CN109825573A - A kind of multiple gene detection kit and its application method for antidepressant medication guide - Google Patents
A kind of multiple gene detection kit and its application method for antidepressant medication guide Download PDFInfo
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Abstract
The invention discloses a kind of multiple gene detection kits and its application method for antidepressant medication guide.The present invention uses multiplex PCR binding fragment length/mass analysis method, simultaneously and rapidly the metabolism of qualitative detection and antidepressant, transport, 7 single nucleotide polymorphism (SNP) sites on 4 genes such as target spot effect relevant CYP2D6, CYP2C19, ABCB1 and FKBP5.Detecting step: (1) mouth desquamated cells are acquired and are stored in cell collection card, or acquires blood sample and extracts nucleic acid；(2) cell collection card or nucleic acid described in step 1 is used to carry out multiplexed PCR amplification for template；(3) by PCR product segment length/SNP site of quality separated in synchronization 7, the PCR product of 3 human DNA reference genes and 1 PCR reaction internal reference；(4) interpretation of result interpretation.Advantage of the invention be quickly, high sensitivity, reproducible, high specificity, flux it is high, at low cost.
Description
Technical field
The present invention relates to a kind of multiple gene detection kits, and in particular to a kind of for antidepressant medication guide
Multiple gene detection kit and its application method.
Background technique
Depression (depression) is a kind of common disturbance of emotion disease that can be suffered from all the life, betides each year
The characteristics of age grade section is in recurrent exerbation, intermittent phase serviceable condition.The World Health Organization (WHO) announce data show, depression
Disease incidence shows an increasing trend year by year, and global patients with depression is more than 300,000,000, and wherein major depressive disorder patient has 89,000,000 people.
Global annual about 800,000 because depression is committed suiside, and suicide has become second cause of death of 15~29 years old age group crowd.WHO in
It is pointed out in statement-of-health in 2001, depression proportion maximum (11.9%) in global disease burden, far more than glycosuria
Disease and hypertension chronic diseases.Expect the year two thousand twenty, functional disability caused by severe depression will rise to the 2nd of the total class of disease, only
Inferior to ischemic heart disease.
Taking drugs is the main means for treating depression.Antidepressant object mainly has monoamine oxidase inhibitors
(MAOI), tricyclic antidepressants (TCAs) antidepressant and selective serotonin (5-HT) recycle inhibitor three categories.With pharmacy
The development of industry, such as ten thousand daraf(reciprocal of farad) stars, nefazodone new drug emerge one after another, but clinical application at present it is most be still tricyclic antidepressants
(TCAs) antidepressant and selective serotonin (5-HT) recycle inhibitor.Although current drug is to treat depressive disorder most
Effective method, but still have the problems such as efficient low and adverse reaction of drug is serious.Studies have shown that single antidepressants
Object treats depressive disorder, even if only 30~45% patient obtains the complete of clinical symptoms under the conditions of the administration of enough pedicure journeys
Direct release, and there are deferred reactions for remission；There is 10% patient to treat equal nothing for any kind of antidepressant object
Effect.
In recent years, as pharmacogenomics develop rapidly, antidepressant object especially tricyclic antidepressants (TCAs) antidepression
The mechanism of drug action of drug and selective serotonin (5-HT) recycling inhibitor is more and more clear.Pharmacogenomics are aobvious
Show, big main of drug individual difference is the reason is that hereditary difference, i.e., there are gene pleiomorphisms between different patients.Human gene
80% or more gene pleiomorphism of group is all single nucleotide polymorphism (Single Nucleotide polymorphism, SNP).
SNP refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level, including base transition, transversion,
Missing and insertion.Most of antidepressant object is all metabolized through CYP2D6 or CYP2C19.Drug metabolic enzyme gene SNP site hair
Raw mutation can make enzymatic activity change, and cause prodrug or intermediate active production concentration too high or too low, to make patient
Unsatisfactory curative effect, or even serious toxicity occurs.SNP site relevant to drug transport and drug target effect mutates
Drug response is had a major impact.Currently, neuropsychopharmacology drug surveillance treatment common recognition guide (2017) and pharmGKB
Database has disclosed the metabolism, transhipment and target gene of part antidepressant object.By to drug metabolism, transhipment and target spot
Relevant SNP site is detected, and can determine whether reaction of the patient to different pharmaceutical, is the customized a set of therapeutic regimen of patient,
Therapeutic efficiency is improved, mitigates patient medical burden and pain, saves a large amount of hospitals and social resources (being shown in Table 1).
1 antidepressant medication guide related gene of table
Currently on the market, there are mainly three types of SNP parting detection techniques: PCR sequencing PCR, fluorescence quantitative PCR method, gene chips:
(1) fluorescence quantitative PCR method
Quantitative fluorescent PCR uses fluorescent quenching and double end-labellings, and the probe of specificity is designed for SNP site,
With high sensitivity, the high and specific high advantage of accuracy.But quantitative fluorescent PCR flux is low, to detect multiple SNP simultaneously
Site needs to be in charge of detection, and complicated for operation, amount of samples is big, it is difficult to adapt to clinical demand.In addition, quantitative fluorescent PCR is difficult to set
Internal reference Quality Control is set, not can avoid false positive and false negative.
(2) gene chips
Genetic chip is that the DNA fragmentation (gene probe) of ten hundreds of particular sequences is had rule by micro-processing technology
The arrangement of rule ground is fixed on the supports such as silicon wafer, slide, and the two-dimentional DNA probe array of one of composition utilizes this kind of chip and mark
The biological sample of note is hybridized, can gene expression profile biological information to sample carry out fast qualitative and quantitative analysis.Its is excellent
Point is that flux is high, easy to operate；The disadvantage is that testing cost is expensive, poor repeatability, sensitivity are lower.The type of chip is more, difficult
The universal of biochip technology is also limited to formulate a unified quality control standard.
(3) PCR sequencing PCR
Sanger PCR sequencing PCR is SNP parting goldstandard, can not only detect known SNP, can also find unknown SNP.But
Sanger PCR sequencing PCR is complicated for operation, higher cost.Site sequencing one by one is needed when sequencing site is more, time-consuming, adds up valence
Lattice are relatively expensive.Two generation sequencing technologies are realized to be sequenced in synthesis, has high-throughput, efficient advantage, however two generations surveyed
Sequence Platform Price is expensive, popularization degree is low, is not mature enough as the application of SNP detection technique clinically.
Since gene SNP quantity relevant to antidepressant medication guide is more, the above technology all has obvious limitation
Property, therefore it is difficult to apply to the detection of antidepressant medication guide multiple gene.
Currently, it is domestic there is no it is multiple in relation to the antidepressant medication guide based on multiplex PCR and CE isolation technics
The report of genetic test scheme.
Summary of the invention
The present invention provides one kind quickly, high sensitivity, reproducible, accuracy is high, high specificity, flux are high, cost
Low antidepressant medication guide multiple gene detection kit and its application method.It is characterized in that, using multiplex PCR knot
Segment length/mass analysis method is closed, simultaneously and rapidly qualitative detection relevant to antidepressant medication 7 in a reaction tube
A single nucleotide polymorphism (SNP) site.This kit joined 3 in the PCR reaction system for detecting above-mentioned 7 SNP sites
A human gene group DNA's (huDNA) internal reference and 1 PCR reaction internal reference (as shown in table 2), it is synchronous to carry out PCR amplification, for monitoring
Nucleic acid extraction and PCR reaction process can avoid false negative and false positive.
2 antidepressant medication guide multiple gene detection kit detection site of table and primer
This kit includes following components: Primer composition MDD Primer Mix, PCR reaction solution and positive reference substance,
Ultrapure water.Positive reference substance includes plasmid mixture corresponding to above-mentioned 7 SNP sites and reference gene, detects for SNP
Quality after system testing and each primer are ordered controls.PCR reaction solution includes following components: ultrapure water, 2 × PCR buffer,
Archaeal dna polymerase, dNTP.
It is as follows using this kit detecting step:
(1) acquisition mouth desquamated cells are stored in cell collection card, or acquire blood sample and extract nucleic acid.Wherein, it protects
The mouth desquamated cells being stored on cell collection card can be not required to carry out nucleic acid extraction, be directly used in PCR amplification, save nucleic acid
The time of extraction；
(2 carry out multiplexed PCR amplification using cell collection card or the nucleic acid of extraction as template.PCR reaction condition are as follows: 95 DEG C
5min；94 DEG C of 10s, 61 DEG C of 1min；70 DEG C of 30s are recycled 29 times；60 DEG C, 3min；4 DEG C until collect PCR product；
(3) PCR product segment length/SNP site of quality separated in synchronization 7 and 4 internal references are pressed.The present invention is using capillary electricity
Swimming separation PCR product: electrophoresis Sample is prepared in 96 hole sample panels, takes high-purity 8.7 μ L of formamide, standard items SIZE-5000.3 μ
L, 1 μ L of PCR product mix centrifugation.Prepared electrophoresis Sample is placed in 3500 Genetic Analysers, carries out hair by operating instruction
Thin electrophoresis；
(4) interpretation of result is carried out according to the fragment length of designed each detection site.
According to detection peak figure, the genotype of each SNP site can get, in conjunction with the corresponding clinical reference letter of each gene
Breath, judges reaction of the patient to various antidepressants, to instruct the personalized of antidepressant to use, such as table 3.1~3.4
It is shown.
The corresponding clinical reference information of 3.1 CYP2D6 gene of table
The mutation type in the site a.*5 is deletion mutant.DW represents at least one chromosome and deletion mutation does not occur；
DD represents two chromosomes and deletion mutation occurs.
The corresponding clinical reference information of 3.2 CYP2C19 gene of table
The corresponding clinical reference information of 3.3 ABCB1 gene of table
The corresponding clinical reference information of 3.4 FKBP5 gene of table
Compared with prior art, present invention has the advantage that
The present invention is based on 3500 Genetic Analysers founded it is a kind of 4 relevant to antidepressant medication with synchronous detection
The detection scheme of 7 SNP sites on gene；Specificity and accuracy can reach qPCR level；It can in a short time (2.5 hours)
It is completed at the same time the detection of multiple 7 SNP sites of sample；DNA internal reference and react internal reference use can monitoring of DNA extraction and PCR it is anti-
The efficiency answered, avoids false negative and false positive.
In conclusion the present invention provides a kind of synchronizations to detect on 4 genes relevant to antidepressant object medication 7
The detection scheme of SNP site, have quickly, high sensitivity, reproducible, high specificity, flux be high, the advantages such as at low cost.
Detailed description of the invention
Fig. 1 is the mouth desquamated cells capture card sample of a patients with depression (without nucleic acid extraction, directly progress PCR)
Testing result；
Fig. 2 is the testing result of the whole blood sample of a patients with depression.
Specific embodiment
In order to better understand the content of the present invention, it is described further combined with specific embodiments below with attached drawing.Ying Li
Solution, these embodiments are only used for that the present invention is further described, rather than limit the scope of the invention.In addition, it should also be understood that,
After having read the contents of the present invention, person skilled in art makes some nonessential changes or adjustment to the present invention, still belongs to
In protection scope of the present invention.
Primer composition MDD Primer Mix described in Examples 1 and 2 is described in table 2 for expanding each of 7 SNP sites
Each 2 primers of 3 primers and 4 reference genes: SEQ ID NO.1~SEQ ID NO.29.
PCR reaction solution described in Examples 1 and 2 includes following components: ultrapure water, 2 × PCR buffer, archaeal dna polymerase and
dNTP。
Positive reference substance MDD POS described in Examples 1 and 2 is includes 7 SNP sites and 4 reference gene institutes described in table 2
Corresponding plasmid mixture.
Embodiment 1
The present embodiment directly carries out multi-PRC reaction by template of mouth desquamated cells capture card, finally uses electrocapillary phoresis
Method separates sample, the specific steps are as follows:
1. production is used for the multiple gene detection kit of antidepressant medication guide, including following components:
1) Primer composition MDD Primer Mix；
2) PCR reaction solution；
3) positive reference substance MDD POS；
4) ultrapure water.
2. collecting sample
The mouth desquamated cells that a patients with depression is acquired using buccal swab, are stored on cell collection card.
3. carrying out PCR reaction by template of cell collection card
1) reagent and sample are added in 96 hole sample panels/eight union of PCR by table 4.
4 PCR reaction system of table
| Reagent | Measure/the hole (μ L) |
| PCR reaction solution | 14 |
| Primer composition | 2 |
| Cell collection card | 0 |
| Water | 4 |
| Total | 20 |
2) prepared PCR system is mixed and is centrifuged, carry out PCR reaction by the program of table 5:
5 PCR amplification program of table
| Step | Program | Time |
| 1 | 95℃ | 5min |
| 2 | 94℃ | 10s |
| 3 | 61℃ | 1min |
| 4 | 70℃ | 30s |
| 5 | N/A | Repeat 2~4 steps 29 time |
| 6 | 60℃ | 3min |
| 7 | 4℃ | It lasts up to and collects PCR product |
4. electrocapillary phoresis separates sample
1) electrophoresis Sample is prepared
Electrophoresis Sample is prepared in 96 hole sample panels by table 6.
2) electrocapillary phoresis separates sample
Sample panel is placed in 3500DX Genetic Analyser, " fragment " electrophoresis method is selected, electrophoresis is carried out, is detailed in
ABI3500 operational manual.
6 electrophoresis Sample of table is prepared
| Reagent | Measure/the hole (μ L) |
| Hi-Di | 8.7 |
| SIZE-500 | 0.3 |
| PCR product | 1 |
| Total | 10 |
5. interpretation of result
The position occurred according to each characteristic peak and quantity, determine genotype, to judge patient to various antidepressants
Reaction, provide medication guide.Fig. 1 is the mouth desquamated cells capture card pattern detection peak figure of a patients with depression, 7 He of table
Table 8 is respectively genotype results and drug direction.
As shown in Figure 1, abscissa is PCR fragment size, ordinate is fluorescence signal intensity, is according to the position of characteristic peak
It can get the genotype in each site.The genotype in the site CYP2C19*2 is AA；The genotype in the site CYP2D6*5 is DW；FKBP5
The genotype in the site gene rs4713916 is GG；The genotype in the site ABCB1 gene rs1045642 is CT；CYP2C19*17
The genotype of point is CC；The genotype in the site CYP2C19*3 is GG；The genotype in the site CYP2D6*10 is CT.Wherein 4 positions
Point gene type be different from wild type: the site CYP2D6*5, the site rs4713916 of FKBP5 gene, ABCB1 the position rs1045642
Point and the site CYP2D6*10, influence therapeutic effect, need to adjust therapeutic scheme；Other sites are wild type, can be by recommendation
Dosage takes medicine (see Table 7 for details, table 8).
The testing result of 7 one patients with depression mouth desquamated cells capture card samples of table
The antidepressant medication guide of 8 one patients with depression of table
Embodiment 2
The present embodiment acquires a patients with depression whole blood sample and extracts nucleic acid, carries out using the nucleic acid of extraction as template more
Weight PCR reaction, finally separates sample with electrocapillary phoresis method, the specific steps are as follows:
1. production is used for the multiple gene detection kit of antidepressant medication guide, reagent constituents such as embodiment 1
It is described.
2. collecting sample
It acquires the whole blood sample of a patients with depression and extracts nucleic acid.
3. carrying out PCR reaction by template of the nucleic acid of extraction
1) reagent and sample are added in 96 hole sample panels/eight union of PCR by table 9.
9 PCR reaction system of table
2) prepared PCR system is mixed and is centrifuged, carry out PCR reaction, PCR program is as described in Example 1.
3) electrocapillary phoresis separates sample, and operating procedure is as described in Example 1.
4. interpretation of result
The position occurred according to each characteristic peak and quantity, determine genotype, to judge patient to various antidepressants
Reaction, provide medication guide.Fig. 2 is that the whole blood sample of a mankind detects peak figure, and table 10 and table 11 are respectively genotype knot
Fruit and drug direction.
As shown in Fig. 2, abscissa is PCR fragment size, ordinate is fluorescence signal intensity, is according to the position of characteristic peak
It can get the genotype in each site.The genotype in the site CYP2C19*2 is GA；The genotype in the site CYP2D6*5 is DW；FKBP5
The genotype in the site gene rs4713916 is GA；ABCB1 gene rs1045642 is that the genotype in site is CT；CYP2C19*
The genotype in 17 sites is CC；The genotype in the site CYP2C19*3 is GG；The genotype in the site CYP2D6*10 is TT.Wherein 5
A loci gene type be different from wild type: the site CYP2D6*5, the site CYP2C19*2, FKBP5 gene the site rs4713916,
The site rs1045642 and the site CYP2D6*10 of ABCB1, influences therapeutic effect, needs to adjust therapeutic scheme；Other sites
For wild type, can take medicine by recommended dose (see Table 10 for details, table 11).
The genetic test result of 10 1 patients with depression of table
The antidepressant medication guide of 11 1 patients with depression of table
The SNP detection method that the present invention uses is based on multiplex PCR and Capillary Electrophoresis (CE) isolation technics.Same anti-
Multipair specific gene amplimer and internal control primer is added in Ying Guanzhong simultaneously, obtains gene amplification fragment not of uniform size, makes
It is separated with Capillary Electrophoresis, and then analyzes SNP genotype.Detection method of the present invention and kit can quickly have
Effect ground detects multiple SNP sites, overcomes defect existing for conventional method, has the advantage that
1, high-throughput: to be able to achieve synchronous detection up to 7 SNP sites.
2, accuracy is high: PCR product is separated using CE technology, it can be by non-specific amplification product, primer dimerization
Body and specific amplification products separation, utmostly reduce false positive.
3, high sensitivity: the DNA sample that this system energy detection level is reacted down to 1ng/ has hypersensitivity.
4, method is easy, uses economy: the present invention provides a full set of experiment such as reagent, multiple PCR primer design, interpretation of result
Scheme；Testing cost is low, is conducive to large-scale promotion.
Above description is not the limitation to invention, and the present invention is also not limited to the example above.The common skill of the art
For art personnel in the essential scope of invention, the variations, modifications, additions or substitutions made also should belong to protection scope of the present invention.
Sequence table
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Claims (9)
1. a kind of multiple gene detection kit for antidepressant medication guide, which is characterized in that including such as following table institute
The primer of the simultaneously and rapidly qualitative detection shown 7 SNP sites relevant with antidepressant medication and 4 reference genes combines
Object: SEQ ID NO.1~SEQ ID NO.29, PCR reaction solution, positive reference substance and ultrapure water；7 SNP sites are as follows:
* 2, * 3 and * 17 sites of CYP2C19 gene, * 5 and * 10 sites of CYP2D6 gene, the site rs4713916 of FKBP5 gene,
The site rs1045642 of ABCB1 gene；
2. a kind of multiple gene detection kit for antidepressant medication guide as described in claim 1, feature
It is, 7 SNP sites are in addition to the site CYP2D6*5, other SNP sites only have 1 base mutation, for each
SNP site, which devises 3 primers wherein 2 primers are complementary with wild type gene and mutated genes respectively, to be combined, and 1 shared
Primer forms primer pair with wild type and mutant primers respectively, and amplifying segment length has the PCR of 2~10 base differences to produce
Object.
3. a kind of multiple gene detection kit for antidepressant medication guide as claimed in claim 2, feature
It is, CYP2D6*5 site mutation type is that deletion mutation judges deletion fragment upstream and downstream design primer by clip size
Whether the site CYP2D6*5 occurs deletion mutation.
4. a kind of multiple gene detection kit for antidepressant medication guide as described in claim 1, feature
It is, 3 human gene group DNA's internal references is added in multi-PRC reaction and a PCR reacts internal reference.
5. a kind of multiple gene detection kit for antidepressant medication guide as described in claim 1, feature
It is, the positive reference substance includes plasmid mixture corresponding to 7 SNP sites and 4 reference genes.
6. a kind of multiple gene detection kit for antidepressant medication guide as described in claim 1, feature
It is, the PCR reaction solution includes following components: ultrapure water, 2 × PCR buffer, archaeal dna polymerase and dNTP.
7. a kind of application method of the multiple gene detection kit for antidepressant medication guide, which is characterized in that packet
It includes following steps: (1) acquiring mouth desquamated cells and be stored in cell collection card, or acquire blood sample and extract nucleic acid；(2) it adopts
Cell collection card or nucleic acid described in step 1 are that template carries out multiplexed PCR amplification；(3) same by PCR product segment length/quality
Step 7 SNP sites of separation and 4 internal references；(4) interpretation of result interpretation.
8. a kind of user of the multiple gene detection kit for antidepressant medication guide as claimed in claim 7
Method, which is characterized in that the mouth desquamated cells are stored on cell collection card, it may be unnecessary to which nucleic acid extraction is directly used in PCR
Amplification.
9. a kind of user of the multiple gene detection kit for antidepressant medication guide as claimed in claim 7
Method, which is characterized in that the PCR reaction condition are as follows: 95 DEG C of 5min；94 DEG C of 10s, 61 DEG C of 1min；70 DEG C of 30s are recycled 29 times；60
℃3min；4 DEG C until collect PCR product.
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| CN110511993A (en) * | 2019-09-06 | 2019-11-29 | 江苏先声医疗器械有限公司 | For detecting primer sets, application, product and the method for children's drug metabolism associated SNP positions |
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| CN112921100A (en) * | 2019-12-06 | 2021-06-08 | 宁波海尔施基因科技有限公司 | Kit and method for detecting human pressure sensitivity genotype |
| CN112921099A (en) * | 2019-12-06 | 2021-06-08 | 宁波海尔施基因科技有限公司 | Kit and method for detecting human scientific gas potential genotype |
| 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 |
| CN113528638A (en) * | 2021-02-22 | 2021-10-22 | 北京市理化分析测试中心 | Primer group for amplifying gene locus of children personalized medication, primer group for detection and application of primer group |
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| CN112921099A (en) * | 2019-12-06 | 2021-06-08 | 宁波海尔施基因科技有限公司 | Kit and method for detecting human scientific gas potential genotype |
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| 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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Application publication date: 20190531 |