CN112921100A - Kit and method for detecting human pressure sensitivity genotype - Google Patents
Kit and method for detecting human pressure sensitivity genotype Download PDFInfo
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Abstract
The invention discloses a kit and a method for detecting human pressure sensitivity genotype. The invention adopts multiple PCR amplification and electrophoresis methods to analyze and identify the allelic polymorphism (SNP) of 5 genes: MAOA, PPP1R1B, COX10, CADM2 and COMT, comprising the steps of: a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample; b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification; c) running an amplification program; d) and carrying out electrophoretic analysis on the amplification products, and judging according to a peak pattern. And analyzing and interpreting the result. The invention can synchronously detect the SNP of a plurality of genes related to a testee, and realizes the simplicity, high efficiency and specificity of detection. The analysis provides reference for the resistance of the testee to the pressure.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a kit and a method for detecting human pressure sensitivity genotype.
Background
Modern society life rhythm is fast, and the life requires highly, and the work load is heavy, and a lot of working clans face huge psychological pressure generally. The psychological stress can further influence daily work, study and physical health through feedback. The emotional manifestations of high stress are irritability, impatience, anxiety, tension, apathy, anxiety and collapse. Anger is one of the fundamental emotions inherent to animals and humans of all races. Anger is often combined with aggressive behavior and is a common manifestation of stress. Molecular genetics has demonstrated that about 50% of variations in stress-related behavior are caused by genes, and studies of those genetic loci that affect stressful emotions have begun. These genes include Monoamine oxidase A (MAOA), DARPP-32 (dopamine and cAMP regulated phosphoprotein, 32kD, also known as PPP1R1B) gene function polymorphisms, COX10, CADM2, and catechol-O-methyltransferase (COM), among others.
MAOA proteins are responsible for the breakdown of brain signaling molecules, including serotonin, after they have lost function. If it fails to work properly, the accumulation of these failed neurotransmitters can cause abnormal mood and violent behavior. Under external stimulation, the rs1465108 polymorphic A allele of the gene is easy to generate negative urgency. Genotyping results of rs1465108 SNP of MAOA gene by researchers showed that 58.5% of 277 participants were GG genotype, 22.0% were GA genotype and 19.5% were AA genotype. The presence of the a allele showed a higher aggressiveness in all MAOA genotypes.
The PPP1R1B protein is present in the brain in neurons that receive dopaminergic projections, is centrally located in signal transduction pathways, and is involved in a variety of physiological functions and pathological processes. The polymorphic T allele carrier of the rs907094 gene is easier to anger, has less gray matter in the amygdala of the brain and has higher dopamine level than a non-T allele carrier. Amygdala is the part of the brain responsible for controlling mood. Recent research results underscore the importance of DARPP-32, a key regulatory molecule in the dopamine signaling pathway. A significant correlation between rs907094 and anger was found in the sample from a caucasian in n 838 healthy Germany. The C → T single nucleotide polymorphism is located on intron 5 of the DARPP-32 gene. Carriers of the T allele showed significantly higher anger scores than participants without the T allele (F (1, 837) ═ 9.52, p ═ 0.002).
COX10 gene is expressed in various tissues, and is most expressed in heart, skeletal muscle and testis. The enzyme encoded by this gene is the last important enzyme of the mitochondrial respiratory chain. COX10 deletion can cause cytochrome C oxidase deletion, inhibit mitochondrial aerobic respiration, and arrest cell growth. The rs17608059 polymorphic C allele type of the gene has more persistence than the behavior of T type and higher stress tolerance. The CADM2 gene has close relation with the information transmission among brain cells. CADM2 is very active in both the prefrontal and cingulate cortex regions of the brain, which in themselves play a significant role in the thought response of the human brain. The rs12494658 polymorphic C allele of the gene is more persistent than the T-type behavior and has higher stress tolerance.
The protein encoded by COMT (catechol-O-methyltransferase gene) is very active in both the prefrontal and cingulate cortex regions of the brain and has a close relationship with the transfer of information between brain cells. The rs4680 polymorphic variation of the gene causes the heat stability of the protein to be changed. The carrier of the A allele has higher exploratory property and lower COMT activity, so that the dopamine level is higher, the dopamine is sensitive to pain and pressure, the pain and the pressure are easy to be frustrated, but the information processing at ordinary times is more effective; g allele, lower exploratory, higher COMT activity, lower dopamine levels, better tolerance to pain, tougher, better performance under stress, but slightly reduced executive and cognitive abilities at ordinary times. Behavioral phenotypes are often complex, reflecting the role of a variety of different genes. Nevertheless, there is increasing evidence that key genetic variations can alter the activity circuits within specific neurons and thus affect specific cognitively-emotional phenomena. An example is the COMT gene, with a common variant at codon 158. Those with increased valine (Val158) allele had more COMT activity and lower prefrontal extracellular dopamine (Met158) substitution compared to methionine. The Val158 allele may be an aversive stimulus associated with dominance in the treatment (warrior strategy), while the Met158 allele may be associated with memory and attention tasks (fear strategy). Under conditions of increased dopamine release (e.g., stress), individuals with the Val158 allele may have improved dopaminergic transmission and better performance, while individuals with the Met158 allele may have less efficient neurotransmission and perform worse. Some evidence suggests that the Val158 allele is associated with schizophrenia, whereas the Met158 allele is associated with anxiety.
At present, in the market, there are three major SNP typing detection techniques: sequencing method, fluorescent quantitative PCR method, gene chip method:
(1) fluorescent quantitative PCR method
The fluorescent quantitative PCR adopts fluorescent quenching and double-end labeling technology, and specific probes are designed aiming at SNP sites, so that the fluorescent quantitative PCR has the advantages of high sensitivity, high accuracy, high specificity and the like. However, the fluorescent quantitative PCR flux is low, and if a plurality of SNP sites are to be detected simultaneously, tube-by-tube detection is required, the operation is complex, the sample dosage is large, and the clinical requirement is difficult to adapt. In addition, the fluorescent quantitative PCR is difficult to set internal reference quality control, and false positive and false negative cannot be avoided.
(2) Gene chip method
The gene chip is a two-dimensional DNA probe array formed by regularly arranging and fixing tens of thousands of DNA fragments (gene probes) with specific sequences on supports such as a silicon chip, a glass slide and the like by a micromachining technology, and can carry out rapid qualitative and quantitative analysis on biological information of a gene expression profile of a sample by hybridizing the chip with a marked biological sample. Its advantages are high flux and simple operation; the defects are high detection cost, poor repeatability and lower sensitivity. The variety of the chip is large, and the difficulty in establishing a uniform quality control standard also limits the popularization of the gene chip technology.
(3) Sequencing method
The Sanger sequencing method is an SNP typing gold standard, and can detect known SNPs and discover unknown SNPs. However, the Sanger sequencing method is complex in operation and high in cost. When the number of sequencing sites is large, sequencing needs to be carried out one by one, the time consumption is long, and the accumulated price is relatively expensive. The second-generation sequencing technology realizes sequencing while synthesis, and has the advantages of high throughput and high efficiency, but the second-generation sequencing platform is expensive and low in popularity, and is not mature enough to be used as an SNP detection technology in clinic.
At present, no report about a multiplex gene detection scheme related to pressure sensitivity based on multiplex PCR and capillary electrophoresis separation technology exists in China.
Disclosure of Invention
The invention aims to provide a kit and a method for detecting human pressure sensitivity genotype, wherein the kit can detect related genes of a testee and give analysis.
Specifically, the invention discloses a kit and a method for detecting a human pressure sensitivity genotype, which comprises the following steps:
a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample;
b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification;
c) running an amplification program;
d) carrying out electrophoretic analysis on the amplification product, and judging according to a peak pattern;
the kit comprises the following components: primer Mix, PCR reaction solution and DNA/RNA-free water.
Wherein the Primer Mix comprises a Primer group for amplifying 5 gene SNP polymorphic sites and a Primer group for 3 human genome DNA internal references and reaction internal references pcDNA, which are detailed in Table 1.
TABLE 1 SNP detection sites, primer sequences and PCR amplification fragment lengths of the kit
The kit can carry out synchronous multiplex PCR amplification on a detected sample, and can accurately detect the genotypes of detected genes CADM2, COX10, PPP1R1B, MAOA and COMT through the design of SNP primers: heterozygotes or homozygotes; the pcDNA is used for detecting a reaction system, monitoring whether the reaction system is effective or not and whether amplification is normal or not; 3 human genome DNA is internally referred to for detecting human samples, so that the human samples are guaranteed to be effective.
Wherein the PCR reaction solution comprises the following components: 2 XPCR buffer, DNA polymerase, dNTPs, potassium chloride, magnesium chloride, etc.
The PCR amplification reaction conditions of the kit are shown in Table 2.
TABLE 2 PCR amplification reaction conditions of the present invention
The amplification product of the kit is analyzed by electrophoresis; the preferred electrophoresis is capillary electrophoresis.
The kit can simultaneously amplify a plurality of sites, realizes the simplicity, high efficiency and specificity of detection, provides reference for the sensitivity and the anti-pressure capability of a testee to pressure by analyzing an amplification map, and is shown in Table 3.
TABLE 3 detection site-character reference information of the present invention
Drawings
FIG. 1 is a negative control DNA/RNA-free water amplification map of the kit. Only characteristic peaks of pcDNA appear.
FIG. 2 is an amplification profile of a sample of exfoliated cells from the oral cavity of subject 1. The CADM2 has the genotype C, COX10 of C, PPP1, R1B of CT and MAOA of G, COMT of GA, and has a pcDNA peak and characteristic peaks of human genomic DNA (huDNA) internal ginseng-1, human genomic DNA (huDNA) internal ginseng-3 and human genomic DNA (huDNA) internal-8.
Fig. 3 is an amplification map of a sample of exfoliated cells from the oral cavity of subject 2. The CADM2 genotype is T, COX10 is C, PPP1, R1B is CT, MAOA is GA, COMT is GA; the pcDNA peak and characteristic peaks of human genome DNA (huDNA) internal reference-1, human genome DNA (huDNA) internal reference-3 and human genome DNA (huDNA) internal-8 appear.
FIG. 4 is an amplification map of a DNA sample extracted from blood of subject 2, as shown in FIG. 3.
The specific implementation method comprises the following steps:
for a better understanding of the present invention, reference is made to the following detailed description and accompanying drawings. It is to be understood that these examples are for further illustration of the invention and are not intended to limit the scope of the invention. Moreover, it should be understood that the invention is not limited to the above-described embodiments, but is capable of various modifications and changes within the scope of the invention.
The detection kit for detecting whether the inherent stress resistance capability exists comprises a box body and reagents stored in the box body, and comprises the following steps:
a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample;
b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification;
c) running an amplification program;
d) carrying out electrophoretic analysis on the amplification product, and judging according to a peak pattern;
the kit comprises the following components: primer Mix including Primer set, PCR reaction solution and DNA/RNA-free water.
The PCR reaction system Primer Mix comprises a Primer group for amplifying 5 SNP polymorphic sites, 3 personal genome references and pcDNA reaction references, and the details are shown in Table 1.
Wherein the PCR reaction solution comprises the following components: 2 XPCR buffer, DNA polymerase, dNTPs, potassium chloride, magnesium chloride, etc.
In the embodiment, the samples are DNA/RNA-free water, an oral cavity wall cast-off cell collecting card of the testee 1, an oral cavity wall cast-off cell collecting card of the testee 2 and a blood DNA extracting sample of the testee 2.
In the implementation case, the electrophoresis is to perform electrophoresis on the amplification product through capillary electrophoresis, and an amplification map is given and analyzed by combining analysis software, so as to judge the sensitivity and the pressure resistance of the testee to the pressure.
Example one
(1) The selected samples were: DNA/RNA-free water.
(2) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(3) Adding the sample
According to the instructions, using a pipette to take a corresponding volume of DNA/RNA-free water, and adding into the reaction system which is divided.
(4) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(5) Detection of amplification product on 3500DX genetic analyzer
A sample mixture consisting of deionized formamide and an internal molecular weight standard in the system (Size-500) { (1. mu. LSize-500+ 12. mu.L deionized formamide) × (number of samples) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kV, sample injection time: 15s, electrophoresis time 1210-. The results of the detection are shown in FIG. 1.
(6) Analyzing data
The experiment uses DNA/RNA-free water, and the electrophoresis pattern should not have target peaks of each gene locus, and only has characteristic peaks of the pcDNA gene, as follows: the peak appearance corresponding to the pcDNA site is: singlet, showing pcDNA in the map;
as shown in FIG. 1, the electrophoretically analyzed DNA/RNA-free water-amplified product showed only the peak of the internal reference pcDNA of the reaction.
The experiment can effectively verify the amplification effectiveness of the reaction reagent and has no external pollution source.
Example two
(1) The selected samples were: samples of cells exfoliated from the oral wall of subjects 1 and 2.
(2) Sample collection
The collection type is as follows: cells are shed from the oral wall.
The acquisition method comprises the following steps: the saliva collecting rod is adopted to wipe the inner wall of the oral cavity back and forth for 4 times, the reverse side of the saliva collecting rod is used to wipe the inner wall of the oral cavity back and forth for 4 times, the saliva collecting rod is taken out, the saliva collecting rod is repeatedly pressed on a saliva sample collecting card, cells on the inner wall of the saliva are transferred to the saliva sample collecting card, and the collected saliva sample collecting card is dried in a pollution-free area.
Valid samples: the area of the saliva sample acquisition card with the pink area changed into light pink or white is the effective saliva sample area.
The sample selecting method comprises the following steps: manual punch sampling was performed using a dabber plastic punch (1.0 mm).
(3) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(4) Adding a sample: and taking 1-2 effective samples in the saliva card by using a puncher, and adding the effective samples into the prepared reaction system.
(5) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(6) Detection of amplification product on 3500DX genetic analyzer
A sample mixture consisting of deionized formamide and an internal molecular weight standard in the system (Size-500) { (1. mu.L Size-500+ 12. mu.L deionized formamide) × (number of samples) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kv, sample injection time: 15s, electrophoresis time 1210-. The detection results are shown in fig. 2 and fig. 3, respectively.
(7) Analyzing data
The experiment uses a sample of a testee, determines the genotype of the testee according to the positions of the target peaks appearing in the map, guides and judges the sensitivity of the testee to pressure, and measures the stress resistance of the testee.
Fig. 2 is an analysis pattern of a saliva sample of the subject 1, and fig. 3 is an analysis pattern of a saliva sample of the subject 2.
The analysis according to FIG. 2 is as follows:
the electrophoresis pattern of the amplification product of the testee 1 shows the following characteristic peaks: the CADM2 genotype is characterized by C, COX10 locus of C, PPP1R1B locus of CT, MAOA locus of G, COMT locus of GA, pcDNA peak, ginseng-1 of human genome DNA (huDNA), ginseng-3 of human genome DNA (huDNA) and characteristic peak of-8 of human genome DNA (huDNA).
Wherein locus C of the CADM2 gene is shown as CADM 21C; COX10 gene site C is shown as COX 10C; PPP1R1B gene site C is PPP1R1B C, site T is PPP1R1B T; MAOA gene site G is shown as MAOA G; COMT gene site G shows COMT G, site A shows COMT A; the pcDNA locus is unimodal; human genomic DNA (huDNA) reference-1 is a single peak, and B1 is shown in the map; human genomic DNA (huDNA) reference-3 is a single peak, and B3 is shown in the map; the human genome DNA (huDNA) has a single peak at the reference-8 locus, and B8 is shown in the map.
Referring to table 3, the CADM2 site, COX10 site, and MAOA site of the test subject No. 1 are homozygotes, and the PPP1R1B site, and the COMT site of the test subject No. 1 are heterozygotes, and these genotypes all show low pressure sensitivity, indicating that the test subject No. 1 has strong anti-pressure ability.
Fig. 3 is an analytical profile of a saliva sample from subject 2.
The electrophoresis pattern of the amplification product of the testee 2 shows the following characteristic peaks: the CADM2 genotype is T, COX10 is C, PPP1, R1B is CT, MAOA is GA, COMT is GA; pcDNA peak, human genome DNA (huDNA) reference-1, human genome DNA (huDNA) reference-3, and human genome DNA (huDNA) reference-8 characteristic peak.
Wherein the CADM2 gene locus T is shown as CADM 21T; COX10 gene site C is shown as COX 10C; PPP1R1B gene site T is shown as PPP1R1B T, site C is shown as PPP1R1B C; the locus G of the MAOA gene is shown as MAOA G, and the locus A is shown as MAOA A; COMT gene site G shows COMT G, site A shows COMT A; the pcDNA locus is unimodal; human genomic DNA (huDNA) reference-1 is a single peak, and B1 is shown in the map; human genomic DNA (huDNA) reference-3 is a single peak, and B3 is shown in the map; the human genome DNA (huDNA) has a single peak at the reference-8 locus, and B8 is shown in the map.
Referring to table 3, subject No. 2 was of the CADM 2T genotype, indicating that subject 2 was more sensitive to pressure than subject No. 1 and less resistant to compression than subject No. 1.
EXAMPLE III
(1) The selected samples were: blood sample of subject 2.
(2) Sample type: a blood sample.
(3) And (3) extracting DNA: and (3) carrying out DNA extraction on the blood sample by using a nucleic acid extractor.
(4) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(5) Adding a sample: according to the instruction, a certain amount of the extracted DNA sample is taken by a pipette and added to the reaction system.
(6) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(7) Detection of amplification product on 3500DX genetic analyzer
A sample mixture consisting of deionized formamide and an internal molecular weight standard in the system (Size-500) { (1. mu.L Size-500+ 12. mu.L deionized formamide) × (number of samples) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kv, sample injection time: 15s, electrophoresis time 1210-. The results of the measurements are shown in FIG. 4.
(8) Analyzing data
FIG. 4 is an analysis chart of a DNA sample extracted from blood of a subject 2, wherein the analysis chart lacks a CADM 2C peak and a COX 10T peak, and other peaks are all the same as those in FIG. 3 and only have peak height differences, which indicates that the detection kit is also suitable for the DNA sample extracted from the blood sample.
Claims (7)
1. A kit and method for detecting a human pressure sensitivity genotype comprising the steps of:
a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample;
b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification;
c) running an amplification program;
d) carrying out electrophoretic analysis on the amplification product, and judging according to a peak pattern;
the PCR reaction system comprises a primer group for amplifying 5 gene SNP polymorphic sites, 3 personal genome DNA internal references and 1 PCR reaction internal reference, and the primer group is shown in Table 1, and the following primer sequence directions are from 5 'end to 3' end.
TABLE 1 SNP detection sites, primer sequences and PCR amplification fragment lengths of the kit
2. The kit and method for detecting the genotype of human pressure sensitivity according to claim 1, wherein the exfoliated cells in the mouth are exfoliated cells on the inner wall of the mouth, and are preserved on a cell collection card and can be directly used for PCR amplification.
3. The kit and method for detecting the genotype of human pressure sensitivity according to claim 1, wherein the blood sample is a blood sample of a subject or a blood sample collected from a blood storage card, and the extracted DNA is directly used for PCR amplification.
4. The kit and the method for detecting the human scientific gas potential genotype of claim 1, wherein the reaction system comprises a Primer Mix including a Primer group, a PCR reaction solution and DNA/RNA-free water.
5. The kit and method for detecting human pressure sensitivity genotype of claim 1, wherein the Primer Mix in the reaction system comprises primers for detecting all genotypes of SNPs of CADM2, COX10, PPP1R1B, MAOA and COMT, and comprises pcDNA and 3 human genomic DNA internal reference primers.
6. The kit and method for detecting the human pressure-sensitive genotype as claimed in claim 1, wherein the PCR reaction solution in the reaction system comprises 2 XPCR buffer, DNA polymerase, dNTPs, potassium chloride, magnesium chloride, etc.
7. The kit and method for detecting a human pressure sensitivity genotype as claimed in claim 1, wherein the amplification products are analyzed by electrophoresis; preferably the electrophoresis is capillary electrophoresis.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1635142A (en) * | 2003-12-26 | 2005-07-06 | 中国医学科学院基础医学研究所 | Reagent kit for forecasting susceptibility of intolerance type dementia preaecox and primer used thereby |
| CN102373266A (en) * | 2010-08-09 | 2012-03-14 | 上海奇芯基因科技发展有限公司 | Detection chip and detection method of children customized education genes |
| CN102712956A (en) * | 2009-10-30 | 2012-10-03 | 普罗米修斯实验室股份有限公司 | Methods for diagnosing irritable bowel syndrome |
| CN104364654A (en) * | 2012-05-11 | 2015-02-18 | 弗莱德哈钦森癌症研究中心 | Methods for predicting and detecting cancer risk |
| CN106086179A (en) * | 2016-06-16 | 2016-11-09 | 北京东方亚美基因科技研究院有限公司 | A kind of gene tester assessing child's natural endowment ability |
| CN109082466A (en) * | 2018-08-21 | 2018-12-25 | 宁波海尔施基因科技有限公司 | A kind of multiple gene detection kit and its application method detecting CACNA1S gene pleiomorphism |
| CN109207579A (en) * | 2018-09-06 | 2019-01-15 | 宁波海尔施基因科技有限公司 | A kind of Multiple detection kit and application thereof detecting malignant fever tumor susceptibility gene |
| CN109825573A (en) * | 2019-03-20 | 2019-05-31 | 宁波海尔施基因科技有限公司 | A kind of multiple gene detection kit and its application method for antidepressant medication guide |
| CN109952100A (en) * | 2016-09-15 | 2019-06-28 | 普瑞尼亚医疗发展有限公司 | Puli more fixed for treating the purposes of anxiety and depression |
-
2019
- 2019-12-06 CN CN201911242248.8A patent/CN112921100A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1635142A (en) * | 2003-12-26 | 2005-07-06 | 中国医学科学院基础医学研究所 | Reagent kit for forecasting susceptibility of intolerance type dementia preaecox and primer used thereby |
| CN102712956A (en) * | 2009-10-30 | 2012-10-03 | 普罗米修斯实验室股份有限公司 | Methods for diagnosing irritable bowel syndrome |
| CN102373266A (en) * | 2010-08-09 | 2012-03-14 | 上海奇芯基因科技发展有限公司 | Detection chip and detection method of children customized education genes |
| CN104364654A (en) * | 2012-05-11 | 2015-02-18 | 弗莱德哈钦森癌症研究中心 | Methods for predicting and detecting cancer risk |
| CN106086179A (en) * | 2016-06-16 | 2016-11-09 | 北京东方亚美基因科技研究院有限公司 | A kind of gene tester assessing child's natural endowment ability |
| CN109952100A (en) * | 2016-09-15 | 2019-06-28 | 普瑞尼亚医疗发展有限公司 | Puli more fixed for treating the purposes of anxiety and depression |
| CN109082466A (en) * | 2018-08-21 | 2018-12-25 | 宁波海尔施基因科技有限公司 | A kind of multiple gene detection kit and its application method detecting CACNA1S gene pleiomorphism |
| CN109207579A (en) * | 2018-09-06 | 2019-01-15 | 宁波海尔施基因科技有限公司 | A kind of Multiple detection kit and application thereof detecting malignant fever tumor susceptibility gene |
| CN109825573A (en) * | 2019-03-20 | 2019-05-31 | 宁波海尔施基因科技有限公司 | A kind of multiple gene detection kit and its application method for antidepressant medication guide |
Non-Patent Citations (4)
| Title |
|---|
| DAVID S. CHESTER等: "Monoamine oxidase A (MAOA) genotype predicts greater aggression through impulsive reactivity to negative affect", 《BEHAVIOURAL BRAIN RESEARCH》, vol. 283, 28 January 2015 (2015-01-28), pages 97 - 101 * |
| MARTIN REUTER等: "The biological basis of anger: Associations with the gene coding for DARPP-32(PPP1R1B) and with amygdala volume", 《BEHAVIOURAL BRAIN RESEARCH》, vol. 202, 1 April 2009 (2009-04-01), pages 179 - 183, XP026127164, DOI: 10.1016/j.bbr.2009.03.032 * |
| SK SERVICE等: "A genome-wide meta-analysis of association studies of Cloningers Temperament Scales", 《TRANSL PSYCHIATRY》, vol. 2, 15 May 2012 (2012-05-15), pages 116 * |
| 刘继文等: "心理健康问题流行病学研究进展", 《新疆医科大学学报》, vol. 41, no. 11, 30 November 2018 (2018-11-30), pages 1342 - 1346 * |
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