CN110079596A - A kind of multiple gene detection kit and its application method for antimanic agents medication guide - Google Patents
A kind of multiple gene detection kit and its application method for antimanic agents medication guide Download PDFInfo
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Abstract
The invention discloses a kind of multiple gene detection kits and its application method for antimanic agents medication guide.The present invention uses multiplex PCR binding fragment length/mass analysis method, simultaneously and rapidly 2 single nucleotide polymorphism (SNP) sites on qualitative detection GSK3B and NTRK2 gene relevant to antimanic agents medication.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 2, the PCR product of 3 human genome DNA's 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 antimanic agents medication guide
Multiple gene detection kit and its application method.
Background technique
Mania is a kind of affective disorder disease, the essential characteristic of the disease be patient's flight of ideas, it is in high spirits,
Speech movement increases more usually, and main clinical manifestation is irritability and gets excited.Mania often has paralepsy concurrently,
Therefore mania has been cited as one kind of bipolar disorder.Recurrent exerbation, severe patient can be presented in most of color of light in manic patients
It can also be with psychotic symptoms such as illusion, vain hopes.
Drug therapy is to treat the main method of mania.With being gradually revealed to mania morbidity physiological mechanism, resist
The development at full speed that the exploitation of mania class drug also obtains.It is hot-tempered to be clinically often used the joint other drugs treatment of mood stabilizer
Mad disease.Mood stabilizers include lithium salts, carbamazepine and valproate.Lithium salts is most important antimanic agents, but it is treated
Window is narrow, and overdose will lead to serious adverse reaction, and dose is too low that therapeutic effect can be made bad.Studies have shown that GSK3B base
Because polymorphism influences the metabolism of lithium salts, NTRK2 gene pleiomorphism influences the therapeutic effect of valproate, should be according to Patient genotype
Appropriate adjustment drug dose.
80% or more gene pleiomorphism of human genome 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.Gene SNP site mutation relevant to drug can make corresponding enzymatic activity
It changes, to influence drug metabolism, transhipment or targeted integration, causes curative effect of medication bad, or even serious poison pair occurs instead
It answers.Therefore, by being detected to the relevant SNP site of antimanic medication, it is the customized a set of therapeutic regimen of patient, can mentions
High therapeutic efficiency mitigates the medical burden and pain of color of light in manic patients, saves a large amount of hospitals and social resources (being shown in Table 1).
1 antimanic agents 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, genetic chip
Method:
(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 simultaneously
SNP 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
Internal reference Quality Control is arranged, false positive and false negative not can avoid.
(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 antimanic agents medication is more, the above technology all has obvious limitation, because
This is difficult to apply to the multiple gene detection of antimanic agents medication guide.
Currently, domestic there is no in relation to the more of the antimanic agents medication guide based on multiplex PCR and CE isolation technics
The report of genetic test scheme again.
Summary of the invention
The present invention provides one kind quickly, high sensitivity, reproducible, accuracy is high, high specificity, flux are high, cost
The multiple gene detection kit and its application method of low antimanic agents medication guide.It is characterized in that, using multiplex PCR
Binding fragment length/mass analysis method, simultaneously and rapidly qualitative detection is related to antimanic agents medication in a reaction tube
2 single nucleotide polymorphism (SNP) sites.This kit is added in the PCR reaction system for detecting above-mentioned 2 SNP sites
3 human gene group DNA's (huDNA) internal references and 1 PCR reaction internal reference (as shown in table 2), synchronous progress PCR amplification, are used for
Nucleic acid extraction and PCR reaction process are monitored, can avoid false negative and false positive.
The multiple gene detection kit detection site and primer of 2 antimanic agents medication guide of table
This kit includes following components: Primer composition Mania Primer Mix, PCR reaction solution and positive control
Product, ultrapure water.Positive reference substance includes plasmid mixture corresponding to above-mentioned 2 SNP sites and reference gene, is examined for SNP
Quality control after examining system test and every time primer order.PCR reaction solution includes following components: ultrapure water, 2 × PCR buffering
Liquid, 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, 15min；4 DEG C until collect PCR product；
(3) PCR product segment length/SNP site of quality separated in synchronization 2 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, is carried out by operating instruction
Electrocapillary phoresis；
(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 information of each gene
(table 3.1~3.2) instructs the personalized of antimanic agents to use.
The corresponding clinical reference information of 3.1 GSK3B gene of table
The corresponding clinical reference information of 3.2 NTRK2 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 2 relevant to antimanic agents medication with synchronous detection
The detection scheme of 2 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 2 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 2 genes relevant to antimanic agents medication 2
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 color of light in manic patients (without nucleic acid extraction, directly progress PCR)
Testing result；
Fig. 2 is the testing result of the whole blood sample of a color of light in manic patients.
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 Mania Primer Mix described in Examples 1 and 2 is described in table 2 for expanding 2 SNP sites
Each 2 primers of each 3 primers and 4 reference genes: SEQ ID NO.1~SEQ ID NO.14.
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 Mania POS described in Examples 1 and 2 is to include 2 SNP sites and 4 reference genes described in table 2
Corresponding plasmid mixture.
Embodiment 1
The present embodiment acquires the mouth desquamated cells of a color of light in manic patients, directly carries out using cell collection card 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 antimanic agents medication guide, including following components:
1) Primer composition Mania Primer Mix；
2) PCR reaction solution；
3) positive reference substance Mania POS；
4) ultrapure water.
2. collecting sample
The mouth desquamated cells that a color of light in manic patients 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
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, in conjunction with the corresponding clinical reference information of each gene,
Judge reaction of the patient to various antimanic agents, provides medication guide.Fig. 1 is the mouth desquamated cells of a color of light in manic patients
Capture card pattern detection peak figure.Table 7 is the genotype results of the patient, and table 8 is the medication guide of the patient.
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 patient GSK3B gene rs334558 is AA, influences drug therapy effect
Fruit need to adjust therapeutic scheme；The genotype in the site NTRK2 gene rs2769605 is CT, can take medicine and (be detailed according to recommended dose
Table 7 and table 8).
7 one phrenetic's mouth desquamated cells capture card sample genotype call results of table
| Serial number | Detect gene | Detection site | Testing result |
| 1 | GSK3B | Rs334558 (G > A) | AA |
| 2 | NTRK2 | Rs2769605 (C > T) | CT |
The antimanic agents medication guide of 8 one phrenetics of table
Embodiment 2
The present embodiment acquires a color of light in manic patients 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 antimanic agents medication guide, reagent constituents such as embodiment 1
It is described.
2. collecting sample
It acquires the whole blood sample of a phrenetic and extracts nucleic acid.
3. carrying out PCR reaction by template of the nucleic acid of extraction
Reagent and sample are added in 96 hole sample panels/eight union of PCR by table 9.
9 PCR reaction system of table
| Reagent | Measure/the hole (μ L) |
| PCR reaction solution | 14 |
| Primer composition | 2 |
| Nucleic acid | 2 |
| Water | 2 |
| Total | 20 |
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 antimaniacal anti-
It answers, provides medication guide.Fig. 2 is that the whole blood sample of a phrenetic detects peak figure.Table 10 is the genotype results of the patient,
Table 11 is the medication guide of the patient.
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 patient GSK3B gene rs334558 is GA, can be according to recommended dose
Medication；The genotype in the site NTRK2 gene rs2769605 is CT, and can take medicine (see Table 10 for details and table 11) according to recommended dose.
10 1 phrenetic's mouth desquamated cells capture card sample genotype call results of table
| Serial number | Detect gene | Detection site | Testing result |
| 1 | GSK3B | Rs334558 (G > A) | GA |
| 2 | NTRK2 | Rs2769605 (C > T) | CT |
The antimanic agents medication guide of 11 1 phrenetics of table
| Serial number | Medicine name | Pharmaceutical relevant gene | Drug response | Drug direction |
| 1 | Lithium salts | GSK3B | Therapeutic effect is preferable | It is used according to recommended dose |
| 2 | Valproate | NTRK2 | Therapeutic effect is preferable | It is used according to recommended dose |
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 the synchronous multiple SNP sites of detection.
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
<110>Ningbo Health Gene Technologies Co., Ltd.
<120>a kind of multiple gene detection kit and its application method for antimanic agents medication guide
<130> 2019-03-06
<160> 14
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Claims (8)
1. a kind of multiple gene detection kit for antimanic agents medication guide, which is characterized in that including such as following table institute
The primer of the simultaneously and rapidly qualitative detection shown 2 SNP sites relevant with antimanic agents medication and 4 reference genes combines
Object: SEQ ID NO.1~SEQ ID NO.14, PCR reaction solution, positive reference substance and ultrapure water；2 SNP sites are as follows:
The site rs334558 of GSK3B gene, the site rs2769605 of NTRK2 gene；
2. a kind of multiple gene detection kit for antimanic agents medication guide as described in claim 1, feature
It is, devises 3 primers for each of 2 SNP sites SNP site, wherein wild primers and saltant type
Primer is complementary with wild type gene and mutated genes respectively to be combined, and 1 shared primer draws with wild type and saltant type respectively
Object forms primer pair, amplifies the PCR product that fragment length has 2~10 base differences.
3. a kind of multiple gene detection kit for antimanic agents 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.
4. a kind of multiple gene detection kit for antimanic agents medication guide as described in claim 1, feature
It is, the positive reference substance includes plasmid mixture corresponding to 2 SNP sites and 4 reference genes.
5. a kind of multiple gene detection kit for antimanic agents 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.
6. a kind of application method of the multiple gene detection kit for antimanic agents 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 2 SNP sites of separation and 4 internal references；(4) interpretation of result interpretation.
7. a kind of user of the multiple gene detection kit for antimanic agents medication guide as claimed in claim 6
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.
8. a kind of user of the multiple gene detection kit for antimanic agents medication guide as claimed in claim 6
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, circulation 29
It is secondary；60℃ 15min；4 DEG C until collect PCR product.
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Cited By (1)
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| CN111944896A (en) * | 2020-09-01 | 2020-11-17 | 吉林大学 | Detection substance for single nucleotide polymorphism of genes Gsk3 beta, SFRP4, LPR5 and PLPPR5 related to non-traumatic femoral head necrosis risk and application thereof |
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| US20080152589A1 (en) * | 2006-09-08 | 2008-06-26 | Peter Schofield | Diagnostics and Therapeutics of Neurological Disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111944896A (en) * | 2020-09-01 | 2020-11-17 | 吉林大学 | Detection substance for single nucleotide polymorphism of genes Gsk3 beta, SFRP4, LPR5 and PLPPR5 related to non-traumatic femoral head necrosis risk and application thereof |
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Application publication date: 20190802 |