CN109371132A - A kit for detecting CYP2D6 gene copy number variation - Google Patents
A kit for detecting CYP2D6 gene copy number variation Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention relates to a kit for detecting CYP2D6 gene copy number variation, which comprises a No. 2 intron, a No. 6 exon, a No. 5 intron and an internal reference gene RPP30 for detecting CYP2D6 gene, and the copy number variation of the CYP2D6 gene is rapidly and accurately measured by using a real-time fluorescence quantification technology. The kit can be used for simultaneously carrying out high-throughput detection on 3 loci of CYP2D6 related genes under the same reaction condition, has strong specificity and high sensitivity, the minimum detection limit is 1 ng/mu L, the time consumption is short, and the result obtained from sample submission can be completed within 2 hours.
Description
Technical field
The present invention relates to CYP2D6 genetic polymorphism detection fields, more specifically it relates to a kind of for detecting CYP2D6 base
Because copying the kit of number variation.
Background technique
CYP2D6 is important one of the member of CYP enzyme family, accounts for the 2-9% of liver enzyme total amount, but participates in 20-30% medicine
The metabolism of object, including beta-blocker, antidepressants, antiarrhymic, antipsychotic drug (the maximum A Li of such as sales volume
Piperazine azoles), antalgesic etc..CYP2D6 is first P450 enzyme being identified by Dominant gene.CYP2D6 is located at No. 22 dyeing
On body, it is about altogether 5400bp containing 9 exons and 8 intrones, total length, is a complete functioning gene.Research
It was found that there are the variations such as polymorphism and gene copy number repetition for CYP2D6 gene.Have now been found that more than 100 kinds CYP2D6 equipotentials
The variation of gene mainly includes single nucleotide variations, the loss of large fragment gene and copy number variation.These variations make
CYP2D6 gene presentation polymorphism, and it is several to determine that the enzymatic activity of its coding albumen shows as missing, reduction, normal or increase etc.
Seed type further equally shows diversity to the metabolism of drug.
The main method of CYP2D6 copy number abnormality detection includes the sequencing of two generations, micro-array chip, fluorescent PCR at this stage
Deng.These methods or time-consuming, it is complicated for operation, or have higher requirements to operator and experimental site or testing cost is higher, or
It is difficult to detect the different mutational sites of multiple genes simultaneously.Therefore, it is necessary to establish a kind of high throughput, high efficiency, low cost
Gene expression dose detection method and product, to realize quickly detection and the generally detection of patient with breast cancer.
Summary of the invention
We have found under study for action, and the common change of Chinese CYP2D6 function is influenced in a variant sites of CYP2D6 gene
It is different to have 8, including (gene copy number increases fast metabolic pattern genetic mutation.Including type allele * 1, allele * 2), it is intermediate
Metabolic pattern genetic mutation (CYP2D6*9, * 10, * 14 and * 41) and slow inactivation genetic mutation (CYP2D6*3, * 4 and * 5).
These variations of CYP2D6 gene can cause the difference of enzymatic activity and enzyme quantity, cause curative effect insufficient or the production of toxic side effect
It is raw, the final individual difference for generating curative effect of medication.Therefore, the polymorphism research of CYP2D6 has great importance to clinic,
Through becoming the hot spot studied at present.The copy number variation of CYP2D6 gene is even more important, because of copy number caused by Duplication
Increase and gene delection caused by copy number reduction be to influence CYP2D6 enzymatic activity to increase and reduced major reason.Detection
Single nucleotide polymorphism, oligonucleotides insertion and the missing of CYP2D6 gene are able to detect the missing and drop of CYP2D6 enzymatic activity
It is low, but the increase of only gene copy number can determine the increase of CYP2D6 enzymatic activity.Therefore, CYP2D6 gene copy number is examined
Survey is of great significance in clinical application.Microflow control technique is micro-volume reaction to be realized with micro-processing technology, and can realize high pass
Manual operations and testing cost is greatly reduced in quantitative response, significantly improves efficiency.
Based on the above research, the present invention provides a kind of for detecting the kit of CYP2D6 gene copy number variation, wraps
Include the PCR amplification primer of multiple sections for detecting CYP2D6 gene and the primer and spy of fluorescence probe and reference gene
Needle.
In a preferred embodiment, the section of CYP2D6 gene detected includes the 2nd of CYP2D6 gene including
One or more of son, the 6th exon, the 5th introne.
In a preferred embodiment, the primer pair for detecting intron 2 is visited as shown in SEQ ID NO 1 and 2
Needle is as shown in SEQ ID NO 3.
In a preferred embodiment, the primer pair for detecting the 6th exon is visited as shown in SEQ ID NO 4 and 5
Needle is as shown in SEQ ID NO 6.
In a preferred embodiment, the primer pair for detecting the 5th introne is visited as shown in SEQ ID NO 7 and 8
Needle is as shown in SEQ ID NO 9.
In a preferred embodiment, the reference gene is RPP30 gene.
In a preferred embodiment, for detecting the primer pair of RPP30 gene as shown in SEQ ID NO 10 and 11,
Probe is as shown in SEQ ID NO 12.
Using micro-fluidic detection of nucleic acids instrument MF800, and using the high-throughput micro-fluidic chip battle array of 12 rows × 12 column design
Column technology is realized it is accurate, quick and economical while detection CYP2D6 gene copy number variation 3 sites and 1 internal reference base
Cause, each samples of each column can be with the probe in 3 copy number sites in micro-fluidic array and 1 reference genes
Probe reaction.
It can be under the same reaction condition simultaneously to 3 and the site of CYP2D6 related gene using kit of the invention
Carry out high-throughput detection.The high specificity of detection, high sensitivity, lowest detection are limited to 1ng/ μ L, and the used time is short, from specimen transfer to
Obtaining a result can complete within 2 hours.Suitable for the detection and screening to CYP2D6 gene.
Detailed description of the invention
Fig. 1 is that the intro 2 detected using the kit in embodiment analyzes the amplification curve diagram of result.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
1. the detection in site
1.1 detection techniques and instrument
In order to fast high-flux detect these mutation, we use microflow control technique, including micro-fluidic chip and
Two parts of microfluidic platform.
Micro-fluidic chip is integrated in microfluidic channel, valve and reaction warehouse on one chip, and air pressure, temperature control are passed through
System processed and system of fluorescence analysis are automatically performed reaction system mixing, realize up to 9216 PCR reactions.Greatly reduce craft
The step of sample-adding and time complete whole process about 2 hours.
It is all accurate on micro-fluidic chip to be provided with many microchannels (sample, reagent, control liquid channel) and micro- reaction warehouse, in advance
It first is separately added into reagent, sample and control liquid in injection port, the different valve switch on chip can be realized by pressure control, essence
Quasi- control solution flows in chip channel, mixes in reaction warehouse, 9216 nL grades of PCR reaction warehouse at most may be implemented,
Carry out real-time fluorescence quantitative PCR reaction.
The microfluidic platform that we use (corresponds to Fluidigm's by the micro-fluidic nucleic acid augmentative instrument of Ascend MF600 type
Juno it) is formed with the micro-fluidic detection of nucleic acids instrument of Ascend MF800 type (Biomark of corresponding Fluidigm).
The difference of the micro-fluidic nucleic acid augmentative instrument of Ascend MF600 type (Juno) PCR and common amplification instrument essentially consists in miniflow
Control technology, principle are as follows:
Microfluidic channel, valve and reaction warehouse are integrated on a chip by micro-fluidic nucleic acid augmentative instrument using chip,
Reaction system mixing and PCR reaction are automatically performed by micro-fluidic nucleic acid augmentative instrument pneumatic control system and temperature control system.
The performance of micro-fluidic nucleic acid augmentative instrument built-in PC control system adjustable and monitoring instrument, identification and memorization COMS clip
Bar code.Using touch LCD display, customized and selection needs test procedures.Instrument use chip by microfluidic channel,
Valve and reaction warehouse are integrated on a chip, by pneumatic control system control sample and reagent micro-fluidic chip each
The indoor accurate flowing of small reaction and mixing, temperature control module realize quick, accurate, uniform heating/drop during PCR
Temperature realizes micro-fluidic PCR reaction on chip.Replace manual operation completely, realize reaction system mixing and PCR reaction it is complete from
Dynamicization.Micro-fluidic nucleic acid augmentative instrument includes pneumatic control system and temperature control control module, and pneumatic control system passes through vacuum pump
Accurate flowing of the pressure control liquid in micro-fluidic chip can be used as chip sample and reagent pretreatment controller, embedded
The performance of PC adjustable and monitoring instrument；Temperature control module controls quick, accurate, uniform heating/cooling during PCR.User
Using touch LCD display, customized and selection needs test procedures.
Micro-fluidic detection of nucleic acids instrument principle is similar to real-time fluorescence quantitative PCR instrument.Principle is as follows: micro-fluidic detection of nucleic acids instrument
Microfluidic channel, valve and reaction warehouse are integrated on a chip by institute using chip, can be automatically performed PCR reaction and result point
Analysis.When PCR reacts just beginning, amount of reagent is sufficient, and the concentration of template and product is all sufficiently low, and product is not competed with primer, expands
Increasing is increased with constant exponential rate.Late phase reaction rate is no longer exponentially increased, and amplification increases in variable linear, into linear
Rise period.In plateau, rate of amplification levels off to zero.
1.2 detection primers and probe design
High-throughput quickly detection can be achieved in the combination of micro-fluidic chip and platform.It is all anti-but on a chip
It answers the reaction condition of system to be consistent, needs to be comprehensively considered in terms of primer and probe, realize all 4 kinds of detections reactions
Effective and accurate testing result can be obtained under the same reaction condition.This brings to disposably detecting different sites
Great difficulty.
We have spent the plenty of time to study and experimental verification, and it is disposable right to realize to devise following primer and probe
Above-mentioned 4 sites effectively accurately detect.Primer and probe it is as shown in table 1.1 detection primer of table and probe
| Number | Title | Sequence |
| SEQ ID NO 1 | intro 2 FW | CCCTGCTGCAGCAAAGTTCAT |
| SEQ ID NO 2 | intro 2 RW | TGAGCCCAGGTGGATG |
| SEQ ID NO 3 | intro 2 probe | GGTGCCCAGCCTGTACGCTTG |
| SEQ ID NO 4 | Exon 6 FW | CAAAGTTCATCCCTGCTGCAG |
| SEQ ID NO 5 | Exon 6 RW | TCACCAGGATCTGGGAAAC |
| SEQ ID NO 6 | Exon 6 probe | GGCCCGATTAGCTTCTAATCCGGCCCGATTA |
| SEQ ID NO 7 | intro 5 FW | ACCATCCACCACCCACTCC |
| SEQ ID NO 8 | intro 5 RW | GGTGGATGCACAAAGAGTGG |
| SEQ ID NO 9 | intro 5 probe | TTAGCTTCTAATCCGGCCCTGGTCTCCCGCA |
| SEQ ID NO 10 | RPP30 FW | AGATCAGGACCTGCGAGCG |
| SEQ ID NO 11 | RPP30 RW | CTCGGGCTGTCTCCACAAGT |
| SEQ ID NO 12 | RPP30 probe | AACTGACCTGAACCCTCT |
1.3 reaction systems composition and reaction condition.
The template can be whole blood.
Reaction condition is as shown in table 2.
2 reaction condition of table
3 specific detection examples
4 pairs of primers and 4 probes are synthesized by Invitrogen；192.24 chip is purchased from Fluidigm company；ROX reference dye
Purchased from Life company；Specific PCR reaction solution is purchased from Applied Biosystems company.
Instrument: micro-fluidic nucleic acid augmentative instrument (Juno), micro-fluidic detection of nucleic acids instrument (Biomark), 2720 type PCR amplifications
Instrument, turbula shaker, centrifuge.
This kit is used to have confirmed that total 18 Whole Blood Genomic DNAs of copy number variation, 1 CYP2D6 base with clinic
Because the control sample singly copied is template, establishes micro-fluidic fluorescent PCR and detect 3 CYP2D6 genes and reference RPP gene
Reaction system, the standard finally judged as a result with amplification curve diagram after circulation terminates.
Primed probe mix X is prepared, wherein X indicates number 1-4, respectively represents first to fourth site.Divide according to table 3
Not Pei Zhi 24 sites primed probe mix.By primed probe mix X according to 10 × reagent is prepared shown in table 4, it is respectively labeled as
Assay 1-4.Sample premixed liquid is prepared by table 5, sample premixed liquid and template are then configured to end reaction system (table 6).
The preparation of 3 primed probe mix of table
The preparation of 4 10 × reagent of table
The preparation of 5 sample premixed liquid of table
The preparation of 6 end reaction system of table
| Component | Reaction volume (μ L) |
| DNA | 1.6 |
| Sample premixed liquid | 2.4 |
| Total volume | 4.0 |
Reaction system is uploaded on micro-fluidic chip, carries out carrying out fluorescent PCR detection reaction in microfluidic platform, instead
Answer condition as shown in table 2.
Taking CYP2D6 gene is the sample that singly copies as control sample.Test object and control sample respectively carry out 4 weights
Multiple PCR reaction, obtains the Ct value of real-time quantitative PCR amplification.The weighted value of CYP2D6 is determined using reference gene；Weighted value=
Sample CYP2D6 Average Ct values ÷ sample reference gene RPP30 Average Ct values.It is calculated using the CYP2D6 copy number of control crowd
The gene copy number of test object；α=test object weighted value ÷ control sample weighted value.If α < 0.2, test object
CYP2D6 copy number is 0；If α is 0.5-1.3, the CYP2D6 copy number of test object is 1；If α is 1.5-2.3, detection
The CYP2D6 copy number of object is 2；If α is 2.5-3.3, the CYP2D6 copy number of test object is 3；If α is 3.5-
4.3, the CYP2D6 copy number of test object is 4；It needs to detect again if α is other values.
Fig. 1 is such as table 7 after the statistics of 2 site intro point.The results show that kit test result of the invention and practical phase
Symbol.
The testing result of 7 18 samples of table counts
The distribution frequency of 8 18 pattern detection results of table
| Sample number (example) | Frequency | |
| Copy number is 0 | 1 | 5.6% |
| Copy number is 1 | 5 | 27.8% |
| Copy number is 2 | 9 | 50.0% |
| Copy number is 3 | 3 | 16.7% |
| Copy number is 4 | 0 | 0% |
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
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<213>artificial sequence (Artificial Sequence)
<400> 7
accatccacc acccactcc 19
<210> 8
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
accatccacc acccactcc 19
<210> 9
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ttagcttcta atccggccct ggtctcccgc a 31
<210> 10
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
agatcaggac ctgcgagcg 19
<210> 11
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
agatcaggac ctgcgagcg 19
<210> 12
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
aactgacctg aaccctct 18
Claims (7)
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| CN201811453537.8A CN109371132A (en) | 2018-11-30 | 2018-11-30 | A kit for detecting CYP2D6 gene copy number variation |
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| CN201811453537.8A CN109371132A (en) | 2018-11-30 | 2018-11-30 | A kit for detecting CYP2D6 gene copy number variation |
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| CN109371132A true CN109371132A (en) | 2019-02-22 |
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| 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 |
| CN114277110A (en) * | 2021-12-21 | 2022-04-05 | 杭州瑞普基因科技有限公司 | Kit for detecting copy number and/or amplification of FGF19 gene, detection method and use |
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