CN107841555A - The primer sets in the detection gene polymorphic site related to drug metabolism enzymatic activity and its application and application its product and detection method - Google Patents
The primer sets in the detection gene polymorphic site related to drug metabolism enzymatic activity and its application and application its product and detection method Download PDFInfo
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Abstract
The invention provides a kind of primer sets for detecting the gene polymorphic site related to drug metabolism enzymatic activity and its application and apply its product and detection method, it is related to gene engineering technology field, primer sets provided by the present invention for detecting the gene polymorphic site related to drug metabolism enzymatic activity, including for detecting CYP2C19 gene C YP2C19*2 sites, CYP2C19*3 sites, ABCB1 gene 3435C sites, the primer pair and probe in PON1 gene rs854560 sites, the gene polymorphic site related to drug metabolism enzymatic activity not of the same race can be detected simultaneously, detection is comprehensive, it is widely used, testing result can be used for clinical guidance medication.Reagent and kit provided by the present invention for detecting medicine poor metabolizer, including primer sets provided by the invention or reagent, cost is low, efficiency high, can quickly and accurately detect medicine poor metabolizer, high sensitivity, high specificity.
Description
Technical field
The present invention relates to gene engineering technology field, and the base related to drug metabolism enzymatic activity is detected more particularly, to a kind of
Because of the primer sets of polymorphic site and its application and apply its product and detection method.
Background technology
Drug metabolism processes are completed by a series of enzymatic reactions, and the enzyme of participation has two major classes：Microsomal enzyme and non-particulate
Body enzyme.Microsomal enzyme, which is primarily present in the positions such as liver, lung, kidney, small intestine, placenta, skin, also certain presence, micro- with liver
Plastochondria enzymatic activity highest, the metabolism of the exogenous materials such as medicine is mainly catalyzed, so also known as drug metabolic enzyme, abbreviation drug metabolizing enzyme.
Drug metabolic enzyme plays important catalytic action in the metabolism of medicine.After medicine enters human body, on the one hand influence body and produce
Raw pharmacological action, while metabolism disposal is also carried out by body, most drug is mainly lived by metabolic conversion to lose its pharmacology
Property, and excreted as water-soluble high material, also there is small part medicine, just produced after the metabolism of drug metabolic enzyme
Pharmaceutical activity.The active relatively low crowd of internal drug metabolic enzyme be referred to as medicine poor metabolizer (Poor Metabolizers,
PMs).The medicine of pharmaceutical activity is just produced after the same metabolism by drug metabolic enzyme, is given for drug metabolism weak person
During medicine, in order to avoid drug resistance or situation that is reactionless and causing influence curative effect of medication produce, drug user is entered before administration
The detection of row drug metabolism enzymatic activity or the detection of drug metabolism weak person are particularly important.
At present, the detection of drug metabolism enzymatic activity is carried out to drug user or the method for the detection of drug metabolism weak person is mainly
Traditional PCR method, this method waste time and energy, it is necessary to detected one by one to different sites, and the detection site being directed to is less,
It is difficult to ensure that clinical needs.
Therefore, one kind is developed multiple sites can detect simultaneously, it is time saving and energy saving, and specificity is high, testing result
More accurate method is particularly important.
In view of this, it is special to propose the present invention.
The content of the invention
First purpose of the present invention is to provide a kind of gene polymorphic related to drug metabolism enzymatic activity for detection
The primer sets in site, to alleviate the technology that can not be carried out to multiple sites present in prior art while detect, waste time and energy
Problem.
Second object of the present invention is that providing a kind of above-mentioned primer sets is preparing for detecting medicine poor metabolizer
Product in application, lack a kind of primer sets for being capable of effective detection medicine poor metabolizer present in prior art to alleviate
Technical problem.
Third object of the present invention is to provide a kind of reagent for being used to detect medicine poor metabolizer, and the of the invention the 4th
Individual purpose is to provide a kind of kit for being used to detect medicine poor metabolizer, lacks one kind present in prior art to alleviate
Being capable of the reagent of effective detection medicine poor metabolizer or the technical problem of kit.
The 5th purpose of the present invention is to provide a kind of method for detecting medicine poor metabolizer, to alleviate in the prior art
The existing detection method to drug metabolism weak person wastes time and energy, is difficult to ensure that the clinical technical problem needed.
The invention provides a kind of primer sets for being used to detect the gene polymorphic site related to drug metabolism enzymatic activity, institute
Stating primer sets includes following 4 groups of primer pairs or at least one set of primer pair or probe in 4 groups of probes；
The primer pair includes：For detecting the primer pair in CYP2C19 gene C YP2C19*2 sites, for detecting
The primer pair in CYP2C19 gene C YP2C19*3 sites, the primer pair for detecting ABCB1 gene 3435C sites and for detecting
The primer pair in PON1 gene rs854560 sites；
The probe includes：For detecting the wild type detection probe and saltant type in CYP2C19 gene C YP2C19*2 sites
Detection probe, the wild type detection probe for detecting CYP2C19 gene C YP2C19*3 sites and saltant type detection probe, use
In the wild type detection probe and saltant type detection probe in detection ABCB1 gene 3435C sites and for detecting PON1 genes
The wild type detection probe and saltant type detection probe in rs854560 sites；
The primer pair for being used to detect CYP2C19 gene C YP2C19*2 sites includes SEQ ID NO.1 and SEQ ID
Nucleotide sequence shown in NO.2；
The primer pair for being used to detect CYP2C19 gene C YP2C19*3 sites includes SEQ ID NO.3 and SEQ ID
Nucleotide sequence shown in NO.4；
The primer pair for being used to detect ABCB1 gene 3435C sites includes SEQ ID NO.5 and SEQ ID NO.6 institutes
The nucleotide sequence shown；
The primer pair for being used to detect PON1 gene rs854560 sites includes SEQ ID NO.7 and SEQ ID NO.8
Shown nucleotide sequence；
The wild type detection probe for being used to detect CYP2C19 gene C YP2C19*2 sites has such as SEQ ID NO.9
Shown nucleotide sequence, saltant type detection probe have the nucleotide sequence as shown in SEQ ID NO.10；
The wild type detection probe for being used to detect CYP2C19 gene C YP2C19*3 sites has such as SEQ ID
Nucleotide sequence shown in NO.11, saltant type detection probe have the nucleotide sequence as shown in SEQ ID NO.12；
The wild type detection probe for being used to detect ABCB1 gene 3435C sites has as shown in SEQ ID NO.13
Nucleotide sequence, saltant type detection probe has nucleotide sequence as shown in SEQ ID NO.14；
The wild type detection probe for being used to detect PON1 gene rs854560 sites has such as SEQ ID NO.15 institutes
The nucleotide sequence shown, saltant type detection probe have the nucleotide sequence as shown in SEQ ID NO.16.
Further, the primer sets are made up of 4 groups of primer pairs and 4 groups of probes.
Present invention also offers above-mentioned primer sets to prepare the application in being used to detect the product of medicine poor metabolizer.
Further, the medicine is antiplatelet drug.
Further, the product is reagent or kit.
Present invention also offers a kind of reagent for being used to detect medicine poor metabolizer, including above-mentioned primer sets.
Present invention also offers a kind of kit for being used to detect medicine poor metabolizer, including above-mentioned primer sets or right
It is required that the reagent described in 6.
Further, the kit also includes internal control primer group, dUTP, Buffer, HsTaq, MgCl2And sterilized water.
In addition, present invention also offers a kind of method for detecting medicine poor metabolizer, using above-mentioned primer sets or reagent
Or kit.
Further, methods described includes：Using the primer sets as primer, using the DNA of sample to be tested as template, carry out
Pcr amplification reaction simultaneously carries out Genotyping.
Primer sets provided by the present invention for detecting the gene polymorphic site related to drug metabolism enzymatic activity, including with
In detection CYP2C19 gene C YP2C19*2 sites, CYP2C19*3 sites, ABCB1 gene 3435C sites, PON1 genes
More than one or both of the primer pair in rs854560 sites and probe, it can detect simultaneously not of the same race with drug metabolism enzyme activity
Property related gene polymorphic site, detection is comprehensive, is widely used, testing result can be used for clinical guidance medication.The present invention provides
Be used for detect the reagent of medicine poor metabolizer, including primer sets provided by the invention, detection is quick, accurate.The present invention provides
Be used for detect the kit of medicine poor metabolizer, including primer sets provided by the invention or reagent, cost is low, efficiency high, energy
It is enough quickly and accurately to detect medicine poor metabolizer, high sensitivity, high specificity.Detection medicine poor metabolizer provided by the invention
Method, simple to operate, cost is cheap, take it is short, clinical application range is wide.
Embodiment
Technical scheme is clearly and completely described below in conjunction with embodiment, it is clear that described reality
It is part of the embodiment of the present invention to apply example, rather than whole embodiments.Based on the embodiment in the present invention, the common skill in this area
The every other embodiment that art personnel are obtained under the premise of creative work is not made, belong to the model that the present invention protects
Enclose.
The invention provides a kind of primer sets for being used to detect the gene polymorphic site related to drug metabolism enzymatic activity, bag
Include following 4 groups of primer pairs or at least one set of primer pair or probe in 4 groups of probes：
Primer pair is：
Probe is：
Primer sets provided by the present invention for detecting the gene polymorphic site related to drug metabolism enzymatic activity, Neng Goutong
When the detection gene polymorphic site related to drug metabolism enzymatic activity not of the same race, detection comprehensively, is widely used, testing result can
For clinical guidance medication.Wherein, wild type detection probe can be used for the corresponding original site of detection, and saltant type detection probe can use
In the corresponding mutational site of detection.
In one preferred embodiment, there are FAM modifications at the end of CYP2C19 gene Cs YP2C19*2 site primers probe 5 ',
There are BHQ1 modifications at 3 ' ends；There are VIC modifications at the end of CYP2C19 gene C YP2C19*3 site primers probe 5 ', and there are BHQ1 modifications at 3 ' ends；
There are CY3 modifications at the end of ABCB1 gene 3435C site primers probe 5 ', and there are BHQ1 modifications at 3 ' ends；Examine in PON1 gene rs854560 sites
There are CY5 modifications at the end of probing pin 5 ', and there are BHQ1 modifications at 3 ' ends.Internal reference is β-goblin, its probe sequence is 5 '-
There are ROX modifications at TCTACCCTTGGACCCAGAGGTTCTTTGAGT-3 ' (SEQ ID NO.17), 5 ' ends, and there are BHQ1 modifications at 3 ' ends,
Its primer sequence is：Sense primer (5 ' -3 ')：TCTGATAGCCAGTGACTCTCTCTG (SEQ ID NO.18), anti-sense primer
(5’-3’)：AACACCATCTGCAGTGCACAGAT(SEQ ID NO.19).
Using the primed probe of internal reference respectively mixed with the primer and wild-type probe in each site progress with pipe PCR as
Wild type check groups；Primer and saltant type probe of the primed probe of internal reference respectively at each site are mixed and carried out with pipe PCR
As saltant type check groups, every two pipe is detected as a detection group, and data analysis is carried out after PCR.As internal reference fluorescence ROX
When having amplification curve, illustrate to expand Success in Experiment, then analyze each fluorescence results in wild and mutation group successively, determine sample
The amplification of middle CYP2C19*2, CYP2C19*3, ABCB1 and PON1 this four sites are wild-type genotypes, mutated-genotype or miscellaneous
Close genotype.
In one preferred embodiment, for detecting drawing for the gene polymorphic site related to drug metabolism enzymatic activity
Thing group is made up of above-mentioned 4 groups of primer pairs and 4 groups of probes.
Application simultaneously is used to detect CYP2C19 gene C YP2C19*2 sites, CYP2C19*3 sites, ABCB1 genes
It 3435C sites, the primer pair and probe in PON1 gene rs854560 sites, can ensure that multiple sites to be measured are carried out while examined
Survey, sensitivity is strong, the degree of accuracy is high.Also, detecting influences the medicine generation of gene code in CYP2C19, ABCB1 and PON1 gene
Thank to 4 base position base types of enzymatic activity.The mutation in this 4 sites can solve the weak metabolism of yellow race crowd's related drugs
Person, its testing result can be used for clinical guidance medication.
Present invention also offers the above-mentioned primer for being used to detect the gene polymorphic site related to drug metabolism enzymatic activity
Group is preparing the application in being used to detect the product of medicine poor metabolizer.
In one preferred embodiment, medicine is antiplatelet drug.
Antiplatelet drug, it is the medicine for suppressing hematoblastic Cycloxygenase growth.Antiplatelet drug mainly includes
Thromboxane A2 (TXA2) inhibitor aspirin, P2Y12 receptor antagonists include thiophene pyridine (clopidogrel, prasugrel) and
Non- thiophene pyridines (ticagrelor) and a acceptor inhibitors (the A Xi monoclonal antibodies of glycoprotein (glycoprotein, GP) II b/ III
And tirofiban).And phosphodiesterase inhibitors (such as Dipyridamole and Cilostazol).
In one preferred embodiment, antiplatelet drug is clopidogrel.
Clopidogrel is a kind of antiplatelet drug of novel thiophene arsenic pyridine class, can be used with aspirin combination, be pre-
Anti- and treatment acute coronary syndrome (ACS) ischemic event recurrent exerbation, receive metallic support or drug releasing stent implantation hand
Percutaneous coronary interventions (PCI) patient of art and the standard treatment of acute myocardial infarction AMI.Clopidogrel is prodrug.This
Body is inactive, the rapid metabolization in liver after intestines and stomach absorb of clopidogrel about 50%, and original shape drug concentration is extremely low in blood plasma.
However, in the patient for taking the platelet suppressant drug including clopidogrel, the absolute danger of cardiovascular event recurrent exerbation
It is dangerous still of a relatively high.Individual difference be present in the clinical treatment of clopidogrel, show as clopidogrel Resistant, or even performance
To be reactionless.Which part reason is due to that patient has gene pleiomorphism to clopidogrel metabolic enzyme gene and causes metabolic enzyme
Activity reduce or completely lose, the effect of so as to influence medicine.Therefore, drug metabolism enzyme activity is carried out to drug user before administration
The detection of property or the detection of drug metabolism weak person are particularly important.
In the medicine conversion process of clopidogrel, CYP2C19 enzymes take part in clopidogrel two-step oxidation process and finally turn
Turn to active metabolite, it is known that CYP2C19 always through medicine conversion process all the time.ABCB1 gene code P- glycoprotein
Outer row's transporter, clopidogrel are the matrix of P- glycoprotein, and the bioavilability of clopidogrel can be influenceed by suppressing P- glycoprotein.
PON1 participates in being formed the sulfydryl of clopidogrel active metabolite as a kind of rate-limiting enzyme.
In one preferred embodiment, the said goods are reagent or kit.
Present invention also offers a kind of reagent for being used to detect medicine poor metabolizer, including above-mentioned primer sets.Detection is fast
It is fast, accurate.
Present invention also offers a kind of kit for being used to detect medicine poor metabolizer, including above-mentioned primer sets or examination
Agent.
Kit provided by the present invention for detecting medicine poor metabolizer, including primer sets provided by the invention or examination
Agent, cost is low, efficiency high, can quickly and accurately detect medicine poor metabolizer, high sensitivity, high specificity.
In one preferred embodiment, mentioned reagent box also include internal control primer group, dUTP, Buffer, HsTaq,
MgCl2And sterilized water.
In addition, present invention also offers a kind of method for detecting medicine poor metabolizer, using above-mentioned primer sets or reagent
Or kit.
The method of detection medicine poor metabolizer provided by the invention, simple to operate, cost is cheap, takes short, clinical practice
Scope is wide.
In one preferred embodiment, detecting the method for medicine poor metabolizer includes：Using above-mentioned primer sets as primer,
Using the DNA of sample to be tested as template, carry out pcr amplification reaction and carry out Genotyping.
In one preferred embodiment, wild type PCR amplification system is：
| Reagent | Volume (μ L) |
| dNTP | 2 |
| Buffer | 2.5 |
| HsTaq | 0.15 |
| MgCl2 | 1 |
| CYPC219*2 sense primers | 0.5 |
| CYPC219*2 anti-sense primers | 0.5 |
| CYPC219*3 sense primers | 1 |
| CYPC219*3 anti-sense primers | 1 |
| ABCB1 sense primers | 0.4 |
| ABCB1 anti-sense primers | 0.4 |
| CYPC219*2 wild type detection probes | 0.6 |
| CYPC219*3 wild type detection probes | 0.5 |
| ABCB1 wild type detection probes | 0.5 |
| PON1 wild type detection probes | 0.5 |
| Internal reference upstream primer | 0.5 |
| Internal reference downstream primer | 0.5 |
| Internal reference probe | 0.2 |
| Template | 1 |
| Sterilized water | 11.25 |
Saltant type PCR amplification group systems are：
In one preferred embodiment, performing PCR reaction is entered using ABI 7500, PCR response procedures are：
After PCR reactions, the fluorescence signal in the two reaction tubes just can determine that four different genes of this in sample
The genotype in site.
In order to illustrate more clearly of the present invention, with reference to preferred embodiment, the present invention is described further.
Such as non-specified otherwise, the reagent and instrument used in the embodiment of the present invention are as follows：
(U.S. Applied Biosystems are public using humanized DNA quantification kits for DNA extractings kit used
Department) carry out DNA quantitatively detect (numbering 36508), constant water bath box (Heto Holten, Denmark), tabletop refrigerated centrifuge
(Sigma 3-18K, USA), TaqMan probe are bought from ABI companies, real-time fluorescence quantitative PCR instrument (ABI 7500),
Goldstar TaqMan Mixture and RNase-Free Water (win polygala biotechnology Co., Ltd in Beijing three),
Other chemical reagent such as TE, EDTA, absolute ethyl alcohol etc. are AR.
The design of primer and probe is complete with Primer Express softwares (Applied Biosystems companies of the U.S.)
Into VIC and FA8 is respectively adopted for two probes designed by each SNP site and carries out fluorescence labeling.The instrument used is
7900HT real-time fluorescence quantitative PCRs instrument (Applied Biosystems companies of the U.S.).
The processing of the sample of embodiment 1
In LINYI PEOPLE'S HOSPITAL Cardiological inpatient department, collect after percutaneous coronary stenting and acute coronary artery syndrome is normal
Rule take the clinical case history data of the patient of clopidogrel, and the blood of the ADP inductions of thrombus elastic force recording instrument (TEG) detection
Platelet inhibiting rate, it is selected in the people of patient 100 according to inclusion criteria, and gathers peripheric venous blood about 5mL, separates leucocyte and carry out
DNA is extracted.
DNA extracting LINYI PEOPLE'S HOSPITALs central laboratory completes.
Extract DNA kits：Carried out using humanized DNA quantification kits (Applied Biosystems companies of the U.S.)
DNA is quantitatively detected, including following reagent and material：Buffer AL, Buffer AW1, Buffer AW2, AE, Proteinase K, from
Stem, collecting pipe.
DNA method for extracting (centrifugal column method)：
(1) new sterile centrifugation tube is taken, with liquid-transfering gun plus the μ L of Proteinase K 20；
(2) plus the μ L of leucocyte 200, deficiency are supplied with PBS；
(3) 30 seconds plus the μ L of Buffer AL 200, are shaken, 56 DEG C of waters bath with thermostatic control 10 minutes；
(4) quickly centrifuged after water-bath, 8000rpm is centrifuged 30 seconds；
(5) plus the μ L of absolute ethyl alcohol 200, concussion 30 seconds, 8000rpm are centrifuged 30 seconds；
(6) liquid in pipe is gone in centrifugal column, does not exceed 700 μ L；
(7) 10000rpm is centrifuged 2 minutes, centrifugal column is transferred in new collecting pipe；
(8) 500 μ L, 10000rpm centrifugations of Buffer AW1 2 minutes are added；
(9) centrifugal column is transferred in new collecting pipe, adds 500 μ L, 10000rpm centrifugations of Buffer AW2 5 minutes；
(10) centrifugal column is transferred in new collecting pipe, 10000rpm is centrifuged 3 minutes；
(11) centrifugal column is put into new Eppendorf to manage, adds the μ L of AE 200；
(12) 10000rpm is centrifuged 2 minutes after standing 5 minutes, removes centrifugal column, 4 DEG C of the DNA of dissolving is stored for future use.
The detection of the sample of embodiment 2
Using Real-time quantitative PCR to CYP2C19*2, * 3, ABCB1, PON1, Genotyping is carried out.It is used in experiment
To primer and probe sequence be offer of the present invention.
(1) configuration of PCR reaction systems
DUTP, Buffer, the MgCl added in each reacting hole in kit2, HsTaq, template DNA, interior reference
The PCR reaction systems of thing and internal reference probe are as follows：
Wild type PCR amplification system is：
| Reagent | Volume (μ L) |
| dNTP | 2 |
| Buffer | 2.5 |
| HsTaq | 0.15 |
| MgCl2 | 1 |
| CYPC219*2 sense primers | 0.5 |
| CYPC219*2 anti-sense primers | 0.5 |
| CYPC219*3 sense primers | 1 |
| CYPC219*3 anti-sense primers | 1 |
| ABCB1 sense primers | 0.4 |
| ABCB1 anti-sense primers | 0.4 |
| CYPC219*2 wild type detection probes | 0.6 |
| CYPC219*3 wild type detection probes | 0.5 |
| ABCB1 wild type detection probes | 0.5 |
| PON1 wild type detection probes | 0.5 |
| Internal reference upstream primer | 0.5 |
| Internal reference downstream primer | 0.5 |
| Internal reference probe | 0.2 |
| Template | 1 |
| Sterilized water | 11.25 |
Saltant type PCR amplification group systems are：
PCR response procedures are：
Result after interpretation：
| Genotype | Number of cases |
| CYPC219*2 wild types | 12 |
| CYPC219*2 saltant types | 8 |
| CYPC219*2 heterozygous | 3 |
| CYPC219*3 wild types | 15 |
| CYPC219*3 saltant types | 7 |
| CYPC219*3 heterozygous | 4 |
| ABCB1 wild types | 17 |
| ABCB1 saltant types | 5 |
| ABCB1 heterozygous | 2 |
| PON1 wild types | 20 |
| PON1 saltant types | 5 |
| PON1 heterozygous | 2 |
In summary, provided by the present invention for the primer in the detection gene polymorphic site related to drug metabolism enzymatic activity
Group, the gene polymorphic site related to drug metabolism enzymatic activity not of the same race can be detected simultaneously, detection is comprehensive, is widely used, and examines
Survey result and can be used for clinical guidance medication.The method of detection medicine poor metabolizer provided by the invention, simple to operate, cost is low
Honest and clean, time-consuming short, clinical application range is wide.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, either which part or all technical characteristic are entered
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
SEQUENCE LISTING
<110>Shandong Long Chen Bioisystech Co., Ltd
<120>The primer sets in the detection gene polymorphic site related to drug metabolism enzymatic activity and its application and application its production
Product and detection method
<160> 19
<170> PatentIn version 3.5
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<212> DNA
<213>Artificial sequence
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tcacttacat atggaataat tttcgca 27
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ccaatatatc actatccata agagctag 28
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aatattgaat gaagacatca cgattg 26
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tgtactacag cgcttcgtca 20
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tgtatcttgg tctcctctgc 20
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agcatcgctg agatcatcgc c 21
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aaagaagagt gatgtatagc cccagtttca a 31
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acagagctaa tgaaagccag tccattaggc agtatctcta 40
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ccgggaaccc ata 13
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ttcccaggaa ccc 13
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accccctgga tc 12
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<213>Artificial sequence
<400> 12
gcaccccctg aatc 14
<210> 13
<211> 13
<212> DNA
<213>Artificial sequence
<400> 13
cctcacgatc tct 13
<210> 14
<211> 14
<212> DNA
<213>Artificial sequence
<400> 14
ccctcacaat ctct 14
<210> 15
<211> 18
<212> DNA
<213>Artificial sequence
<400> 15
tttggcagga acaggagc 18
<210> 16
<211> 22
<212> DNA
<213>Artificial sequence
<400> 16
tggaataaac tggaaccatg tt 22
<210> 17
<211> 30
<212> DNA
<213>Artificial sequence
<400> 17
tctacccttg gacccagagg ttctttgagt 30
<210> 18
<211> 24
<212> DNA
<213>Artificial sequence
<400> 18
tctgatagcc agtgactctc tctg 24
<210> 19
<211> 23
<212> DNA
<213>Artificial sequence
<400> 19
aacaccatct gcagtgcaca gat 23
Claims (10)
1. a kind of primer sets for being used to detect the gene polymorphic site related to drug metabolism enzymatic activity, it is characterised in that described
Primer sets include following 4 groups of primer pairs or at least one set of primer pair or probe in 4 groups of probes；
The primer pair includes：For detecting the primer pair in CYP2C19 gene C YP2C19*2 sites, for detecting CYP2C19 bases
Primer pair because of the primer pair in CYP2C19*3 sites, for detecting ABCB1 gene 3435C sites and for detecting PON1 genes
The primer pair in rs854560 sites；
The probe includes：For detecting wild type detection probe and the saltant type detection in CYP2C19 gene C YP2C19*2 sites
Probe, the wild type detection probe for detecting CYP2C19 gene C YP2C19*3 sites and saltant type detection probe, for examining
Survey the wild type detection probe and saltant type detection probe and for detecting PON1 genes in ABCB1 gene 3435C sites
The wild type detection probe and saltant type detection probe in rs854560 sites；
The primer pair for being used to detect CYP2C19 gene C YP2C19*2 sites includes SEQ ID NO.1 and SEQ ID NO.2
Shown nucleotide sequence；
The primer pair for being used to detect CYP2C19 gene C YP2C19*3 sites includes SEQ ID NO.3 and SEQ ID NO.4
Shown nucleotide sequence；
The primer pair for being used to detect ABCB1 gene 3435C sites is included shown in SEQ ID NO.5 and SEQ ID NO.6
Nucleotide sequence；
The primer pair for being used to detect PON1 gene rs854560 sites is included shown in SEQ ID NO.7 and SEQ ID NO.8
Nucleotide sequence；
The wild type detection probe for being used to detect CYP2C19 gene C YP2C19*2 sites has as shown in SEQ ID NO.9
Nucleotide sequence, saltant type detection probe has nucleotide sequence as shown in SEQ ID NO.10；
The wild type detection probe for being used to detect CYP2C19 gene C YP2C19*3 sites has such as SEQ ID NO.11 institutes
The nucleotide sequence shown, saltant type detection probe have the nucleotide sequence as shown in SEQ ID NO.12；
The wild type detection probe for being used to detect ABCB1 gene 3435C sites is with the core as shown in SEQ ID NO.13
Nucleotide sequence, saltant type detection probe have the nucleotide sequence as shown in SEQ ID NO.14；
The wild type detection probe for being used to detect PON1 gene rs854560 sites has as shown in SEQ ID NO.15
Nucleotide sequence, saltant type detection probe have the nucleotide sequence as shown in SEQ ID NO.16.
2. primer sets according to claim 1, it is characterised in that the primer sets are by 4 groups of primer pairs and 4 groups of probe groups
Into.
3. primer sets as claimed in claim 1 or 2 are preparing the application in being used to detect the product of medicine poor metabolizer.
4. application according to claim 3, it is characterised in that the medicine is antiplatelet drug.
5. application according to claim 3, it is characterised in that the product is reagent or kit.
6. a kind of reagent for being used to detect medicine poor metabolizer, it is characterised in that including the primer sets described in claim 1 or 2.
7. a kind of kit for being used to detect medicine poor metabolizer, it is characterised in that including the primer described in claim 1 or 2
Reagent described in group or claim 6.
8. kit according to claim 7, it is characterised in that the kit also include internal control primer group, dUTP,
Buffer、HsTaq、MgCl2And sterilized water.
A kind of 9. method for detecting medicine poor metabolizer, it is characterised in that using the primer sets or power described in claim 1 or 2
Profit requires the kit described in reagent or claim 7 described in 6.
10. according to the method for claim 9, it is characterised in that methods described includes：Using the primer sets as primer, with
The DNA of sample to be tested is template, carries out pcr amplification reaction and carries out Genotyping.
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Application publication date: 20180327 |