in CYP2 families, many Endogenous Substrates, and clinically
About 2% medicine is all metabolized by its catalysis.
The CYP2C19 assignments of genes gene mapping are in No. 10 chromosome q24.1-q24.3 zone of people, and it is all sequentially comprising aobvious outside 9
Son, encodes 490 amino acid.CYP2C19 enzymes are also known as S- mephenytoin hydroxylases, it has now been found that it at least has 14 kinds of mutation
Gene, 28 kinds of allelotypes.The gene for wherein encoding normal enzymatic activity is CYP2C19*1, and CYP2C19 allele is main
It is * 1, * 2, * 3......*17.And CYP2C19*2 and CYP2C19*3 allele accounts for the weak metabolic phenotype of Asians (PM)
More than 99%.At the kinase inactive of * 2, * 3 allele coding, the extron 5 the 681st of CYP2C19*2 allelic variations
Base morph (G/A) form an aberrant splicing point so that CYP2C19 enzymes lose catalytic activity, thus cause
Incidence of the slow metabolism in Chinese be about 30%.CYP2C19*3 allele is in the 636th generation G/A of extron 4
Mutation, generates termination codon in advance, and albumen synthesis is terminated, and loses CYP2C19 enzymatic activitys.
Research shows that 99% Asians poor metabolizer and 88% can be explained in two kinds of mutation of CYP2C19*2 and CYP2C19*3
White people poor metabolizer phenotype.Mainly include following several classes through the CYP2C19 medicines being metabolized：It is proton pump inhibitor, anti-insane
Epilepsy, sedative and antidepressants, anticoagulation etc..Therefore when CYP2C19 turns into the main metabolic enzyme of above medicine, without gene
The pharmacokinetic parameters of type individual will be affected differently.Ordinary circumstance, fast metabolic pattern metabolic drug is than very fast.It is not likely to produce not
Good reaction, and slow inactivation metabolic drug is slow, is easily caused the generation of adverse reaction.Therefore should be appropriate for fast metabolic patient
Increasing dose to reach effective blood drug concentration, and reduction dose that should be appropriate for slow metabolic patient is to prevent medicine
Adverse reaction.
Accordingly, it would be desirable to by detecting influence Reasonable adjustment medicine of the CYP2C19 gene pleiomorphisms to related drugs blood concentration
The dosage of thing, so that clinically individuation is really moved towards in the application of related drugs, to improve clinical efficacy, is reduced bad anti-
Should.
2.CYP2C19 gene pleiomorphism detecting method brief introductions
Method at present for detection SNP mutation detection is a lot, mainly with method include：Direct sequencing, ARMS-
PCR methods, digestion (PCR-RLFR) method, DNA chip method, TaqMan-MGB probe typings etc..
All more or less there is such as reagent testing cost height, complex operation and be also easy to produce pollution in these methods, produce in popularization
The defects such as raw certain false negative and false positive.
Such as：1) direct sequencing, the detection method needs to carry out PCR amplifications to fragment to be detected, then uses DNA sequencer
Sequencing, is the goldstandard of SNP detections.The sequencing peak of homozygous SNP site is single peak type, and the sequencing of heterozygous SNP site
Peak is set peak, thus is easy to be made a distinction.Have the disadvantage that course of reaction needs processing product of uncapping, easily cause aerosol
Pollution, and whole reaction process is time-consuming and uneconomical.
2) ARMS-PCR methods, allele specific amplification method, also referred to as amplification refractory mutation system, for known mutations
Gene is detected.The method is by designing two 5 ' end primers, and one complementary with normal DNA, and one complementary with mutant DNA, right
In homozygous mutant, it is separately added into both primers and 3 ' end primers carries out two parallel PCR, only mutant DNA is complete complementary
Primer just may extend away and obtain pcr amplification product, because the 3` ends that mispairing is located at primer then cause PCR to extend, then be referred to as
ARMS.This method carries out electrophoresis after terminating by using PCR reactions, and judged result is brought from projection bar is whether there is, though this method
Expensive instrument, but electrophoretic procedures are not needed so, add the chance of PCR pollutions, and time and effort consuming.
3) digestion (PCR-RLFR) method, using the specificity of the restriction enzyme site of restriction enzyme, with two kinds or two kinds with
On restriction enzyme act on the DNA segment of same process PCR amplification enrichment, if there is SNP site, digestion segment
Difference then occurs in length and quantity, according to the result of electrophoresis it may determine that whether SNP site.But the technology is using restricted
Factor be SNP site must the recognition site containing the limitation restriction endonuclease, and method is also relatively complicated.
4) gene chip hybridization method, passes through the detection kit of the shapings of CFDA certifications, Shanghai hundred on the market at present
" CYP2C19 genotype detections kit (the DNA microarray chip method) " of proud biotech inc, the kit is adopted
Detected with PCR- gene chip hybridization methods, aggregated PCR (PCR) technology expands corresponding CYP2C19 genes piece
Section, and hybridize with specific dna probe on chip, to distinguish the genotype for determining CYP2C19.The detection method, however it remains
Complex operation, open pipe operation, the problem of easily causing pollution, in addition hybridization hybrid chip make complicated, it is necessary to the consumptive material such as slide, significantly
Improve testing cost.Whole detection cycle needs 3-4 hour, inefficient.
5) PCR-Taqman MGB probes typing, because its is simple to operate, parting is accurate, whole stopped pipe operation, detection week
The features such as phase is short, is the SNP classifying methods for comparing main flow at present.Also detected on the market by the shaping of CFDA certifications at present
Kit, such as Wuhan You Zhiyou bio tech ltd " (PCR- is glimmering for mankind's CYP2C19 genetic polymorphism detections kit
Light probe method) ", however require that two Taqman-MGB probes, and the synthesis price of MGB probes is general T aqman probes
Several times, considerably increase testing cost, and MGB probes are to belong to LIFE house journals of U.S. technology, can on raw material by
More limitation.
Therefore, how to solve during detection CYP2C19 gene pleiomorphisms the problem of exist, carry out it is a kind of simple and easy to apply,
Detection method that is quality-high and inexpensive, being limited without special expensive instrument, as urgent problem to be solved.
The content of the invention
In consideration of it, the present invention provides the method and kit of a kind of new detection mankind's CYP2C19 gene pleiomorphisms, to solve
The detecting instrument price that certainly conventional detection technique and finished product detection kit are present is high, complex operation, easily pollution, testing cost
Height, the problems such as clinical practice promotes difficult.
The present invention is in order to solve the above technical problems, a kind of brand-new detection method technical scheme provided, new detection skill
The design of art method comprises the following steps：
(1) according to CYP2C19 gene purpose SNP sites, oppositional allele separately designs ARMS primers, the ARMS
3 ' ends of primer are mutational site, and by any bit missense mutation in the nucleotides of mutational site upstream 1-3；
(2) after the design allele ARMS primers for each SNP, respectively in two specific ARMS primers
5 ' ends introduce one section with human genome sequencing without homologous artificial sequence oligodeoxynucleotide, and formation is containing one section of universal sequence label
SNP site specificity ARMS primer sequences；For two allele-specific ARMS primers of a SNP site, 5 ' ends are drawn
The sequence label entered be it is different, it is specific；
(3) according to the general artificial sequence of the introducing, design one and universal sequence intermediate segment sequence complete complementary
The Taqman probes of pairing, the specific fragment of specific recognition ARMS primers amplification.The Taqman probes, correspondence is not equal
The specific fragment of position gene loci, is marked with different fluorescence radiation groups respectively, such as one end of Taqman probes 5 ' quilt
Flag F AM groups, 3 ' end mark correspondence quencher BHQ1, then then 5 ' ends are labeled HEX groups to another probe, and 3 ' ends are visited
For answering quencher BHQ1.
(4) sample DNA is extracted, fluorescent quantitation is carried out using the probe designed in the primer pair and step 3 designed in step 2
PCR is expanded, and detection site genotype is judged according to fluorescence signal.
It is preferred that, in the step (1), the ARMS designed for CYP2C19 gene C .681 sites GG allelotypes draws
Thing is that penultimate carries out missense mutation, the ARMS primers designed for CYP2C19 gene C .681 sites AA allelotypes
Missense mutation is carried out for antepenulatimate；It is for the ARMS primers of CYP2C19 gene C .636 sites GG allelotypes design
Antepenulatimate carries out missense mutation, is to fall for the ARMS primers that CYP2C19 gene C .636 sites AA allelotypes are designed
Number second carries out missense mutation.
It is preferred that, in the step (2), the ARMS designed for CYP2C19 gene C .681 sites GG allelotypes draws
Thing introduces the sequence of the general-distinctive embedment primer formed after universal sequence as shown in SEQ NO.1；For CYP2C19 genes
C.681 the ARMS primers of site AA allelotypes design introduce the sequence of the general-distinctive embedment primer formed after universal sequence
Row are as shown in SEQ NO.2；General sequence is introduced for the ARMS primers that CYP2C19 gene C .636 sites GG allelotypes are designed
Sequence after row is as shown in SEQ NO.3；The ARMS primers designed for CYP2C19 gene C .636 sites AA allelotypes draw
Enter the sequence after universal sequence as shown in SEQ NO.4.
Further preferably, C.681 site wild-type amplification ARMS primer SEQ NO.1 and the C.636 expansion of site wild type
Increase ARMS primer SEQ NO.3, the universal sequence of institute's primer is duplicate, and sequence is as shown in SEQ NO.5；C.681 site
Saltant type expand ARMS primer SEQ NO.2 and C.636 site mutation type expand ARMS primer SEQ NO.4, institute's primer it is general
Sequence is duplicate, and sequence is as shown in SEQ NO.6；
It is preferred that, in the step (3), the specific recognition of design C.681 site GG allelotypes amplified fragments
The Taqman probe sequences complete one of Taqman probe sequences and specific recognition C.636 site GG allelotypes amplified fragments
Cause, all as shown in SEQ NO.7；The Taqman probes of the specific recognition of design C.681 site AA allelotypes amplified fragments
The Taqman probe sequences of sequence and specific recognition C.636 site AA allelotypes amplified fragments are completely the same such as SEQ
Shown in NO.8.
Further preferably, Taqman probe SEQ NO.7,5 ' ends are marked with FAM fluoresceins, and 3 ' ends are marked with
BHQ1 quenchers；Taqman probe SEQ NO.8,5 ' ends are marked with HEX fluoresceins, and 3 ' ends are marked with BHQ1 quenching bases
Group.
Further preferably, above-mentioned two specific probes, belong to and the pairing of universal sequence intermediate segment complete complementary
Generic detection probe, be applicable to other it is any want using the method SNP site detection, probe be general T aqman spy
Pin.
Another technical scheme proposed to solve above-mentioned technical problem of the present invention is to provide a kind of based on the above method
Mankind's CYP2C19 genetic polymorphism detection kits.
The kit includes pcr amplification reaction liquid, c.681 site amplimer probe mixed liquor, and c.636 site is expanded
Primed probe mixed liquor, positive control solution, negative controls.
The pcr amplification reaction liquid includes PCR buffer solutions, dNTPs, HotStart Taq archaeal dna polymerases.
C.681 the site amplimer probe mixed liquor includes the specific forward primer of c.681 site wild type (G)
As shown in SEQ NO.1 and for saltant type (A) specific upstream ARMS primers as shown in SEQ NO.2, and c.681 site
The specific forward primer of wild type (G) and the shared anti-sense primer such as SEQ NO.9 of (A) specific forward primer of saltant type
It is shown；For C.681 site GG allelotypes amplified fragments Taqman probe sequences as shown in SEQ NO.5, for
C.681 the Taqman probe sequences of site AA allelotypes amplified fragments are as shown in SEQ NO.6.
C.636 the site amplimer probe mixed liquor includes the specific forward primer of c.636 site wild type (G)
As shown in SEQ NO.3 and for saltant type (A) specific upstream ARMS primers as shown in SEQ NO.4, and c.636 site
The specific forward primer of wild type (G) and the shared anti-sense primer such as SEQ NO.10 of (A) specific forward primer of saltant type
It is shown；For C.636 site GG allelotypes amplified fragments Taqman probe sequences as shown in SEQ NO.5, for
C.636 the Taqman probe sequences of site AA allelotypes amplified fragments are as shown in SEQ NO.6.
Above-mentioned primer probe sequence such as table 1 below：
Above-mentioned technical proposal it is further preferred, c.681 the specific forward primer SEQ NO.1 of site wild type (G) are whole
Concentration and (A) specific upstream ARMS primer SEQ NO.2 final concentrations for saltant type, and c.681 site wild type (G)
Specific forward primer and saltant type the shared anti-sense primer SEQ NO.7 final concentrations of (A) specific forward primer and
C.681 the Taqman probes SEQ NO.5 final concentrations of site GG allelotypes amplified fragments and c.681 site AA allele
The Taqman probe sequence SEQ NO.6 final concentrations ratio 2: 2: 4: 1: 1 of type amplified fragments.
It is further preferred in above-mentioned technical proposal, the corresponding primer and probe final concentration ratio in the c.636 site with
C.681 it is consistent.
The positives comparison liquid of mentioned reagent box be containing c.681 G ＞ A site wild plasmids and the mutant plasmids and
C.636 the G ＞ A site wild plasmids and mutant plasmids mixed liquor.
Above-mentioned negative controls are pure water.
One kind of above-mentioned technical proposal is preferably, the composition of the detection kit of the mankind CYP2C19 gene pleiomorphisms
Composition and concentration are as shown in table 2：
The beneficial effects of the present invention are：
(1) method and kit for a kind of new detection mankind's CYP2C19 gene pleiomorphisms that the present invention is provided, compared to biography
ARMS methods are organically combined and detected by the Taqman-MGB sonde methods detection SNP site of system, this invention with Taqman probes, profit
The specificity that allele is carried out with ARMS primers distinguishes amplification, can greatly increase allele-specific primers and distinguish different
The ability of allele；One section of artificial nucleosides is introduced respectively for two special ends of ARMS primers 5 ' of same detection site simultaneously
Acid sequence, is used as the site of the specific recognition of Taqman probes.Therefore detected for different SNP site partings, two
Taqman probes can be introduced arbitrarily, general probe can be detected as SNP partings, while being compared with general T aqman probes
Taqman-MGB probes, greatly reduce research and development and the testing cost of SNP partings.
(2) method and kit of a kind of new detection mankind's CYP2C19 gene pleiomorphisms of the invention, overcome existing
Detection technique in, troublesome operation is time-consuming, and Cost Problems, only needs traditional fluorescent dye, and any high cost detection is not related to
Reagent, greatlys save cost, improves efficiency.
(3) method and kit of a kind of new detection mankind's CYP2C19 gene pleiomorphisms of the invention, whole stopped pipe enters
OK, the possibility that pollution is produced is reduced, while introducing perfect quality control system, it is ensured that the accuracy of testing result.
(4) a kind of method of new detection mankind's CYP2C19 gene pleiomorphisms of the invention and kit research and development are simple, behaviour
Facilitate, use condition is cheap, it is adaptable to large-scale clinical application.
Object of the present invention, feature, and advantage will be in following embodiment, accompanying drawing, and claims
In detailed exposure.
Brief description of the drawings
Fig. 1 is the detection principle diagram of new detection mankind's CYP2C19 gene pleiomorphism methods according to the embodiment of the present invention
Fig. 2 is sample 1c.681 site primers result (wild type).
Fig. 3 is sample 1c.636 site primers result (homozygous mutant).
Fig. 4 is sample 2c.681 site primers result (homozygous mutant).
Fig. 5 is sample 2c.636 site primers result (wild type).
Fig. 6 is sample 3c.681 site primers result (heterozygous mutant).
Fig. 7 is sample 3c.636 site primers result (heterozygous mutant).
Embodiment
The present invention is specifically described below by embodiment, it is necessary to it is pointed out here that be that following examples are only used
It is further described in the present invention, it is impossible to be interpreted as limiting the scope of the invention, person skilled in art can
To make some nonessential modifications and adaptations to the present invention according to the invention described above content.The side of unreceipted actual conditions in text
Method, the Science Press generally write according to normal condition such as J. Pehanorm Brookers etc. version in 2002《Molecular Cloning: A Laboratory refers to
South》Condition described in one book, or the condition proposed by manufacturer.Unless otherwise defined, all specialties used in text
It is used for science identical with meaning known to those skilled in the art of the present technique.In addition, any similar or equal to described content
Deng method and material all can apply in the present invention.
Embodiment 1
The specific detecting step of the kit for detecting mankind's ApoE gene pleiomorphisms of the present embodiment is as follows：
1st, DNA is extracted.
Sample 1,2,3 (sample 1,2,3 is EDTA anticoagulated whole bloods entirely) is extracted using blood sample DNA extraction kit, specifically
Operation is referring to DNA kit extract product specifications.
2nd, sample DNA quality testing.
Obtain after sample DNA, by determining the Ratio control sample quality of concentration and OD260/OD280, ultimately join anti-
Answer the sample in system, OD260/OD280 ratio obtains peak optimization reaction result between 1.7~2.0, concentration is 5~
20ng/ μ l are more suitable.
3rd, PCR reacts.
1) 20 μ L PCR reaction systems are prepared：
Calculate for c.681 (the PCR reaction tubes number=sample number+positive control+the moon of PCR reaction tubes number needed for site primer
Property control), that is, detect 3 samples c.681 site, it is necessary to 5 PCR reaction tubes.
5 fluorescent PCR pipes are taken respectively, all add 10 μ L pcr amplification reaction liquid, 2 μ L c.681 site respectively thereto
The PCR level pure water of amplimer probe mixed liquor and 6 μ L, each μ L of pipe 18；2 μ L samples to be tested DNA, 3 positive controls are taken respectively
The μ L of liquid 2, the μ L of negative controls 2, are added in correspondence PCR pipe, cover PCR pipe lid, be marked according to added template.
Same method, prepares the 5 pipes detection reaction solution c.636 needed for site primer, PCR pipe lid is covered, according to added
Template is marked.
2) performing PCR reaction is entered
PCR amplification programs are as shown in table 3.
Above PCR reactions are carried out on grand stone SLAN-48P quantitative real time PCR Instruments.
4th, interpretation of result and interpretation.
1st, kit availability deciding：
1) positive control：FAM, VIC channel C t value≤32, amplification curve have obvious Exponential growth stage.
2) negative control：FAM, VIC passage are without amplification curve, or amplification curve is straight line or slight oblique line, without substantially
Exponential growth stage, no Ct values or Ct value >=38.
2nd, the judgement of sample site genotype
According to 1 judge testing result it is effective after, the result to sample detection pipe judges, according to formula " Δ Ct values=
FAM Ct values-HEX is worth " the Δ Ct values of effective detection sample are calculated, and pattern detection result is carried out according to following steps and table 5
Interpretation, determines the genotype of sample.
Table 5：Result judgement
C.681 site primer result is shown in Fig. 2 (681 site wild type), CYP2C19 genes to the CYP2C19 genes of sample 1
C.636 site primer result is shown in Fig. 3 (636 site homozygous mutant), according to criterion, and the CYP2C19 genes of sample 1 belong to *
3/*3 types.
C.681 site primer result is shown in Fig. 4 (681 site homozygous mutant), CYP2C19 bases to the CYP2C19 genes of sample 2
Because c.636 site primer result is shown in Fig. 5 (636 site wild type)；According to criterion, the CYP2C19 genes of sample 2 belong to *
2/*2 types.
C.681 site primer result is shown in Fig. 6 (681 site heterozygous mutant), CYP2C19 bases to the CYP2C19 genes of sample 3
Because c.636 site primer result is shown in Fig. 7 (636 site heterozygous mutant), according to criterion, the CYP2C19 genes of sample 3 category
In * 2/*3 types.
Obviously, above-described embodiment is only intended to clearly illustrate example of the present invention, and is not to the present invention
The restriction of embodiment.For those of ordinary skill in the field, it can also be made on the basis of the above description
Its various forms of changes or variation.There is no necessity and possibility to exhaust all the enbodiments.And these belong to this hair
Among the obvious changes or variations that bright spirit is extended out is still in protection scope of the present invention.
Claims (10)
1. a kind of method for detecting mankind's CYP2C19 gene pleiomorphisms, it is characterised in that comprise the following steps：
(1) according to CYP2C19 gene purpose SNP sites, oppositional allele separately designs ARMS primers, the ARMS primers
3 ' ends be mutational site, and by any bit missense mutation in the nucleotides of mutational site upstream 1-3；
(2) after the design allele ARMS primers for each SNP, respectively at 5 ' ends of two specific ARMS primers
One section is introduced with human genome sequencing without homologous artificial sequence oligodeoxynucleotide, the SNP positions containing one section of universal sequence label are formed
Point specificity ARMS primer sequences；For two allele-specific ARMS primers of a SNP site, the sequence that 5 ' ends are introduced
Column label be it is different, it is specific；
(3) according to the general artificial sequence of the introducing, design one is matched with universal sequence intermediate segment sequence complete complementary
Taqman probes, specific recognition ARMS primers amplification specific fragment；The Taqman probes, the different equipotential bases of correspondence
Because of the specific fragment in site, different fluorescence radiation groups are marked with respectively, and such as one end of Taqman probes 5 ' is labeled
FAM groups, 3 ' end mark correspondence quencher BHQ1, then then 5 ' ends are labeled HEX groups to another probe, and 3 ' ends are by probe pair
Answer quencher BHQ1；
(4) sample DNA is extracted, quantitative fluorescent PCR is carried out using the probe designed in the primer pair and step 3 designed in step 2
Amplification, detection site genotype is judged according to fluorescence signal.
2. the method for detection mankind's CYP2C19 gene pleiomorphisms according to claim 1, it is characterised in that：The step
(2) in, introduce what is formed after universal sequence for the ARMS primers that CYP2C19 gene C .681 sites GG allelotypes are designed
The sequence of general-distinctive embedment primer is as shown in SEQNO.1；For the design of CYP2C19 gene C .681 sites AA allelotypes
ARMS primers introduce the sequence of general-distinctive embedment primer formed after universal sequence as shown in SEQ NO.2；For
The sequence such as SEQ NO.3 that the ARMS primers of CYP2C19 gene C .636 sites GG allelotypes design are introduced after universal sequence
It is shown；Sequence after the ARMS primers introducing universal sequence designed for CYP2C19 gene C .636 sites AA allelotypes is such as
Shown in SEQ NO.4；C.681 site wild-type amplification ARMS primer SEQ NO.1 and, C.636 wild-type amplification ARMS in site draws
Thing SEQ NO.3, the universal sequence of institute's primer is duplicate, and sequence is as shown in SEQ NO.5；C.681 site mutation type expands
Increase ARMS primer SEQ NO.2 and C.636 site mutation type expands ARMS primer SEQ NO.4, and the universal sequence of institute's primer has been
As complete, sequence is as shown in SEQ NO.6.
3. the method for detection mankind's CYP2C19 gene pleiomorphisms according to claim 1, it is characterised in that：The step
(3) in, the Taqman probe sequences and specific recognition of specific recognition C.681 site GG allelotypes amplified fragments
C.636 the Taqman probe sequences of site GG allelotypes amplified fragments are completely the same, all as shown in SEQ NO.7；Specificity
Taqman probe sequences and the specific recognition C.636 site AA equipotentials of identification C.681 site AA allelotypes amplified fragments
The Taqman probe sequences of genotype amplified fragments are completely the same as shown in SEQ NO.8.
4. the method for detection mankind's CYP2C19 gene pleiomorphisms according to claim 1, it is characterised in that：The step
(2) in, two specific probes belong to the generic detection probe matched with universal sequence intermediate segment complete complementary, fit
For other any detections for wanting the SNP site using the method, probe is general T aqman probes.
5. the method for detection mankind's CYP2C19 gene pleiomorphisms according to claim 1, it is characterised in that：Including specific
Detecting step is as follows：
(1) DNA is extracted
Sample 1 is extracted using blood sample DNA extraction kit, 2,3, wherein sample 1,2,3 be EDTA anticoagulated whole bloods；
(2) sample DNA quality testing
Obtain after sample DNA, by determining the Ratio control sample quality of concentration and OD260/OD280, ultimately join reactant
Sample in system, OD260/OD280 ratio obtains peak optimization reaction result between 1.7~2.0, and concentration is 5~20ng/
μl；
(3) PCR reacts, including step is as follows：
(a) 20 μ L PCR reaction systems are prepared：
For c.681 PCR reaction tubes number needed for site primer, (PCR reaction tubes number=sample number+positive control+feminine gender is right for calculating
According to), that is, detect the c.681 site of 3 samples, it is necessary to 5 PCR reaction tubes；
5 fluorescent PCR pipes are taken respectively, all add the c.681 site amplification of 10 μ L pcr amplification reaction liquid, 2 μ L respectively thereto
The PCR level pure water of primed probe mixed liquor and 6 μ L, each μ L of pipe 18；2 μ l samples to be tested DNA, the μ of 3 positive control solution 2 are taken respectively
L, the μ L of negative controls 2, are added in correspondence PCR pipe, cover PCR pipe lid, be marked according to added template；
Same method, prepares the 5 pipes detection reaction solution c.636 needed for site primer, PCR pipe lid is covered, according to added template
It is marked；
(b) performing PCR reaction is entered
Wherein, PCR amplification steps include：95 DEG C of holding 5min；Circulation 1 time；95 DEG C of holding 10s, then 60 DEG C of holding 40s, are followed
Ring 45 times, wherein collecting fluorescence when keeping 40s for 60 DEG C；
Wherein, the PCR reactions are carried out on quantitative real time PCR Instrument.
6. a kind of detection kit of the method for detection mankind's CYP2C19 gene pleiomorphisms based on described in claim 1-5, its
It is characterised by, including：Pcr amplification reaction liquid, c.681 site amplimer probe mixed liquor, c.636 site amplimer probe
Mixed liquor, positive control solution and negative controls.
7. detection kit according to claim 6, it is characterised in that：
The pcr amplification reaction liquid includes PCR buffer solutions, dNTPs, HotStart Taq archaeal dna polymerases；
C.681 the site amplimer probe mixed liquor includes the specific forward primer of c.681 site wild type (G) such as
Shown in SEQ NO.1 and for saltant type (A) specific upstream ARMS primers as shown in SEQ NO.2, and c.681 site open country
The specific forward primer of raw type (G) and the shared anti-sense primer such as SEQ NO.9 institutes of (A) specific forward primer of saltant type
Show；For C.681 site GG allelotypes amplified fragments Taqman probe sequences as shown in SEQ NO.5, for C.681
The Taqman probe sequences of site AA allelotype amplified fragments are as shown in SEQ NO.6；
C.636 the site amplimer probe mixed liquor includes the specific forward primer of c.636 site wild type (G) such as
Shown in SEQ NO.3 and for saltant type (A) specific upstream ARMS primers as shown in SEQ NO.4, and c.636 site open country
The specific forward primer of raw type (G) and the shared anti-sense primer such as SEQ NO.10 institutes of (A) specific forward primer of saltant type
Show；For C.636 site GG allelotypes amplified fragments Taqman probe sequences as shown in SEQ NO.5, for C.636
The Taqman probe sequences of site AA allelotype amplified fragments are as shown in SEQ NO.6；
The positives comparison liquid of kit is to contain c.681 G ＞ A site wild plasmids and the mutant plasmids and described
C.636 G ＞ A site wild plasmids and mutant plasmids mixed liquor；
The negative controls are pure water.
8. detection kit according to claim 7, it is characterised in that：C.681 the specific upstream of site wild type (G)
Primer SEQ NO.1 final concentrations and (A) specific upstream ARMS primer SEQ NO.2 final concentrations for saltant type, and c.681
The specific forward primer of site wild type (G) and the shared anti-sense primer SEQ of (A) specific forward primer of saltant type
The Taqman probes SEQ NO.5 final concentrations of NO.7 final concentrations and c.681 site GG allelotypes amplified fragments with c.681
The Taqman probe sequence SEQ NO.6 final concentrations ratio 2: 2: 4: 1: 1 of site AA allelotype amplified fragments.
9. the detection kit according to claim 7 or 8, it is characterised in that：The corresponding primer in the c.636 site and spy
Pin final concentration ratio with it is c.681 consistent.
10. the detection kit according to claim 6-9, it is characterised in that the concentration including each component is as follows：
The pcr amplification reaction liquid includes：2 × PCR buffer solutions, 0.25mM dNTP and 0.1U HotStart Taq DNA；
C.681 the site amplimer probe mixed liquor includes：0.5 μM of GG type-special primer SEQ NO.1；0.5 μM
AA type-special primer SEQ NO.2；1 μM of c.681 site general reverse primer SEQ NO.3；0.25 μM of GG types are general to be visited
Pin SEQ NO.7；And 0.25 μM of AA type general probe SEQ NO.8；
C.636 the site amplimer probe mixed liquor includes：0.5 μM of GG type-special primer SEQ NO.4；0.5 μM
AA type-special primer SEQ NO.5；1 μM of c.636 site general reverse primer SEQ NO.6；0.25 μM of GG types are general to be visited
Pin SEQ NO.7；And 0.25 μM of AA type general probe SEQ NO.8；
The positive control solution includes：C.681 G ＞ A sites GG types wild plasmid, c.636 AA types mutant plasmids, G ＞ A
Site GG types wild plasmid and AA type mutant plasmids mixed liquors, wherein the concentration of each plasmid is 0.0005ng/ μ L；
The negative controls are pure water.
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| CN111363792A (en) * | 2019-10-31 | 2020-07-03 | 北京安智因生物技术有限公司 | Kit and method for detecting gene polymorphism based on shared primer probe and application |
| CN110885884A (en) * | 2019-12-16 | 2020-03-17 | 昆明和合医学检验所有限公司 | Method for synchronously detecting polymorphism of CYP2C19 gene 2, 3, 17 locus gene |
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