CN106498053A - A kind of method for fast and accurately detecting CYP2D6 gene copy number variations - Google Patents
A kind of method for fast and accurately detecting CYP2D6 gene copy number variations Download PDFInfo
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Abstract
The invention discloses a kind of method for fast and accurately detecting CYP2D6 gene copy number variations：(1) primer of detection CYP2D6 gene pleiomorphisms is prepared；(2) RPP30 gene primers and probe are prepared；(3) quantitative fluorescent PCR reaction：Copy number variation using the method detection detection CYP2D6 genes of the double quantitative PCR of single tube；(4) sample that CYP2D6 genes are single copy is taken as control sample.Detection object and control sample respectively carry out the PCR reactions of 4 repetitions, obtain the Ct values of real-time quantitative PCR amplification；(5) fluorescent quantitative PCR result analysis.Real-time quantitative fluorescence PCR test method of the present invention does not have PCR post processings, being capable of time-consuming in a large number and manpower.Test from DNA extraction to fluorescent PCR and terminate, and data analysiss, all processes are only needed 4 hours, the real experiment operating time is less than 2 hours.
Description
Technical field
The present invention relates to for determining from small group or the method for individuality CYP2D6 copy number changes, the method can
Changed with the copy number of the detection CYP2D6 genes of quickly, efficiently and accurately, the concurrently application in biology and medical science now.
Background technology
CYP2D6 is first and is identified by the P450 enzymes of Dominant gene.CYP2D6 is located on No. 22 chromosome, altogether
Containing 9 exons and 8 introns, total length is about 5400bp, is a complete functioning gene.Modern study is sent out
Existing, in liver, the content of CYP2D6 only accounts for the 2% of P450 liver protein total amounts, but the clinic that it but participates in metabolism about 25% is used
Medicine, including antidepressants, anti-arrhythmic, psychosis and analgesic etc..Have now been found that more than 100 kinds of CYP2D6 etc.
The variation of position gene, mainly includes single nucleotide variations, the loss of large fragment gene and copy number variation.These variations make
CYP2D6 genes assume polymorphism, and determine the enzymatic activity of its encoding proteins show as lacking, reduce, normal or increase etc. several
Type, further equally shows multiformity to the metabolism of medicine,.
Have now been found that CYP2D6 genes have more than 100 variant sites, but affect the normal of Chinese's CYP2D6 functions
See that variation there are 8, including fast metabolic pattern genovariation (gene copy number increases), intermediate supersession type genovariation (CYP2D6*
9th, * 10, * 14 and * 41) and slow inactivation genovariation (CYP2D6*3, * 4 and * 5).These variations of CYP2D6 genes can cause
Enzymatic activity and the difference of enzyme quantity, cause the generation of curative effect deficiency or toxic and side effects, the final individual diversity for producing curative effect of medication
Different.Therefore, the polymorphic Journal of Sex Research of CYP2D6 has great importance to clinic, has become the focus of current research.
The copy number change of CYP2D6 genes is even more important, because the copy number that gene redundancy causes increases and gene delection
It is the major reason for affecting CYP2D6 enzymatic activitys to increase and reduce that the copy number for causing is reduced.The monokaryon glycosides of detection CYP2D6 genes
Sour polymorphism, oligonucleotide insertion and disappearance can detect disappearance and the reduction of CYP2D6 enzymatic activitys, but only gene copy
Several increases can determine the increase of CYP2D6 enzymatic activitys.Therefore, CYP2D6 gene copy numbers detection has weight in clinical practice
Want meaning.
The product of at present both at home and abroad detection CYP2D6 gene mutation, the xTAG liquid-phase chips of such as Luminex, Roche
The susceptivenesss such as AmpliChip CYP450 chips, direct sequencing, PCR- single-strand conformation polymorphism analysis (SSCP) detection are not
Height, testing result are repeatable poor.These methods need to expend a large amount of manpowers, while required detection time is longer, from nucleic acid
Extract result to obtain more than 8 hours, i.e., more than 1 day normal working hours.In addition these methods all fast and accurately cannot be surveyed
Determine the copy number change of CYP2D6 genes.At present, accurately the method for identification CYP2D6 copy number changes has numeral
Round pcr (101821619 B of patent CN) and the standard curve law technology (104263820 A of patent CN) based on quantitative PCR,
But two methods reagent cost is higher, should not promote.
In this application, we detect the copy number change of CYP2D6 genes using real-time fluorescence PCR method.Real-time fluorescence
The sensitivity of PCR methods is high, and typing accurately, simple and efficient to handle, easily popularize, it is easy to promotes the use of by instrument.Meanwhile, at us
Research in, for CYP2D6 genes, we employ multiple probes, can accomplish that result accuracy is high, detect convenient and efficient,
Also large sample amount can be used for quickly detecting simultaneously.
Content of the invention
Based on the technical problem that background technology is present, shortcoming of the present invention for prior art, real-time quantitative fluorescence PCR are examined
Survey method does not have PCR post processings, being capable of time-consuming in a large number and manpower.Test from DNA extraction to fluorescent PCR and terminate, and data
Analysis, all processes are only needed 4 hours, and the real experiment operating time is less than 2 hours.
Traditional real-time quantitative fluorescence PCR detection technique and product are often designed just for single site, it is impossible to while
Obtain multiple site informations.The accurate letter of CYP2D6 copy number changes can accurately not be obtained in the case of detection unit point
Breath.What the present invention was detailed analyzes CYP2D6 genes and its homogenic sequence difference, is further directed to CYP2D6 genes
Specific DNA sequence, respectively near the Second Exon of CYP2D6 genes, the 6th include sub-district and the 9th exon 1 sets respectively
A pair of detection primers and probe are counted such that it is able to obtain multiple site informations of CYP2D6 genes, have been accurately identification
CYP2D6 genes have laid good scientific basic.Additionally, the method is set up on the basis of common fluorescent PCR, with operation
Simple and efficient, with low cost, it is easy to the advantage of popularization.
Technical scheme is as follows：
1st, the primer of one group of detection CYP2D6 gene pleiomorphism, it is characterised in that：Including 3 sections of CYP2D6 genes
Pcr amplification primer thing and fluorescent probe.CYP2D6 gene pairss answer probe FAM to carry out 5 ' end labellings.
3 groups of primers and probe sequence of concrete CYP2D6 genes are as follows：
2nd, in order to study the copy number variation of CYP2D6 genes, from human genome single copy gene RPP30
(ribonuclease P/MRP 30kDa subunit) is used as reference sequences.RPP30 gene pairss answer probe HEX to carry out 5 ' ends
Labelling.
RPP30 gene primers and probe are respectively：
3rd, quantitative fluorescent PCR reaction：
Using the copy number variation of the method detection detection CYP2D6 genes of the double quantitative PCR of single tube, concrete reaction system
For 10 μ L, comprising 8ng gene DNAs, 5 μ L 2x TaqMan mix, RPP30 gene primers and probe and one group of CYP2D6 gene
Primer and probe (primer and probe final concentration are 0.2 μM), then moisturizing is to 10 μ L.For each sample, 4 weights are carried out
Multiple.PCR amplification conditions carry out thermal starting for 95 DEG C of 10min, then 95 DEG C of 15s degeneration, 60 DEG C of annealing and amplification 1min, and altogether 40
Individual circulation.
4th, sample that CYP2D6 gene be single copy is taken as control sample.Detection object and control sample respectively carry out 4
The PCR reactions of repetition, obtain the Ct values of real-time quantitative PCR amplification.
5th, fluorescent quantitative PCR result parser：
(1) weighted value of CYP2D6 is determined using reference gene.
Weighted value=sample CYP2D6 Average Ct values ÷ sample reference gene RPP30 Average Ct values
(2) gene copy number of detection object is calculated using the CYP2D6 copy numbers of control crowd.
α=detection object weighted value ÷ control sample weighted value
If α<0.2, the CYP2D6 copy numbers of detection object are 0；
If α is 0.5-1.3, the CYP2D6 copy numbers of detection object are 1；
If α is 1.5-2.3, the CYP2D6 copy numbers of detection object are 2；
If α is 2.5-3.3, the CYP2D6 copy numbers of detection object are 3；
If α is 3.5-4.3, the CYP2D6 copy numbers of detection object are 4；
If α is other values need to detect again.
The invention has benefit that：
(1) present invention devises brand-new CYP2D6 genes and single copy base using real-time fluorescence quantitative PCR detection technique
Because internal control primer and fluorescent probe are used for the detection of copy number change.
(2) application claims test crowd and control crowd carry out multigroup repetition and are detected in same PCR plate,
Employ the 384 orifice plate reaction systems of 16x24.Can accurately, fast and efficiently carry out the copy number inspection of multiple patient samples
Survey.
(3) present invention greatly reduces the cost of CYP2D6 gene copy numbers detection, improves gene copy number detection
Accuracy.
(4) principle of the invention may apply to existing any fluorescence real-time quantitative PCR instrument, convenient use, it is easy to
Interior popularization on a large scale.
(5) as a result detection method of the present invention be able to can be used with the copy number of simple and quick detection CYP2D6 genes
Instruct in the personalized medicine of following multi-medicament, such as beta-blocker, antidepressants, anti-arrhythmic, psychosiss
Medicine, analgesic etc., corresponding medicament categories have Aripiprazole, atomoxetine, venlafaxine, Risperidone, plug support bromo-amine suction,
Tamoxifen, timolol, fluoxetine, olanzapine, cevimeline, tolterodine, terbinafine, tramadol, clozapine, Mei Tuo
Luo Er, Propranolol, carvedilol, Propafenone, thioridazine, protriptyline, codeine, pimozide, tetrabenazine, she
Pan Li ketone etc..
Description of the drawings
Fig. 1 is the flow chart of the present invention.
Specific embodiment
Embodiment 1 prepares the primer of detection CYP2D6 gene pleiomorphisms：
Pcr amplification primer thing and fluorescent probe including 3 sections of CYP2D6 genes.CYP2D6 gene pairss answer probe FAM
Carry out 5 ' end labellings.
3 groups of primers and probe sequence of concrete CYP2D6 genes are as follows：
Embodiment 2 prepares RPP30 gene primers and probe：
In order to study the copy number variation of CYP2D6 genes, from human genome single copy gene RPP30
(ribonuclease P/MRP 30kDa subunit) is used as reference sequences.RPP30 gene pairss answer probe HEX to carry out 5 ' ends
Labelling.
RPP30 gene primers and probe are respectively：
3 quantitative fluorescent PCR of embodiment reacts：
Using the copy number variation of the method detection detection CYP2D6 genes of the double quantitative PCR of single tube, concrete reaction system
For 10 μ L, comprising 8ng gene DNAs, 5 μ L 2x TaqMan mix, RPP30 gene primers and probe and one group of CYP2D6 gene
Primer and probe (primer and probe final concentration are 0.2 μM), then moisturizing is to 10 μ L.For each sample, 4 weights are carried out
Multiple.PCR amplification conditions carry out thermal starting for 95 DEG C of 10min, then 95 DEG C of 15s degeneration, 60 DEG C of annealing and amplification 1min, and altogether 40
Individual circulation.
Embodiment 4：
Sample that CYP2D6 genes are single copy is taken as control sample.Detection object and control sample respectively carry out 4 weights
Multiple PCR reactions, obtain the Ct values of real-time quantitative PCR amplification.
5 fluorescent quantitative PCR result parser of embodiment：
(1) weighted value of CYP2D6 is determined using reference gene.
Weighted value=sample CYP2D6 Average Ct values ÷ sample reference gene RPP30 Average Ct values
(2) gene copy number of detection object is calculated using the CYP2D6 copy numbers of control crowd.
α=detection object weighted value ÷ control sample weighted value
If α<0.2, the CYP2D6 copy numbers of detection object are 0；
If α is 0.5-1.3, the CYP2D6 copy numbers of detection object are 1；
If α is 1.5-2.3, the CYP2D6 copy numbers of detection object are 2；
If α is 2.5-3.3, the CYP2D6 copy numbers of detection object are 3；
If α is 3.5-4.3, the CYP2D6 copy numbers of detection object are 4；
If α is other values need to detect again.
It can be seen that, compared with prior art, the technology have testing result accurately and reliably, flux is higher, experimental design is flexible,
Simple and efficient to handle, detection speed is fast, easy to spread, and with low cost.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, should all be included within the scope of the present invention.
Claims (4)
1. the primer of one group of detection CYP2D6 gene pleiomorphism, it is characterised in that：PCR including 3 sections of CYP2D6 genes expands
Increase primer and fluorescent probe, CYP2D6 gene pairss answer probe FAM to carry out 5 ' end labellings；
3 groups of primers of the CYP2D6 genes and probe sequence are as follows：
The RPP30 gene primers and probe are respectively：
RPP30-P-F 5’-AGATTTGGACCTGCGAGCG-3’
RPP30-P-R 5’-GAGCGGCTGTCTCCACAAGT-3’
RPP30-P 5’HEX-TTCTGACCTGAAGGCTCT-3'BHQ-1.
2. a kind of fast and accurately detect CYP2D6 gene copy number variations method, it is characterised in that step is as follows：
(1) primer of detection CYP2D6 gene pleiomorphisms is prepared, it is characterised in that：PCR including 3 sections of CYP2D6 genes expands
Increase primer and fluorescent probe, CYP2D6 gene pairss answer probe FAM to carry out 5 ' end labellings；
3 groups of primers of the CYP2D6 genes and probe sequence are as follows：
(2) RPP30 gene primers and probe are prepared, and the RPP30 gene primers and probe are respectively：
RPP30-P-F 5’-AGATTTGGACCTGCGAGCG-3’
RPP30-P-R 5’-GAGCGGCTGTCTCCACAAGT-3’
RPP30-P 5’HEX-TTCTGACCTGAAGGCTCT-3'BHQ-1；
(3) quantitative fluorescent PCR reaction：
Copy number variation using the method detection detection CYP2D6 genes of the double quantitative PCR of single tube；
(4) sample that CYP2D6 genes are single copy is taken as control sample.Detection object and control sample respectively carry out 4 repetitions
PCR reaction, obtain real-time quantitative PCR amplification Ct values；
(5) fluorescent quantitative PCR result analysis.
3. the method for fast and accurately detecting CYP2D6 gene copy number variations as claimed in claim 2, it is characterised in that：
The concrete reaction system of step (3) is 10 μ L, comprising 8ng gene DNAs, 5 μ L 2x TaqMan mix, RPP30 gene primers
With the primer and probe (primer and probe final concentration are 0.2 μM) of probe and one group of CYP2D6 gene, then moisturizing is to 10 μ
L.For each sample, 4 repetitions are carried out.PCR amplification conditions carry out thermal starting for 95 DEG C of 10min, and then 95 DEG C of 15s become
Property, 60 DEG C are annealed and amplification 1min, altogether 40 circulations.
4. the method for fast and accurately detecting CYP2D6 gene copy number variations as claimed in claim 2, it is characterised in that：
Fluorescent quantitative PCR result parser is：
The weighted value for determining CYP2D6 using reference gene；
Weighted value=sample CYP2D6 Average Ct values ÷ sample reference gene RPP30 Average Ct values
The gene copy number of detection object is calculated using the CYP2D6 copy numbers of control crowd；
α=detection object weighted value ÷ control sample weighted value
If α<0.2, the CYP2D6 copy numbers of detection object are 0；
If α is 0.5-1.3, the CYP2D6 copy numbers of detection object are 1；
If α is 1.5-2.3, the CYP2D6 copy numbers of detection object are 2；
If α is 2.5-3.3, the CYP2D6 copy numbers of detection object are 3；
If α is 3.5-4.3, the CYP2D6 copy numbers of detection object are 4；
If α is other values need to detect again.
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Cited By (7)
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| CN108342457A (en) * | 2018-04-01 | 2018-07-31 | 佛山市顺德区辉锦创兴生物医学科技有限公司 | The detection kit of HLA-B5801 allele and its application |
| CN109371132A (en) * | 2018-11-30 | 2019-02-22 | 广东腾飞基因科技股份有限公司 | It is a kind of for detecting the kit of CYP2D6 gene copy number variation |
| CN112522385A (en) * | 2020-12-11 | 2021-03-19 | 长沙金域医学检验实验室有限公司 | Kit for detecting DNM2 gene copy number variation |
| CN113151441A (en) * | 2021-04-12 | 2021-07-23 | 湖南菲思特精准医疗科技有限公司 | Gene detection kit for beta receptor antagonist medication and method and application thereof |
| CN113186267A (en) * | 2021-06-07 | 2021-07-30 | 上海康黎诊断技术有限公司 | Primer-probe combination, kit and method for detecting human CYP2D6 copy number variation and genotyping |
| CN113186266A (en) * | 2021-06-07 | 2021-07-30 | 上海康黎诊断技术有限公司 | Method for detecting copy number variation of human CYP2D6 gene |
| CN113337593A (en) * | 2021-05-13 | 2021-09-03 | 广西金则医学科技发展有限公司 | Primer probe combination, kit and gene detection method for SSRIs and tricyclic antidepressant precise medication |
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| CN108342457A (en) * | 2018-04-01 | 2018-07-31 | 佛山市顺德区辉锦创兴生物医学科技有限公司 | The detection kit of HLA-B5801 allele and its application |
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| CN109371132A (en) * | 2018-11-30 | 2019-02-22 | 广东腾飞基因科技股份有限公司 | It is a kind of for detecting the kit of CYP2D6 gene copy number variation |
| CN112522385A (en) * | 2020-12-11 | 2021-03-19 | 长沙金域医学检验实验室有限公司 | Kit for detecting DNM2 gene copy number variation |
| CN113151441A (en) * | 2021-04-12 | 2021-07-23 | 湖南菲思特精准医疗科技有限公司 | Gene detection kit for beta receptor antagonist medication and method and application thereof |
| CN113337593A (en) * | 2021-05-13 | 2021-09-03 | 广西金则医学科技发展有限公司 | Primer probe combination, kit and gene detection method for SSRIs and tricyclic antidepressant precise medication |
| CN113186267A (en) * | 2021-06-07 | 2021-07-30 | 上海康黎诊断技术有限公司 | Primer-probe combination, kit and method for detecting human CYP2D6 copy number variation and genotyping |
| CN113186266A (en) * | 2021-06-07 | 2021-07-30 | 上海康黎诊断技术有限公司 | Method for detecting copy number variation of human CYP2D6 gene |
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