CN107841557A - Drug taxol curative effect related gene genetic polymorphism detection kit and detection method - Google Patents
Drug taxol curative effect related gene genetic polymorphism detection kit and detection method Download PDFInfo
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- CN107841557A CN107841557A CN201610834400.1A CN201610834400A CN107841557A CN 107841557 A CN107841557 A CN 107841557A CN 201610834400 A CN201610834400 A CN 201610834400A CN 107841557 A CN107841557 A CN 107841557A
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Abstract
The invention provides a kind of drug taxol curative effect related gene（CYP2C8 and GSTP1）Genetic polymorphism detection kit and detection method, the kit include PCR buffer solutions, specific ARMS detection primers, Quality Control primer.Present invention also offers a kind of taxol curative effect related gene（CYP2C8 and GSTP1）Gene pleiomorphism detecting method.The inventive method can rapidly and accurately detect taxol curative effect related gene（CYP2C8 and GSTP1）Gene pleiomorphism, there is high sensitivity, high specificity, method is simple, it is as a result accurate the features such as.
Description
Technical field：
The present invention relates to biotechnology and medical domain, more particularly to drug taxol curative effect related gene CYP2C8
(GSTP1) genetic polymorphism detection kit and detection method.
Background technology：
The metabolism of most of medicine is carried out in liver.Cytochrome P 450 Enzyme (CYP or P450) is main in liver
One of metabolism enzyme system.CYP is microsome mixed function monooxygenase, and it is outer to participate in medicine, precarcinogen, poisonous substance, mutagen etc.
The biological oxidation process of source thing.It is found that CYP gene pleiomorphisms affect depression, mental disease, tumour, cardiovascular and cerebrovascular, stomach
Ulcer, the individual drugs reaction of the therapy of serious disease medicine such as pain and insane pain.As research deepens continuously, some medicines may be used also
According to CYP genotype, the adjustment of dosage is carried out, to optimize the medication effect of different genotype carrier and safety
Property.The medicine through I phase enzymatics of CYP2C8 metabolism at least 1%, among these including the significant antineoplastic of clinical treatment
The taxol of thing one, interindividual variations of the CYP2C8 taxol 6a through baseization activity cause the clinic of taxol to be controlled up to 38 times
Therapeutic effect and drug safety are uneven.CYP2C8 has found many single nucleotide polymorphisms at present, causes CYP2C8 etc.
The generation of position gene, i.e. CYP2C8*1-* 10.Wherein CYP2C8*3 is the most important allele of CYP2C8, wherein common prominent
Become gene (G416A).Mutated-genotype patient substantially reduces to the scavenging capacity of the antineoplastics such as taxol, about wild type
The 15% of patient, therefore should suitably reduce dosage.
Gene pleiomorphism is detected furthermore with PCR-RFLP, it is found that carrying GSTP1 (A313G) gene mutation causes corresponding enzyme
Inactive or low activity, so that individual reduces to the clearance rate of chemotherapeutic, efficacy time increases, thus AG/GG genotype
Patient has good response to chemotherapeutics.
Have much on detection method of gene mutation, scholars have carried out numerous studies to this.Oneself side through report
Method includes direct sequencing, DHPLC, PCR-SSCP/RFLP, Scorpions ARMS, TaqMan PCR, ME-PCR etc..These
Method respectively has advantage and disadvantage, wherein method more conventional in clinical and scientific research is direct sequencing and ARMS
(Amplification refractory mutation system, amplification refractory mutation system) method.
Direct sequencing detectability is limited, its detection sensitivity about 20% or so, and step is complicated, entirely detects
Journey is related to a series of steps such as the deciphering of PCR- electrophoresis-sequencing-sequencing result, and it is laborious to take inch, but is the advantages of this method
It can be found that some new unknown mutations.
ARMS methods are to be combined to create with specific ARMS primers by molecular beacon (probe), ARMS primers
3 ' end designs are matched in mutational site, last base with mutating alkali yl, using the TaqDNA without 3 ' → 5 ' 5 prime excision enzyme activities
Polymerase, 3 ' ends of specific identification primer, when only the end of primer 3 ' is matched completely, could normally expand, work as primer
When mispairing occurs for 3 ' ends, it is impossible to effective amplification.After primer combines with mutagenesis template and extends corresponding product, probe
The fluorophor and quenching group at both ends separate and produce fluorescence.At present, in the market related kit include QIAGEN companies and
Xiamen Ai De companies, it is expensive.
Therefore, this research uses the method that is combined with SYBR dyestuffs of ARMS technologies, develop one kind possess it is entirely autonomous
Intellectual property and can quickly, sensitive and easy detection CYP2C8 (GSTP1) gene pleiomorphism kit.By certainly
Own R ＆ D design ARMS primers and Scorpions probes are changed to SYBR dyestuffs so that testing cost substantially reduces, so more
It is adapted to the detection of Chinese patients CYP2C8 (GSTP1) gene pleiomorphism.In summary, the detection kit and detection method are one
Kind high sensitivity, high specificity, method is simple, as a result accurate CYP2C8 (GSTP1) gene pleiomorphism detecting method.
The content of the invention：
The purpose of the embodiment of the present invention is to provide a kind of high sensitivity, and high specificity, method is simple, as a result accurately purple
China fir alcohol curative effect related gene (CYP2C8 and GSTP1) genetic polymorphism detection kit and detection method.The embodiment of the present invention is
It is achieved in that, a kind of taxol curative effect related gene (CYP2C8 and GSTP1) genetic polymorphism detection kit, the kit
Including PCR buffer solutions, specific mutations detection primer, Quality Control primer.The present invention also provides a kind of taxol curative effect related gene
(CYP2C8 and GSTP1) gene pleiomorphism detecting method, comprises the following steps：(1) provide and draw as described in claim 1
Thing；(2) processing of testing sample and the extraction of template；(3) quantitative fluorescent PCR reaction system is prepared；(4) ARMS guiding regions are utilized
Divide wild type and mutated genes sequence, by the hybridization of SYBR Green dyestuffs and amplified production, detect reaction system
SYBR fluorescence intensities judge testing result.SYBR is detection signal, reaches following needed for the threshold value of setting with SYBR signals
For ring number Ct values as criterion, Ct values are less than 32 for the positive, and Ct values are more than 35 and are between 32 to 35 for feminine gender, Ct values
Weakly positive.
Taxol curative effect related gene (CYP2C8 and GSTP1) genetic polymorphism detection kit of the present invention is contaminated using SYBR
Material method, establish the Multiplex real-time PCR detection method for the mankind CYP2C8 and GSTP1 gene pleiomorphism (as shown in table 1).This
Lot of experiments is passed through in invention, and research and analysis, which successfully filter out, can be used in quick, sensitive, effective detection taxol curative effect phase
The primer combination of correlation gene (CYP2C8 and GSTP1) gene pleiomorphism, and go out to have the advantage that using these primer developments
Be used for detect the method and kit of taxol curative effect related gene (CYP2C8 and GSTP1) gene pleiomorphism.The present invention's
Detection kit and detection method have high sensitivity, and high specificity, method is simple, it is as a result accurate the features such as, therefore the present invention
With substantial technique effect, just with popularization.
Table 1：CYP2C8 and GSTP1 gene pleiomorphism forms
Described specific primer see the table below 2/3.
Table 2：The detection primer of CYP2C8 gene pleiomorphisms
Table 3：The detection primer of GSTP1 gene pleiomorphisms
It is of the present invention detection mankind's CYP2C8 and GSTP1 gene pleiomorphism fluorescence quantifying PCR method include pair
The step of processing of sample to be tested and template extraction, but for the sample from formaldehyde fixation FFPE and the sample from blood plasma
The piece segment DNA that product are obtained still has and the amplification of flesh tissue sample DNA identical and detectability.
The detection kit of the present invention can accurately detect 1% mutant DNA under 50ng wild type gene group DNA backgrounds,
And the specific primer in targeted design difference mutational site, detected using quantitative fluorescent PCR, detection process is stopped pipe
Reaction, substantially reduces pollution.
Brief description of the drawings：
Fig. 1 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects CYP2C8 (416) sample.
Fig. 2 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects CYP2C8 (1196) sample.
Fig. 3 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects GSTP1 (313) sample.
Embodiment：
Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through specific realities different in addition
The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under the spirit of the present invention.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text
Explicitly point out in addition, singulative "one", " one " and " this " include plural form.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can be selected between point and two end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Embodiment 1
1. sample process and nucleic acid extraction
Sample is handled using the DNA extraction kit of commercialization, referring to kit specification, carrying DNA needs for concrete operations
Concentration and purity, its DNA OD are determined with ultraviolet specrophotometer260/OD280Value should 1.8~2.0, concentration should 5~
Between 50ng/ μ L, sample DNA person off quality must not be used to detect, it is proposed that take section to carry out core again less than 5ng/ μ L persons
Acid extraction, defined concentration range is suitably diluted to higher than 50ng/ μ L persons, and the DNA extracted suggests being detected immediately,
Otherwise do not exceeded 6 months in less than -20 DEG C preservations, holding time please.Kit quality-control product is melted using preceding room temperature, and vortex shakes
Swing 10 seconds, 2000rpm centrifuge 15 seconds it is stand-by.
2. reagent configures
2.1 configuration instruction：Detection reaction sets negative quality-control product and positive quality control product.
2.2 configuration process
Reagent was taken out in 30 minutes in advance, room temperature melt, vortex oscillation 10 seconds, 2000rpm centrifuge 15 seconds it is stand-by.It is it is determined that anti-
N, N=sample numbers (n) to be checked+quality-control product number (2)+1 should be counted.The amount for each reagent being added in reactant mixture is calculated, is calculated
Such as table 3 below：
Table 4：PCR reaction system allocation lists.
| Reagent | PCR reaction solutions P | Primed probe mixed liquor | Purified water |
| Reaction solution A (μ L) | 12.5*N | 6.5*N | 4.0*N |
| External control reaction solution B (μ L) | 12.5*N | 6.5*N | 4.0*N |
2 sterile centrifugation tubes are taken to configure above-mentioned 2 reaction systems, vortex oscillation 10 seconds, 2000rpm after reagent all adds
Centrifugation 15 seconds.Then the above-mentioned μ L/ pipes of mixed liquor 23 are dispensed into PCR reaction tubes (sterile and RNase-Free).
3. sample-adding
Table 5：Sample and quality-control product loading table.
Note：STD represents positive quality control product；NTC represents negative quality-control product.
The loading ratio prompted according to table 4, processed sample, positive quality control product P1 and negative quality-control product P2 are added and reacted
Guan Zhong, covers tightly lid (avoiding bubble from producing), and 2000rpm is centrifuged 15 seconds and all got rid of the liquid on tube wall to ttom of pipe, Ran Houli
Carry out pcr amplification reaction.
4.PCR amplification programs are set
Table 6：The setting of PCR response procedures.
5. the deciphering of assay
Divide wild type and mutated genes sequence using ARMS guiding regions, pass through SYBR Green dyestuffs and amplified production
Hybridization, the SYBR fluorescence intensities of reaction system are detected to judge testing result.SYBR is detection signal, is reached with SYBR signals and set
Cycle-index Ct values needed for fixed threshold value are as criterion, and Ct values are less than 32 for the positive, and Ct values are more than 35 for feminine gender, Ct
Value is weakly positive between 32 to 35.
Claims (6)
1. a kind of taxol curative effect related gene (CYP2C8 and GSTP1) genetic polymorphism detection kit, the kit are used for
The primer of gene pleiomorphism is detected, each primer is as follows：
。
2. taxol curative effect related gene (CYP2C8 and GSTP1) genetic polymorphism detection kit as described in requiring right 1,
Characterized in that, the PCR buffer solutions include 3 ' → 5 ' 5 prime excision enzyme activity high-fidelity Taq enzymes, 1.0-5.0mM MgCl2, 1.0-
5.0mM dNTPs, i.e. dATP, dUTP, dGTP, dCTP each 1.0-5.0mM, SYBR Green I.
3. a kind of taxol curative effect related gene (CYP2C8 and GSTP1) gene pleiomorphism detecting method, comprises the following steps：
(1) primer as described in claim 1 is provided；
(2) processing of testing sample and the extraction of template；
(3) quantitative fluorescent PCR reaction system is prepared；
(4) divide wild type and mutated genes sequence using ARMS guiding regions, pass through SYBR Green dyestuffs and amplified production
Hybridization, the SYBR fluorescence intensities of reaction system are detected to judge testing result.SYBR is detection signal, is reached with SYBR signals and set
Cycle-index Ct values needed for fixed threshold value are as criterion, and Ct values are less than 32 for the positive, and Ct values are more than 35 for feminine gender, Ct
Value is weakly positive between 32 to 35.
4. the method for a kind of the detection CYP2C8 and GSTP1 gene pleiomorphisms as described in right 3, it is characterised in that：Step (3) institute
The testing sample stated includes the fresh pathological tissue of surgery excision, and formaldehyde fixes the pathological tissue of FFPE, paraffin section, entirely
Blood, blood plasma, serum, pleural effusion.
A kind of 5. taxol curative effect related gene (CYP2C8 and GSTP1) genetic polymorphism detection side as described in requiring right 3
Method, it is characterised in that each component final concentration and content are as follows in the PCR system in the step (3)：
A kind of 6. taxol curative effect related gene (CYP2C8 and GSTP1) genetic polymorphism detection side as described in requiring right 3
Method, it is characterised in that the PCR reaction conditions in the step (3) are：95 DEG C 10 minutes, 40 circulation：95 DEG C 10 seconds, 60 DEG C
15 seconds.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108676857A (en) * | 2018-06-06 | 2018-10-19 | 杭州艾迪康医学检验中心有限公司 | Detect the primer and method of CYP2C8 point mutation |
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| CN102732600A (en) * | 2011-04-13 | 2012-10-17 | 上海芯超生物科技有限公司 | Genetic evaluation and suit method for individualized tumor therapy |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108676857A (en) * | 2018-06-06 | 2018-10-19 | 杭州艾迪康医学检验中心有限公司 | Detect the primer and method of CYP2C8 point mutation |
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