CN116121358B - Composition for psychotropic drug gene detection, kit and use method - Google Patents
Composition for psychotropic drug gene detection, kit and use method Download PDFInfo
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Abstract
The invention provides a primer and probe composition for detecting a gene polymorphism site of a psychotropic drug, a corresponding kit and a use method, and belongs to the technical field of gene detection. The primer pair, the probe and the kit can detect the polymorphism loci of CYP2D6, CYP2C19, CYP2C 3, CYP2C19, CYP1A2, F, CYP C9, HLA-B1502 and ANKK1 genes simultaneously, and have the characteristics of high sensitivity, low cost, high specificity, simple operation, short detection period and the like, the detection sensitivity can reach 0.1ng, and the detection sensitivity can rapidly and accurately detect the polymorphism of the gene related to the mental individuation.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer and probe composition for detecting a gene polymorphism site of a psychotropic drug, a corresponding kit and a use method thereof.
Background
Mental diseases refer to diseases in which mental activities such as cognition, emotion, mind and behavior appear to different extents due to brain dysfunction under the influence of various biological, psychological and social environmental factors, and are clinically manifested, including developmental disorders such as depression, bipolar disorder, schizophrenia, other psychoses, dementia, intellectual disability and autism.
Drug treatment is currently the primary clinical means of mental illness, and related drugs include apilimidazole, thioridazine, risperidone, olanzapine, bunazone, paliperidone, and the like, but drug efficacy and adverse reactions also exhibit large individual differences, such as poor response to typical and atypical antipsychotics in 40% of schizophrenic patients.
The study of the genome of the medicine shows that the most main reason for the large individual difference of the medicine is the gene polymorphism of the patient. Genetic polymorphism promotes genetic variation of related drug metabolizing enzymes, transporters and receptors, changes the functions and expression levels of the enzymes and the binding state of the drug transporters and the receptors, and thus makes different individuals show differences in the responses to drugs.
The cytochrome P4502D6 (CYP 2D 6) gene is taken as a member of cytochrome P450 protease superfamily, has high gene polymorphism, and can participate in metabolic reaction of antipsychotics such as risperidone.
Amitriptyline, as a tricyclic antidepressant, has a variety of adverse effects such as anticholinergic effects, central nervous system adverse effects, and cardiovascular adverse effects, which are closely related to treatment failure. Cytochrome P450 isozymes CYP2C19 participate in the metabolism of amitriptyline, and genetic variation thereof can lead to individual differences in enzyme activity, which can lead to the appearance of 4 phenotypes of ultrafast metabolizers (ultrarapid metabolizer, UM), fast metabolizers (extensive metabolizer, EM), intermediate metabolizers (INTERMEDIATE METABOLIZER, IM) and slow metabolizers (poormetabolizer, PM) in the population, with CYP2C19 x2 and CYP2C19 x 3 being the two major alleles present in the chinese population that lead to defects in CYP2C19 enzymes. Amitriptyline is a prodrug, is metabolized into active metabolite nortriptyline in vivo, the ratio of plasma amitriptyline to nortriptyline concentration of CYP2C19 PM individuals is obviously increased, adverse reaction is caused, treatment is delayed, and medical resource waste is caused.
Therefore, different treatment schemes are designated according to the genetic variation of metabolic enzymes, transporters and receptors related to individual drug treatment, and the realization of individuation of drug treatment is not only the development direction of current genetics and clinical drug therapeutics, but also has very important social and economic significance.
Human gene polymorphism plays an important role in elucidating human susceptibility to diseases, tolerance to poisons, diversity of clinical manifestations of diseases, and responsiveness to drug treatment. The research of psychology and pharmacogenomics can also provide important basis for individual medication selection of mental diseases and prevention of adverse reactions, thereby improving the accuracy of the prescription of the psychology and reducing treatment failure and adverse reactions caused by incorrect medication.
At present, a plurality of technologies aiming at human gene polymorphism detection are available, and the most commonly used technologies comprise polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP method), sequence-specific PCR, first-generation sequencing, second-generation sequencing, gene chip method and the like, but the technologies have respective application defects, have complicated operation and long detection period, and can not carry out high-flux multi-site simultaneous detection, so that the application of the existing technology for detecting the polymorphism is still not ideal in clinic, and the clinical detection requirement can not be met all the time.
In addition, the existing primers and probes for detecting the polymorphic sites of the neuropsychiatric drug-based genes have the problem of low detection sensitivity.
For example, patent document CN 109777870B [1] provides a kit for guiding medication of mental diseases in human and a detection method thereof, the kit can simultaneously genotype 16 gene loci through one reaction, and can well detect mutation of human genome related to medication of mental diseases, but the amount of DNA used for detection of the kit is 10ng, the detection limit is higher, and the sensitivity is to be improved.
As disclosed in patent document CN 110331214a [2], a kit for guiding medication for mental diseases in humans and a detection method thereof are disclosed, which can simultaneously genotype 16 gene loci through one reaction, including 16 pairs of amplification primers and 16 extension primers for amplifying 16 gene fragments, and can accurately detect mutation of human genome related to medication for mental diseases for almost all test samples based on typing by multiplex PCR and mass spectrometry sequencing technology, but the kit can only detect 10ng of human genomic DNA at the minimum, and its detection sensitivity needs to be further improved.
For example, patent document CN 111808943B [3] provides a method for detecting a gene for mental individuation, which screens out 32 polymorphic sites of genes closely related to 41 antipsychotics, and prepares a primer composition for detecting the 32 polymorphic sites and a detection reagent using the composition, the detection limit of the method is 1-5ng, and the detection sensitivity needs to be further improved.
For example, a PCR system adopted in "mutation detection of schizophrenia susceptibility Gene" [4] of Jinan university's paper is used for mutation detection of DISC1 gene exon sequence, and the detection limit of the method is only 5ng DNA, and the detection sensitivity needs to be improved.
The Wu et al [5] established a method for rapidly detecting CYP2D6 x 10 gene polymorphism, and found that the detection sensitivity of the kit to the gene polymorphism can only reach 30 copies/. Mu.L by adopting a plurality of different kits (goods No. 51104, goods No. YZYMT-028, goods No. 56404, goods No. YZYMT-029 and goods No. R013A) and the detection amount of the genome DNA is about 5ng, and a certain non-specific amplification risk exists, so that the detection sensitivity is required to be further improved.
Therefore, how to provide a method for detecting the gene polymorphism of CYP2D6, CYP2C19, CYP2C 17, CYP2C9, CYP1A2, F, HLA-B1502 and ANKK, which has high sensitivity, low cost, high specificity, simple operation and short detection period, can rapidly and accurately detect the gene polymorphism of the CYP2D6, the CYP2C19, the CYP1A2, and the CYP1A2, is a technical problem to be solved in the invention.
Reference is made to:
[1] CN 109777870B, a kit for guiding the administration of mental diseases in human and detection method thereof, applicant: all companies are medical examination by Shanghai Kangli, publication date: 2019.5.21.
[2] CN 110331214a, a kit for guiding the administration of mental diseases in human and detection method thereof, applicant: all companies are medical examination by Shanghai Kangli, publication date: 2019.10.15.
[3] CN 111808943B, a method for detecting mental individuation medication gene, which comprises the following steps: chongqing Pu Luotong Gene medical institute Co., ltd, publication date: 2020.10.23.
[4] University of Jinan's Shuoshi paper "detection of susceptibility gene mutation for schizophrenia", authors: and a dragon seam, 2013.
[5] Wu Jianyuan, zhang Bai, cai Junlong, etc., establishment and evaluation of a CYP2D6×10 gene polymorphism detection system, J.International journal of inspection medicine, 2022, 7, 43, 14.
Disclosure of Invention
The invention aims to solve the technical problems, and provides a primer and probe composition for detecting a psychotropic gene polymorphism site, a corresponding kit and a use method. The technical purpose of the invention is that: aiming at the problems of high requirements on sample processing, long detection period, complex operation, high cost, low specificity, low sensitivity and the like in the prior art for detecting CYP2D6, CYP2C19, CYP2C 3, CYP2C19, CYP2C9, CYP1A2, F, HLA-B1502 and ANKK gene polymorphism, the primer and probe composition for detecting the psychotropic gene, the kit and the use method thereof are provided, and the kit has the characteristics of high detection sensitivity and specificity for whole blood samples and oral swab samples, low cost, simplicity and convenience in operation, short detection period and the like.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A first object of the present invention is to provide a composition for detecting a psychotropic gene polymorphic site, which comprises at least one of a primer pair and a probe combination for different polymorphic sites:
(1) Primer pair and probe combination for detecting CYP2D6 x10 gene locus: a primer pair 1 consisting of a forward primer shown as SEQ ID NO.1 and reverse primers shown as SEQ ID NO.2-3, and a probe 1 shown as SEQ ID NO. 28;
(2) Primer pair and probe combination for detecting CYP2C19 x 2 gene locus: a primer pair 2 consisting of a forward primer shown as SEQ ID NO.4-5 and a reverse primer shown as SEQ ID NO.6, and a probe 2 shown as SEQ ID NO. 29;
(3) Primer pair and probe combination for detecting CYP2C19 x 3 gene locus: a primer pair 3 consisting of a forward primer shown as SEQ ID NO.7-8 and a reverse primer shown as SEQ ID NO.9, and a probe 3 shown as SEQ ID NO. 30;
(4) Primer pair and probe combination for detecting CYP2C19 x 17 gene locus: a primer pair 4 consisting of a forward primer shown as SEQ ID NO.10-11 and a reverse primer shown as SEQ ID NO.12, and a probe 4 shown as SEQ ID NO. 31;
(5) Primer pair and probe combination for detecting CYP1A2 x 1F gene locus: a primer pair 5 consisting of a forward primer shown as SEQ ID NO.13-14 and a reverse primer shown as SEQ ID NO.15, and a probe 5 shown as SEQ ID NO. 32;
(6) Primer pair and probe combination for detecting CYP2C9 x 3 gene locus: a primer pair 6 consisting of a forward primer shown as SEQ ID NO.16-17 and a reverse primer shown as SEQ ID NO.18, and a probe 6 shown as SEQ ID NO. 33;
(7) Primer pair and probe combination for detecting HLA-B1502 (rs 3909184) gene locus: a primer pair 7 consisting of a forward primer shown as SEQ ID NO.19 and a reverse primer shown as SEQ ID NO.20-21, and a probe 7 shown as SEQ ID NO. 34; primer pair and probe combination for detecting HLA-B1502 (rs 2844682) gene locus: a primer pair 8 consisting of a forward primer shown as SEQ ID NO.22-23 and a reverse primer shown as SEQ ID NO.24, and a probe 8 shown as SEQ ID NO. 35;
(8) Primer pair and probe combination for detecting ANKK gene locus: a primer pair 9 consisting of a forward primer shown as SEQ ID NO.25-26 and a reverse primer shown as SEQ ID NO.27, and a probe 9 shown as SEQ ID NO. 36.
The primer pair and the probe composition provided by the invention can obviously improve the sensitivity of the existing PCR detection method, enhance the reaction specificity and improve the system stability. Experiments prove that the primer pair and the probe composition provided by the invention have the characteristics of high sensitivity, low cost, high specificity, simple operation, short detection period and the like when being used for detecting the gene polymorphism of CYP2D6, CYP2C19, CYP2C 3, CYP2C19, CYP2C9, CYP1A2, F, HLA-B, 1502 and ANKK, and can be used for rapidly and accurately detecting the gene polymorphism of the mental individuation drug. As described in the background art, the detection limit of the primer adopted by the existing detection system is generally 5-10ng, and the detection limit of the detection system formed by the composition can be as low as 0.1ng, so that 100% detection rate of complex and difficult templates such as oral swab samples and blood samples can be achieved, and the sensitivity of the detection system is greatly improved.
Further, in the above composition provided by the invention, the fluorescent group at the 5' end of the probe 1-9 comprises FAM, VIC, HEX, CY or a ROX fluorescent reporter group, preferably FAM, VIC or CY5; the 3' -end quenching group comprises TAMRA, BHQ1, BHQ2, MGB or Dabcy1 fluorescence quenching group, preferably TAMRA or BHQ1.
Further, the composition provided by the invention further comprises inhibitors aiming at different polymorphic sites, wherein the inhibitors have at least one of the following sequence combinations:
(1) Inhibitor 1 for detecting CYP2D6 x 10 gene locus: a combination of sequences as shown in SEQ ID No.37 and SEQ ID No. 38;
(2) Inhibitor 2 for detecting the CYP2C19 x 2 gene locus: a combination of sequences as shown in SEQ ID No.39 and SEQ ID No. 40;
(3) Inhibitor 3 for detecting CYP2C19 x 3 gene locus: a combination of sequences as shown in SEQ ID No.41 and SEQ ID No. 42;
(4) Inhibitor 4 for detecting the CYP2C19 x 17 gene locus: a combination of sequences as shown in SEQ ID No.43 and SEQ ID No. 44;
(5) Inhibitor 5 for detecting CYP1A2 x 1F gene locus: a combination of sequences as shown in SEQ ID No.45 and SEQ ID No. 46;
(6) Inhibitor 6 for detecting CYP2C9 x3 gene locus: a combination of sequences as shown in SEQ ID No.47 and SEQ ID No. 48;
(7) Inhibitor 7 for detecting HLA-B1502 gene locus: a combination of sequences as shown in SEQ ID No.49 and SEQ ID No. 50; a combination of sequences as shown in SEQ ID NO.51 and SEQ ID NO. 52;
(8) Inhibitor 8 for detecting ANKK1 gene locus: the sequence combination is shown as SEQ ID NO.53 and SEQ ID NO. 54.
The combination of the primer, the probe and the inhibitor has the advantages of low sample processing requirement, short detection period, simple operation, low cost, strong specificity and high sensitivity, and can rapidly and accurately detect the polymorphism of the individual psychology medication gene.
Further, the 3' end of the above-mentioned inhibitors 1 to 9 of the present invention is modified with C3 spacer, phosphorylation, thio, MGB or ddC, preferably ddC.
It is a second object of the present invention to provide a kit for detecting a psychotropic gene polymorphism site, comprising the primer pair and probe combination as described above, and a corresponding inhibitor.
Further, the kit also comprises GAPDH internal reference gene primers and probes, PCR reaction liquid, positive quality control products and negative quality control products, wherein:
the sequences of the GAPDH internal reference gene primer and the probe are as follows:
GAPDH-F：GTATGACTGGGGGTGTTGGG
GAPDH-R：GAGGGCCCAAGAGGTTGAAT
GAPDH-P：CCAGCCTGGCAGGGAAGCTCAAG；
The PCR reaction solution comprises hot start taq enzyme, UDG enzyme, buffer solution, magnesium ions and dNTP substances required by the PCR reaction; the method for obtaining the positive quality control product comprises the following steps: constructing a synthetic sequence gene fragment by plasmids according to CYP2D6, CYP2C19, CYP2C 3, CYP2C19, CYP1A2, F, CYP C9, HLA-B1502, ANKK1 and GAPDH related gene sequences published by NCBI database, inserting the fragment into a T vector, converting and extracting plasmids by using escherichia coli DH5 alpha strain, and mixing quality control quality granules in equal proportion to obtain the positive quality control product; the negative quality control product is deionized water treated by DEPC.
It is still another object of the present invention to provide a method for using the kit for detecting a polymorphic site of a psychotropic gene as described above, comprising the steps of:
S1: taking an acquisition sample containing EDTA anticoagulant for DNA extraction;
S2: premixing and split charging gene detection reagents to obtain PCR reaction liquids 1-8 aiming at a plurality of different polymorphic sites respectively, and forming a PCR reaction system together;
S3: adding the DNA obtained in the step S1 into the PCR reaction system of the step S2, and carrying out PCR amplification after centrifugation;
s4: after the reaction is finished, carrying out genotype identification and interpretation;
wherein, the detection sites of the PCR reaction liquids 1-8 are respectively as follows:
| PCR reaction solution | FAM signal | VIC signal | CY5 signal |
| PCR reaction solution 1 | CYP2D6 x 10 wild type | CYP1A2 x 1F wild type | GAPDH gene |
| PCR reaction solution 2 | CYP2D6 x 10 mutant | CYP1A2 x 1F mutant | GAPDH gene |
| PCR reaction solution 3 | CYP2C19 x 2 wild type | CYP2C9 x 3 wild type | GAPDH gene |
| PCR reaction solution 4 | CYP2C19 x 2 mutant | CYP2C9 x 3 mutant | GAPDH gene |
| PCR reaction solution 5 | CYP2C19 x 3 wild type | HLA-B1502 wild type | GAPDH gene |
| PCR reaction solution 6 | CYP2C19 x 3 mutant | HLA-B1502 mutant | GAPDH gene |
| PCR reaction solution 7 | CYP2C 19.17 wild type | ANKK1 wild type | GAPDH gene |
| PCR reaction solution 8 | CYP2C 19-17 mutant | ANKK1 mutant form | GAPDH gene |
Further, the conditions for the PCR amplification in step S3 are as follows:
The conditions for the incubation of the UDG enzyme are: 37 ℃ for 2 minutes;
The conditions for the pre-denaturation are: 95 ℃ for 2 minutes;
the first phase consisted of 5 amplification cycles, provided that: denaturation: 95 ℃ for 15 seconds; annealing: 60 ℃ for 30 seconds; extension: 72 ℃,20 seconds;
The second stage consisted of 35 amplification cycles, provided that: denaturation: 95 ℃ for 15 seconds; annealing: setting fluorescence signal collection at 60 ℃ for 30 seconds; extension: 72℃for 30 seconds.
Further, the genotype identification method comprises the following steps: under the conditions of a PCR reaction system and a circulation program, FAM, VIC and CY5 fluorescence detection signals of each positive quality control product form a logarithmic amplification S-shaped curve; each negative quality control product has no amplification curve or Ct value of 0; the CY5 fluorescence detection signals of each sample to be detected should form a logarithmic amplification S-shaped curve; then observing FAM and VIC fluorescence detection signal amplification curves of the reaction tube of the sample to be detected, and if a logarithmic amplification S-shaped curve is formed, the sample to be detected contains corresponding base polymorphism sites; if the logarithmic amplification "S" type curve is not used or the Ct value is 0, there is no corresponding base polymorphism site.
The beneficial effects of the invention are as follows:
(1) The invention provides a primer pair and probe composition for detecting a psychotropic gene polymorphism site, and a composition containing a corresponding inhibitor, wherein the composition has high detection sensitivity and specificity, and the problem of low sensitivity of the existing primer and probe in the aspect of detecting the psychotropic gene polymorphism site is greatly solved;
(2) According to the invention, 6 related genes and GAPDH reference gene compositions are packaged in an 8-linkage PCR reaction strip in a pre-mixing way, so that the detection is convenient, the multi-gene variation analysis of a reaction system is realized, the cost is reduced, and the detection efficiency is improved;
(3) The kit has the characteristics of high sensitivity, low cost, high specificity, simple and convenient operation, short detection period and the like, the sensitivity can reach 0.1ng, and the polymorphism of the gene related to the individual administration of the mental class can be rapidly and accurately detected.
Drawings
FIG. 1 is a schematic diagram showing the design structure of the probe (left) and the primer (right) of the present invention.
FIG. 2 is a schematic diagram showing the working principle of the probe primer of the present invention.
FIG. 3 shows the amplification curves of positive quality control samples of a psychotropic drug gene test kit and method of use according to the present invention.
FIG. 4 shows the negative quality control amplification curve of a psychotropic gene detection kit and method of use according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be specifically described with reference to the following examples, which are provided for explaining and illustrating the present invention only and are not intended to limit the present invention. Some non-essential modifications and adaptations of the invention according to the foregoing summary will still fall within the scope of the invention.
Example 1: method for designing primer and probe
The following first describes the design concept of the primer and probe of the present invention, and the design structure of the primer and probe of the present invention will be better explained with reference to the accompanying drawings:
as shown in fig. 1 and 2, the technical method of the primer and the probe provided by the invention is as follows:
An improved PCR amplification primer structure, which is q-shaped and 26-44 bases in length, comprising 4 parts from 5 'end to 3':
A first portion of 4-10 bases in length, the sequence of which is a deep sea species sequence, having low homology to human genomic sequences; the preferred base sequence is TAACATA;
a second part, the length of which is 15-20 bases, the sequence of which matches the target gene sequence;
a third portion having a sequence complementary to the first portion and a sequence TATGTTA;
and a fourth part with a length of 3-5 bases, the sequence of which matches the sequence of the target gene.
An improved probe structure, which is circular in shape and 28-42 bases in length, comprising five parts from 5 'end to 3':
a first part having a length of 10 to 15 bases and a sequence matching the target gene sequence;
A second part of 4-6 bases in length and having a sequence of a deep sea species having low homology with the human genome sequence, preferably a base sequence of AGATGT;
a third part, the length of which is 10-15 bases, the sequence of which is matched with the target gene sequence;
A fourth portion having a sequence complementary to the second portion and a sequence ACATCT;
a fifth part having the same sequence as the third part;
The fluorescent group at the 5 'end of the probe is FAM, VIC, HEX, CY or ROX fluorescent report group suitable for fluorescent quantitative PCR analysis, and the quenching group at the 3' end is TAMRA, BHQ1, BHQ2, MGB or Dabcy1 fluorescent quenching group suitable for fluorescent quantitative PCR.
Example 2: primer, probe and inhibitor combination design and use for detecting polymorphic site of neuropsychiatric drug gene
1. Primer, probe and inhibitor combination for detecting CYP2D6 x 10 gene locus
(1) Primer, probe and inhibitor combinations for recognizing CYP2D6 > 10> c:
CYP2D-WR1：
TAACATACAACGCTGGGCTGCACGTATGTTACTACC(SEQ ID NO.2)
CYP2D-F1：
TAACATATCCCTCACCTGGTCGAATATGTTAGCAGT(SEQ ID NO.1)
CYP2D-P1：
FAM-AACCTGCTGCATAGATGTGTGGACTTCCAGAACATCTGTGGACTTCCAGA-TAMRA(SEQ ID NO.28)
CYP2D-WB：
CGCTGGGCTGCACGCTACTCACCAGGCCCCCTGCC-ddC(SEQ ID NO.37)
(2) Primer, probe and inhibitor combinations for recognizing CYP2D6 > 10 t:
CYP2D-MR1：
TAACATACAACGCTGGGCTGCACGTATGTTACTACT(SEQ ID NO.3)
CYP2D-F1：
TAACATATCCCTCACCTGGTCGAATATGTTAGCAGT(SEQ ID NO.1)
CYP2D-P1：
FAM-AACCTGCTGCATAGATGTGTGGACTTCCAGAACATCTGTGGACTTCCAGA-TAMRA(SEQ ID NO.28)
CYP2D-MB：
CGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCC-ddC(SEQ ID NO.38)
the fluorescent group at the 5 'end of the probe is a fluorescent report group which is suitable for fluorescent quantitative PCR analysis and is conventionally used, and can be FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is conventionally used, and can be TAMRA, BHQ1, BHQ2, MGB or Dabcy1, preferably, the fluorescent group at the 5 'end is FAM, and the quenching group at the 3' end is TAMRA. The 3 '-end of the inhibitor is specially modified, preferably C3 spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably ddC is modified at the 3' -end of the inhibitor for specific template amplification.
2. Primer, probe and inhibitor combinations for detecting CYP2C19 x 2 gene locus
(1) Primer, probe and inhibitor combinations for recognizing CYP2C19 x 2>G:
CYP2C19*2-WF2：
TAACATACCCACTATCATTGATTATTTATGTTATCCCG(SEQ ID NO.4)
CYP2C19*2-R：
TAACATATTTTGTTAACATTTTACCTATGTTATTCTC(SEQ ID NO.6)
CYP2C19*2-P1：
FAM-ATTACTTAAAAACCAGATGTTTGCTTTTATGGAAACATCTTTGCTTTTATGGAA-TAMRA(SEQ ID NO.29)
CYP2C19*2-WB：
TTTCCCACTATCATTGATTATTTCCCAGGAAC-ddC(SEQ ID NO.39)
(2) Primer, probe and inhibitor combinations for recognizing CYP2C19 x 2>A:
CYP2C19*2-MF4：
TAACATACCCACTATCATTGATTATTTATGTTATCCCA(SEQ ID NO.5)
CYP2C19*2-R：
TAACATATTTTGTTAACATTTTACCTATGTTATTCTC(SEQ ID NO.6)
CYP2C19*2-P1：
FAM-ATTACTTAAAAACCAGATGTTTGCTTTTATGGAAACATCTTTGCTTTTATGGAA-TAMRA(SEQ ID NO.29)
CYP2C19*2-MB：
TTTCCCACTATCATTGATTATTTCCCGGGAAC-ddC(SEQ ID NO.40)
The fluorescent group at the 5 'end of the probe is a fluorescent reporter group which is suitable for fluorescent quantitative PCR analysis and is commonly used, preferably FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is commonly used, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, more preferably the fluorescent group at the 5 'end is FAM, and the quenching group at the 3' end is TAMRA. The 3 '-end of the inhibitor is specially modified, preferably C3 spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably ddC is modified at the 3' -end of the inhibitor for specific template amplification.
3. Primer, probe and inhibitor combinations for detecting CYP2C19 x3 gene locus
(1) Primer, probe and inhibitor combinations for recognizing CYP2C19 x 3>G:
CYP2C19*3-WF3：
TAACATAGGATTGTAAGCACCCTATGTTACCTGG(SEQ ID NO.7)
CYP2C19*3-R：
TAACATATTTGCCATCTTTTCCAGATATATGTTATTCAC(SEQ ID NO.9)
CYP2C19*3-P1：
FAM-ACAGTCTTTTTTTCAGATGTTGGGAAATCCAAAAACATCTTGGGAAATCCAAAA-TAMRA(SEQ ID NO.30)
CYP2C19*3-WB：
TCAGGATTGTAAGCACCCCCTGAATCCAGG-ddC(SEQ ID NO.41)
(2) Primer, probe and inhibitor combinations for recognizing CYP2C19 x 3>A:
CYP2C19*3-MF3：
TAACATAGGATTGTAAGCACCCTATGTTACCTGA(SEQ ID NO.8)
CYP2C19*3-R：
TAACATATTTGCCATCTTTTCCAGATATATGTTATTCAC(SEQ ID NO.9)
CYP2C19*3-P1：
FAM-ACAGTCTTTTTTTCAGATGTTGGGAAATCCAAAAACATCTTGGGAAATCCAAAA-TAMRA(SEQ ID NO.30)
CYP2C19*3-MB：
TCAGGATTGTAAGCACCCCCTGGATCCAGG-ddC(SEQ ID NO.42)
The fluorescent group at the 5 'end of the probe is a fluorescent reporter group which is suitable for fluorescent quantitative PCR analysis and is commonly used, preferably FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is commonly used, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, more preferably the fluorescent group at the 5 'end is FAM, and the quenching group at the 3' end is TAMRA. The 3 '-end of the inhibitor is specially modified, preferably C3 spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably ddC is modified at the 3' -end of the inhibitor for specific template amplification.
4. Primer, probe and inhibitor combination for detecting CYP2C19 x 17 gene locus
(1) Primer, probe and inhibitor combinations for recognizing CYP2C19 x 17> C:
CYP2C19*17-WF：
TAACATAATTTGTGTCTTCTGTTCTCTATGTTAAAAGC(SEQ ID NO.10)
CYP2C19*17-R：
TAACATATAGCAGATATAAACACCTTTTATGTTAACCAT(SEQ ID NO.12)
CYP2C19*17-P：
FAM-TCAGAAGACCTCAGATGTAGCTCAAATCCCAACATCTAGCTCAAATCCCA-TAMRA(SEQ ID NO.31)
CYP2C19*17-WB：
GTTCTCAAAGTATCTCTGATGTAAGAG-ddC(SEQ ID NO.43)
(2) Primer, probe and inhibitor combinations for recognizing CYP2C19 x 17> t:
CYP2C19*17-MF：
TAACATAATTTGTGTCTTCTGTTCTCTATGTTAAAAGT(SEQ ID NO.11)
CYP2C19*17-R：
TAACATATAGCAGATATAAACACCTTTTATGTTAACCAT(SEQ ID NO.12)
CYP2C19*17-P：
FAM-TCAGAAGACCTCAGATGTAGCTCAAATCCCAACATCTAGCTCAAATCCCA-TAMRA(SEQ ID NO.31)
CYP2C19*17-MB：
GTTCTCAAAGCATCTCTGATGTAAGAG-ddC(SEQ ID NO.44)
The fluorescent group at the 5 'end of the probe is a fluorescent reporter group which is suitable for fluorescent quantitative PCR analysis and is commonly used, preferably FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is commonly used, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, more preferably the fluorescent group at the 5 'end is FAM, and the quenching group at the 3' end is TAMRA. The 3 '-end of the inhibitor is specially modified, preferably C3 spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably ddC is modified at the 3' -end of the inhibitor for specific template amplification.
5. Primer, probe and inhibitor combination for detecting CYP1A 2x 1F gene locus
(1) Primer, probe and inhibitor combinations for recognizing CYP1A2 x 1f > c:
CYP1A2*1F-WF：
TAACATAAAGGGTGAGCTCTGTTATGTTAGGGCC(SEQ ID NO.13)
CYP1A2*1F-R：
TAACATAGCTGAGGGTTGAGATGGTATGTTAAGACA(SEQ ID NO.15)
CYP1A2*1F-P：
VIC-GATGGAGCTTAGTAGATGTCTTTCTGGTATCCACATCTCTTTCTGGTATCC-TAMRA(SEQ ID NO.32)
CYP1A2*1F-WB：
GTGGGCACAGGACGCATGGTAGATGGAG-ddC(SEQ ID NO.45)
(2) Primer, probe and inhibitor combinations for recognizing CYP1A2 x 1f > a:
CYP1A2*1F-MF：
TAACATAAAGGGTGAGCTCTGTTATGTTAGGGCA(SEQ ID NO.14)
CYP1A2*1F-R：
TAACATAGCTGAGGGTTGAGATGGTATGTTAAGACA(SEQ ID NO.15)
CYP1A2*1F-P：
VIC-GATGGAGCTTAGTAGATGTCTTTCTGGTATCCACATCTCTTTCTGGTATCC-TAMRA(SEQ ID NO.32)
CYP1A2*1F-MB：
GTGGGCCCAGGACGCATGGTAGATGGAG-ddC(SEQ ID NO.46)
the fluorescent group at the 5 'end of the probe is a fluorescent reporter group which is suitable for fluorescent quantitative PCR analysis and is commonly used, preferably FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is commonly used, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, more preferably the fluorescent group at the 5 'end is VIC, and the quenching group at the 3' end is TAMRA. The 3 '-end of the inhibitor is specially modified, preferably C3 spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably ddC is modified at the 3' -end of the inhibitor for specific template amplification.
6. Primer, probe and inhibitor combinations for detecting CYP2C9 x 3 gene locus
(1) Primer, probe and inhibitor combinations for recognizing CYP2C9 x 3>A:
CYP2C9-WF4：
TAACATAGTGCACGAGGTCCAGAGTATGTTAATACA(SEQ ID NO.16)
CYP2C9-R：
TAACATAACATGGAGTTGCAGTGTTATGTTAAGGAG(SEQ ID NO.18)
CYP2C9-P1：
VIC-CATGCAGTGACCTGAGATGTTGACATTAAATTCACATCTTGACATTAAATTC-TAMRA(SEQ ID NO.33)
CYP2C9-WB：
ACGAGGTCCAGAGATACCTTGACCTTCTCCCCACCAG-ddC (SEQ ID No. 47) (2) primer, probe and inhibitor combinations for recognizing CYP2C9 x 3>C:
CYP2C9-MF3：
TAACATAGTGCACGAGGTCCAGAGTATGTTAATACC(SEQ ID NO.17)
CYP2C9-R：
TAACATAACATGGAGTTGCAGTGTTATGTTAAGGAG(SEQ ID NO.18)
CYP2C9-P1：
VIC-CATGCAGTGACCTGAGATGTTGACATTAAATTCACATCTTGACATTAAATTC-TAMRA(SEQ ID NO.33)
CYP2C9-MB：
ACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAG-ddC(SEQ ID NO.48)
The fluorescent group at the 5 'end of the probe is a fluorescent reporter group which is suitable for fluorescent quantitative PCR analysis and is commonly used, preferably FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is commonly used, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, more preferably the fluorescent group at the 5 'end is VIC, and the quenching group at the 3' end is TAMRA. The 3 '-end of the inhibitor is specially modified, preferably C3spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably ddC is modified at the 3' -end of the inhibitor for specific template amplification.
7. Primer, probe and inhibitor combination for detecting HLA-B1502 gene locus
(1) Primer, probe and inhibitor combinations for recognition of HLA-B1502 (rs 3909184) > G:
HLA-B*1502-WR1：
TAACATATATACTCTAGAAGGGGGTATGTTACACAC(SEQ ID NO.20)
HLA-B*1502-F1：
TAACATAATTGCTTGAACCTGGGAGTATGTTAGCGAA(SEQ ID NO.19)
HLA-B*1502-P1：
VIC-AAACGGGGGCTCATCAGATGTAGAGAAACACTGGAACATCTAGAGAAACACTGGA-TAMRA(SEQ ID NO.34)
HLA-B*1502-WB1：
GCACAGGTGGGAAAAATAGATTAAACGGGG-ddC(SEQ ID NO.49)
(2) Primer, probe and inhibitor combinations for recognition of HLA-B1502 (rs 3909184) > C:
HLA-B*1502-MR1：
TAACATATATACTCTAGAAGGGGGTATGTTACACAG(SEQ ID NO.21)
HLA-B*1502-F1：
TAACATAATTGCTTGAACCTGGGAGTATGTTAGCGAA(SEQ ID NO.19)
HLA-B*1502-P1：
VIC-AAACGGGGGCTCATCAGATGTAGAGAAACACTGGAACATCTAGAGAAACACTGGA-TAMRA(SEQ ID NO.34)
HLA-B*1502-MB1：
GCACACGTGGGAAAAATAGATTAAACGGGG-ddC(SEQ ID NO.50)
(3) Primer, probe and inhibitor combinations for recognition of HLA-B1502 (rs 2844682) > G:
HLA-B*1502-WF2：
TAACATAGCTTCTCTGAGGTCTCCTATGTTAAAGGG(SEQ ID NO.22)
HLA-B*1502-R2：
TAACATACTCCTCCCCACAAGAGTTGGTATGTTAAACCA(SEQ ID NO.24)
HLA-B*1502-P2：
VIC-GGAGTGGTTCTGGAAGATGTGGAGACCTGCAGCCACATCTGGAGACCTGCAGCC-TAMRA(SEQ ID NO.35)
HLA-B*1502-WB2：
CTTCTCTGAGGTCTCCAAGGAATTGGCCAT-ddC(SEQ ID NO.51)
(4) Primer, probe and inhibitor combinations for recognition of HLA-B1502 (rs 2844682) > a:
HLA-B*1502-MF2：
TAACATAGCTTCTCTGAGGTCTCCTATGTTAAAGGA(SEQ ID NO.23)
HLA-B*1502-R2：
TAACATACTCCTCCCCACAAGAGTTGGTATGTTAAACCA(SEQ ID NO.24)
HLA-B*1502-P2：
VIC-GGAGTGGTTCTGGAAGATGTGGAGACCTGCAGCCACATCTGGAGACCTGCAGCC-TAMRA(SEQ ID NO.35)
HLA-B*1502-MB2：
CTTCTCTGAGGTCTCCAAGGGATTGGCCAT-ddC(SEQ ID NO.52)
The fluorescent group at the 5 'end of the probe is a fluorescent reporter group which is suitable for fluorescent quantitative PCR analysis and is commonly used, preferably FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is commonly used, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, more preferably the fluorescent group at the 5 'end is VIC, and the quenching group at the 3' end is TAMRA. The 3 '-end of the inhibitor is specially modified, preferably C3spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably ddC is modified at the 3' -end of the inhibitor for specific template amplification.
8. Primer, probe and inhibitor combinations for detecting ANKK gene
(1) Primer, probe and inhibitor combinations for identifying ANKK > G:
ANKK1-WF3：
TAACATACATCCTCAAAGTGCTTATGTTAGGTCG(SEQ ID NO.25)
ANKK1-R：
TAACATAGCTGGCCAAGTTGTCTATATGTTAAATTT(SEQ ID NO.27)
ANKK1-P1：
VIC-TGGCCCTCCGCAGCCAGATGTGAAAGCAGGGCATCAACATCTGAAAGCAGGGCATCA-TAMRA(SEQ ID NO.36)
ANKK1-WB：
CACAGCCATCCTCAAAGTGCTGGTCAAGG-ddC(SEQ ID NO.53)
(2) Primer, probe and inhibitor combinations for identifying ANKK > a:
ANKK1-MF4：
TAACATACATCCTCAAAGTGCTTATGTTAGGTCA(SEQ ID NO.26)
ANKK1-R：
TAACATAGCTGGCCAAGTTGTCTATATGTTAAATTT(SEQ ID NO.27)
ANKK1-P1：
VIC-TGGCCCTCCGCAGCCAGATGTGAAAGCAGGGCATCAACATCTGAAAGCAGGGCATCA-TAMRA(SEQ ID NO.36)
ANKK1-MB：
CACAGCCATCCTCAAAGTGCTGGTCGAGG-ddC(SEQ ID NO.54)
The fluorescent group at the 5 'end of the probe is a fluorescent reporter group which is suitable for fluorescent quantitative PCR analysis and is commonly used, preferably FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is commonly used, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, more preferably the fluorescent group at the 5 'end is VIC, and the quenching group at the 3' end is TAMRA. The 3 '-end of the inhibitor is specially modified, preferably C3spacer, phosphorylation, thio, MGB, dideoxycytidine (ddC) and the like, and more preferably ddC is modified at the 3' -end of the inhibitor for specific template amplification.
9. Primer and probe combinations for identifying GAPDH:
GAPDH-F：GTATGACTGGGGGTGTTGGG
GAPDH-R：GAGGGCCCAAGAGGTTGAAT
GAPDH-P：CY5-CCAGCCTGGCAGGGAAGCTCAAG-BHQ1。
The fluorescent group at the 5 'end of the probe is a fluorescent reporter group which is suitable for fluorescent quantitative PCR analysis and is commonly used, preferably FAM, VIC, HEX, CY or ROX, the quenching group at the 3' end is a fluorescent quenching group which is suitable for fluorescent quantitative PCR and is commonly used, preferably TAMRA, BHQ1, BHQ2, MGB or Dabcy1, more preferably the fluorescent group at the 5 'end is CY5, and the quenching group at the 3' end is BHQ1.
The primer pair and the probe provided by the invention can be used for specifically detecting the gene polymorphism of CYP2D6, CYP2C19, CYP2C 3, CYP2C19, CYP1A2, F, CYP, 2C9, HLA-B1502 and ANKK1 in a sample, and are shown in the following table 1.
TABLE 1
Example 3: acquisition of cationic control
The method for obtaining the positive quality control product comprises the following steps: and constructing a synthetic sequence gene fragment by plasmids according to 9 gene sequences of CYP2D6, CYP2C19, CYP2C 3, CYP2C19, CYP2C9, CYP1A2, F, HLA-B1502, ANKK1 and GAPDH published by NCBI database, inserting the fragment into a T vector, converting and extracting plasmids by using an escherichia coli DH5 alpha strain, and mixing quality control quality particles in equal proportion to obtain the positive quality control product.
The sequence of each quality control product is as follows:
CYP2D6 > 10C quality control sequence is shown in SEQ ID NO.55, CYP2D6 > 10> T quality control sequence is shown in SEQ ID NO.56, CYP2C19 > 2>G quality control sequence is shown in SEQ ID NO.57, CYP2C19 > 2>A quality control sequence is shown in SEQ ID NO.58, CYP2C19 > 3>G quality control sequence is shown in SEQ ID NO.59, CYP2C19 > 3>A quality control sequence is shown in SEQ ID NO.60, CYP2C19 > 17> C quality control sequence is shown in SEQ ID NO.61, CYP2C19 > 17> T quality control sequence is shown in SEQ ID NO.62, CYP1A 2> 1F > C quality control sequence is shown in SEQ ID NO.63, CYP1A2 x 1F > A quality control sequence is shown as SEQ ID NO.64, CYP2C9 x 3>A quality control sequence is shown as SEQ ID NO.65, CYP2C9 x 3>C quality control sequence is shown as SEQ ID NO.66, HLA-B1502 (rs 3909184) > G quality control sequence is shown as SEQ ID NO.67, HLA-B1502 (rs 3909184) > C quality control sequence is shown as SEQ ID NO.68, HLA-B1502 (rs 2844682) > G quality control sequence is shown as SEQ ID NO.69, HLA-B1502 (rs 2844682) > A quality control sequence is shown as SEQ ID NO.70, ANKK1> G quality control sequence is shown as SEQ ID NO.71, and ANKK > A quality control sequence is shown as SEQ ID NO. 72.
CYP2D6 > 10> C quality control sequence (SEQ ID NO. 55):
TCCAGGACCTCCTCCCTCACCTGGTCGAAGCAGTATGGTGTGTTCTGGAAGTCCACATGCAGCAGGTTGCCCAGCCCGGGCAGTGGCAGGGGGCCTGGTGGGTAGCGTGCAGCCCAGCGTTGGCGCCGGTGCATCAGGTCCACCAGGAGCAGGAAGATGGCCACTATCACGGCCAGGGGCACCAGTGCTTCTAGCCCCAT
CYP2D6 > 10> T quality control sequence (SEQ ID NO. 56):
TCCAGGACCTCCTCCCTCACCTGGTCGAAGCAGTATGGTGTGTTCTGGAAGTCCACATGCAGCAGGTTGCCCAGCCCGGGCAGTGGCAGGGGGCCTGGTGAGTAGCGTGCAGCCCAGCGTTGGCGCCGGTGCATCAGGTCCACCAGGAGCAGGAAGATGGCCACTATCACGGCCAGGGGCACCAGTGCTTCTAGCCCCAT
CYP2C19 x 2>G quality control sequence (SEQ ID NO. 57):
GCATATTGTATCTATACCTTTATTAAATGCTTTTAATTTAATAAATTATTGTTTTCTCTTAGATATGCAATAATTTTCCCACTATCATTGATTATTTCCCGGGAACCCATAACAAATTACTTAAAAACCTTGCTTTTATGGAAAGTGATATTTTGGAGAAAGTAAAAGAACACCAAGAATCGATGGACATCAACAACCCT
CYP2C19 x 2>A quality control sequence (SEQ ID NO. 58):
GCATATTGTATCTATACCTTTATTAAATGCTTTTAATTTAATAAATTATTGTTTTCTCTTAGATATGCAATAATTTTCCCACTATCATTGATTATTTCCCAGGAACCCATAACAAATTACTTAAAAACCTTGCTTTTATGGAAAGTGATATTTTGGAGAAAGTAAAAGAACACCAAGAATCGATGGACATCAACAACCCT
CYP2C19 x 3>G quality control sequence (SEQ ID NO. 59):
GCTCCATTATTTTCCAGAAACGTTTCGATTATAAAGATCAGCAATTTCTTAACTTGATGGAAAAATTGAATGAAAACATCAGGATTGTAAGCACCCCCTGGATCCAGGTAAGGCCAAGTTTTTTGCTTCCTGAGAAACCACTTACAGTCTTTTTTTCTGGGAAATCCAAAATTCTATATTGACCAAGCCCTGAAGTACAT
CYP2C19 x 3>A quality control sequence (SEQ ID NO. 60):
GCTCCATTATTTTCCAGAAACGTTTCGATTATAAAGATCAGCAATTTCTTAACTTGATGGAAAAATTGAATGAAAACATCAGGATTGTAAGCACCCCCTGAATCCAGGTAAGGCCAAGTTTTTTGCTTCCTGAGAAACCACTTACAGTCTTTTTTTCTGGGAAATCCAAAATTCTATATTGACCAAGCCCTGAAGTACAT
CYP2C19 x 17> C quality control sequence (SEQ ID NO. 61):
ATATTCAGAATAACTAATGTTTGGAAGTTGTTTTGTTTTGCTAAAACAAAGTTTTAGCAAACGATTTTTTTTTTCAAATTTGTGTCTTCTGTTCTCAAAGCATCTCTGATGTAAGAGATAATGCGCCACGATGGGCATCAGAAGACCTCAGCTCAAATCCCAGTTCTGCCAGCTATGAGCTGTGTGGCACCAACAGGTGT
CYP2C19 x 17> T quality control sequence (SEQ ID NO. 62):
ATATTCAGAATAACTAATGTTTGGAAGTTGTTTTGTTTTGCTAAAACAAAGTTTTAGCAAACGATTTTTTTTTTCAAATTTGTGTCTTCTGTTCTCAAAGTATCTCTGATGTAAGAGATAATGCGCCACGATGGGCATCAGAAGACCTCAGCTCAAATCCCAGTTCTGCCAGCTATGAGCTGTGTGGCACCAACAGGTGT
CYP1A2 x 1F > C quality control sequence (SEQ ID NO. 63):
GATGATGTGTGGAGGAGAGAGCCAGCGTTCATGTTGGGAATCTTGAGGCTCCTTTCCAGCTCTCAGATTCTGTGATGCTCAAAGGGTGAGCTCTGTGGGCCCAGGACGCATGGTAGATGGAGCTTAGTCTTTCTGGTATCCAGCTGGGAGCCAAGCACAGAACACGCATCAGTGTTTATCAAATGACTGAGGAAATGAAT
CYP1A2 x 1F > A quality control sequence (SEQ ID NO. 64):
GATGATGTGTGGAGGAGAGAGCCAGCGTTCATGTTGGGAATCTTGAGGCTCCTTTCCAGCTCTCAGATTCTGTGATGCTCAAAGGGTGAGCTCTGTGGGCACAGGACGCATGGTAGATGGAGCTTAGTCTTTCTGGTATCCAGCTGGGAGCCAAGCACAGAACACGCATCAGTGTTTATCAAATGACTGAGGAAATGAAT
CYP2C 9-3>A quality control sequence (SEQ ID NO. 65):
AGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGTAAGTTTGTTTCTCCTACACTGCA
CYP2C 9-3>C quality control sequence (SEQ ID NO. 66):
AGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACCTTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGTAAGTTTGTTTCTCCTACACTGCA
HLA-B1502 (rs 3909184) > G quality control sequence (SEQ ID NO. 67):
GGAGCAGGTGCATGAAGGTGGGTTCCCTCCTGTCTGCTTGGCCAGTCCAGTGGAGTCCAGTGTTTCTCTGATGAGCCCCCGTTTAATCTATTTTTCCCACGTGTGCCCCCTTCTAGAGTATAAATACCTTGAGGGCACTGAGCACATGTTGGCTTTCTGCTATCTCCAGTCTTGCTCAAATCCCCCCACTGTTGCTGCGA
HLA-B1502 (rs 3909184) > C quality control sequence (SEQ ID No. 68):
GGAGCAGGTGCATGAAGGTGGGTTCCCTCCTGTCTGCTTGGCCAGTCCAGTGGAGTCCAGTGTTTCTCTGATGAGCCCCCGTTTAATCTATTTTTCCCACCTGTGCCCCCTTCTAGAGTATAAATACCTTGAGGGCACTGAGCACATGTTGGCTTTCTGCTATCTCCAGTCTTGCTCAAATCCCCCCACTGTTGCTGCGA
HLA-B1502 (rs 2844682) > G quality control sequence (SEQ ID NO. 69):
GTGCTATGGAAAAGAAAATCTTGGGGAAGAAGGGGGATCATGGAGGAGAAGGTAGTGACCATAAAATGGGTCAGGATGGGCTTCTCTGAGGTCTCCAAGGGATTGGCCATAGAGATGGTCATGATCAAAATCAAGAAAAGAACCAGAGAAAAGAGCACAGAGGGTTTGGCCAACGGGACAGTCAGAAAAATAGGGAGTGG
HLA-B1502 (rs 2844682) > A quality control sequence (SEQ ID NO. 70):
GTGCTATGGAAAAGAAAATCTTGGGGAAGAAGGGGGATCATGGAGGAGAAGGTAGTGACCATAAAATGGGTCAGGATGGGCTTCTCTGAGGTCTCCAAGGAATTGGCCATAGAGATGGTCATGATCAAAATCAAGAAAAGAACCAGAGAAAAGAGCACAGAGGGTTTGGCCAACGGGACAGTCAGAAAAATAGGGAGTGG
ANKK1> G quality control sequence (SEQ ID NO. 71):
AGAACATCACGCAAATGTCCACGCCCGCAACAAGGTGGGCTGGACACCCGCCCACCTGGCCGCCCTCAAGGGCAACACAGCCATCCTCAAAGTGCTGGTCGAGGCAGGCGCCCAGCTGGACGTCCAGGATGGAGTGAGCTGCACACCCCTGCAACTGGCCCTCCGCAGCCGAAAGCAGGGCATCATGTCCTTCCTAGAGG
ANKK1> A quality control sequence (SEQ ID NO. 72):
AGAACATCACGCAAATGTCCACGCCCGCAACAAGGTGGGCTGGACACCCGCCCACCTGGCCGCCCTCAAGGGCAACACAGCCATCCTCAAAGTGCTGGTCAAGGCAGGCGCCCAGCTGGACGTCCAGGATGGAGTGAGCTGCACACCCCTGCAACTGGCCCTCCGCAGCCGAAAGCAGGGCATCATGTCCTTCCTAGAGG
Example 4: preparation of PCR reaction solution
The kit adopts 8-linkage PCR reaction strip design, the No. 1-8 tube consists of gene detection reagents, and indicates different gene polymorphic loci of CYP2D6, CYP2C19, CYP2C 3, CYP2C19, CYP1A2, F, CYP, C9, HLA-B1502 and ANKK respectively, and each tube contains reagent components for detecting GAPDH internal reference genes. The reaction solutions of the No. 1-8 tubes correspond to the PCR reaction solutions 1-8, respectively, and the compositions of the PCR reaction solutions 1-8 are shown in tables 2-9 below.
TABLE 2 PCR reaction solution 1
TABLE 3 PCR reaction solution 2
TABLE 4 PCR reaction solution 3
TABLE 5PCR reaction solution 4
TABLE 6 PCR reaction solution 5
TABLE 7 PCR reaction solution 6
TABLE 8 PCR reaction solution 7
Table 9 PCR reaction solution 8
Instrument channel and reaction volume selection:
① Detecting amplification conditions by selecting FAM channels (Reporter: FAM, quantum: TAMRA), VIC channels (Reporter: VIC, quantum: TAMRA) and CY5 channels (Reporter: CY5, quantum: BHQ 1);
② The reaction volume (SampleVolume) was 20. Mu.L.
③ Reference fluorescence (REFERENCE DYE): if ABI series PCR instrument is used, please select "none" at PASSIVE REFERENCE; the specific detection channel arrangement can be described with reference to the use of each instrument.
Experimental example 1: use of gene detection kit
1. Sample collection
10 Patients were collected in this experimental example with oral swabs numbered 1-10, respectively.
The sample was placed in a storage tube containing physiological saline and eluted to form a cell suspension.
2. Sample processing
Sample nos. 1 to 10 were subjected to nucleic acid extraction with a lysate and a stabilizer.
Adding 2 mu L of oral swab sample cell suspension into a 1.5mL centrifuge tube, adding 15 mu L of lysate, vortex mixing, instantaneous centrifuging, incubating at 90 ℃ for 2min, adding 15 mu L of stabilizer, vortex mixing, instantaneous centrifuging, and preserving the cracked DNA solution at 4 ℃ for 1 month, and preserving at-20 ℃ for a long time.
3. Preparation of a reaction system
Reaction System preparation A reaction system was prepared according to the PCR reaction liquids 1 to 8 in example 4.
4. PCR reaction
Adding the DNA extracted from sample No. 1-10 into the prepared reaction system, wherein the added template amount is 0.1-200ng. When the PCR reaction is carried out, the sample to be detected, the positive quality control product and the negative quality control product are required to be detected together in parallel on a machine, and each sample is required to be added with a No. 1-8 tube for reaction.
5. Instrument channel and reaction volume selection
① Selecting FAM channel (Reporter: FAM, quantum: TAMRA), VIC channel (Reporter: VIC, quantum: TAMRA) and CY5 (Reporter: CY5, quantum: BHQ 1) channels to detect amplification conditions;
② The reaction volume (SampleVolume) was 20. Mu.L.
③ Reference fluorescence (REFERENCE DYE): if ABI series PCR instrument is used, please select "none" at PASSIVE REFERENCE; the specific detection channel arrangement can be described with reference to the use of each instrument.
6. PCR reaction procedure
The conditions for the incubation of the UDG enzyme are: 37 ℃ for 2 minutes;
The conditions for the pre-denaturation are: 95 ℃ for 2 minutes;
the first phase consisted of 5 amplification cycles, provided that: denaturation: 95 ℃ for 15 seconds; annealing: 60 ℃ for 30 seconds; extension: 72 ℃,20 seconds;
The second stage consisted of 35 amplification cycles, provided that: denaturation: 95 ℃ for 15 seconds; annealing: setting fluorescence signal collection at 60 ℃ for 30 seconds; extension: 72℃for 30 seconds.
7. Experimental results
And after the reaction program is finished, the result is saved and interpreted.
FAM and VIC fluorescence detection signals in the reaction tubes of the positive quality control products 1-8 form logarithmic amplification S-shaped curves (see figure 3); negative quality controls 1-8 were run without amplification curve (see FIG. 4).
Experimental example 2: sample detection result comparison
The method comprises the steps of selecting 10 peripheral blood samples of patients, extracting nucleic acid, carrying out Sanger sequencing, collecting sample genotype detection data, simultaneously taking 0.1ng of genome DNA, detecting genotypes by using a fluorescence quantification system, wherein the fluorescence quantification system comprises a conventional primer probe and a novel primer probe, the primers used in the conventional primer probe fluorescence quantification system only comprise a second part and a fourth part of the novel primer, the probes used only comprise a first part and a third part (or a fifth part) of the novel probe, no secondary structure is designed for the primers and the probes, and the detection results of the samples with the numbers 1-10 are shown in the following table 10.
The Sanger sequencing results are sample genotype detection gold standard, and the detection results of the fluorescence quantitative system are compared with the detection results, namely, the detection rate of 9 sites of CYP2D6, CYP2C 10, CYP2C19, CYP2C 3, CYP2C19, CYP1A2, F, CYP, 2C9, HLA-B1502 (rs 3909184, rs 2844682) and ANKK1 is 100% by using the novel primer probe fluorescence quantitative system, the detection rate of four sites of CYP2C19, HLA-B1502 (rs 3909184, rs 2844682) is 90%, the detection rate of four sites of CYP2D6, CYP2C19, CYP1A2, F, CYP, 2C9, 3, and ANKK sites of the fluorescence quantitative system is 80%, and the detection rate of the fluorescent quantitative system of the four sites of the conventional primer probe is only 70%.
Table 10
From table 10, the detection limit of the detection system formed by the novel primer probe provided by the invention can be as low as 0.1ng, so that complex and difficult templates such as oral swab samples, blood samples and the like can reach 100% detection rate, and the sensitivity of the detection system is greatly improved.
Claims (13)
1. A composition for detecting a psychotropic gene polymorphic site, comprising at least one of a primer pair and a probe for a different polymorphic site, and an inhibitor combination:
(1) Primer pair, probe and inhibitor combination for detecting CYP2D6 x10 gene locus: a primer pair 1 consisting of a forward primer shown as SEQ ID NO. 1 and a reverse primer shown as SEQ ID NO. 2-3; probe 1 shown in SEQ ID NO. 28; and inhibitor 1 as shown in SEQ ID NO. 37 or SEQ ID NO. 38;
(2) Primer pair, probe and inhibitor combination for detecting CYP2C19 x 2 gene locus: a primer pair 2 consisting of a forward primer shown as SEQ ID NO. 4-5 and a reverse primer shown as SEQ ID NO. 6; probe 2 shown in SEQ ID NO. 29; and inhibitor 2 as shown in SEQ ID NO. 39 or SEQ ID NO. 40;
(3) Primer pair, probe and inhibitor combination for detecting CYP2C19 x 3 gene locus: a primer pair 3 consisting of a forward primer shown as SEQ ID NO. 7-8 and a reverse primer shown as SEQ ID NO. 9; probe 3 shown in SEQ ID NO. 30; and inhibitor 3 as shown in SEQ ID NO. 39 or SEQ ID NO. 40;
(4) Primer pair, probe and inhibitor combination for detecting CYP2C19 x 17 gene locus: a primer pair 4 consisting of a forward primer shown as SEQ ID NO. 10-11 and a reverse primer shown as SEQ ID NO. 12; probe 4 shown in SEQ ID NO. 31; and inhibitor 4 as shown in SEQ ID NO. 39 or SEQ ID NO. 40;
(5) Primer pair, probe and inhibitor combination for detecting CYP1A2 x 1F gene locus: a primer pair 5 consisting of a forward primer shown as SEQ ID NO. 13-14 and a reverse primer shown as SEQ ID NO. 15; probe 5 shown in SEQ ID NO. 32; and inhibitor 5 as shown in SEQ ID NO. 45 or SEQ ID NO. 46;
(6) Primer pair, probe and inhibitor combination for detecting CYP2C9 x 3 gene locus: a primer pair 6 consisting of a forward primer shown as SEQ ID NO. 16-17 and a reverse primer shown as SEQ ID NO. 18; probe 6 shown in SEQ ID NO. 33; and inhibitor 6 as shown in SEQ ID NO. 47 or SEQ ID NO. 48;
(7) Primer pair, probe and inhibitor combination for detecting HLA-B1502 gene locus: a primer pair 7 consisting of a forward primer shown as SEQ ID NO. 19 and a reverse primer shown as SEQ ID NO. 20-21, and a primer pair 8 consisting of a forward primer shown as SEQ ID NO. 22-23 and a reverse primer shown as SEQ ID NO. 24; probe 7 shown as SEQ ID NO. 34 and probe 8 shown as SEQ ID NO. 35; and inhibitor 7 as shown in SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51 or SEQ ID NO. 52; or;
(8) Primer pair, probe and inhibitor combinations for detecting ANKK gene locus: a primer pair 9 consisting of a forward primer shown as SEQ ID NO. 25-26 and a reverse primer shown as SEQ ID NO. 27; probe 9 shown in SEQ ID NO. 36; and inhibitor 8 as shown in SEQ ID NO. 53 or SEQ ID NO. 54;
The inhibitor is used for blocking non-specific template amplification.
2. The composition of claim 1, wherein the 5' fluorescent moiety of the probe 1-9 comprises FAM, VIC, HEX, CY or a ROX fluorescent reporter.
3. The composition of claim 2, wherein the 5' fluorescent group of probes 1-9 is FAM, VIC or CY5.
4. The composition of claim 1 or 2, wherein the quenching group at the 3' end of the probe 1-9 comprises a TAMRA, BHQ1, BHQ2, MGB, or Dabcy1 fluorescence quenching group.
5. The composition of claim 4, wherein the 3' -terminal quenching group of probe 1-9 is TAMRA or BHQ1.
6. The composition of claim 1 or 2, wherein the 3' end of the inhibitor 1-8 is modified with a C3 spacer, phosphorylation, thio, MGB or ddC.
7. The composition of claim 1 or 2, wherein the 3' ends of the inhibitors 1-8 are modified with ddC.
8. A kit for detecting a psychotropic gene polymorphism site, comprising the primer, probe and inhibitor as set forth in claim 1 or 2.
9. The kit of claim 8, further comprising GAPDH internal reference gene primers and probes having the following sequences:
GAPDH-F：GTATGACTGGGGGTGTTGGG
GAPDH-R：GAGGGCCCAAGAGGTTGAAT
GAPDH-P：CCAGCCTGGCAGGGAAGCTCAAG。
10. The kit according to claim 8 or 9, further comprising a PCR reaction solution, a positive quality control and a negative quality control, wherein the PCR reaction solution comprises a hot start taq enzyme, a UDG enzyme, a buffer, magnesium ions and dNTP substances required for the PCR reaction; the method for obtaining the positive quality control product comprises the following steps: constructing a synthetic sequence gene fragment by plasmids according to CYP2D6, CYP2C19, CYP2C 3, CYP2C19, CYP1A2, F, CYP C9, HLA-B1502, ANKK1 and GAPDH related gene sequences published by NCBI database, inserting the fragment into a T vector, converting and extracting plasmids by using escherichia coli DH5 alpha strain, and mixing quality control quality granules in equal proportion to obtain the positive quality control product; the negative quality control product is deionized water treated by DEPC.
11. The method of using a kit for detecting a polymorphic site of a psychotropic gene for non-diagnostic purposes according to any of claims 8-10, comprising the steps of:
S1: taking an acquisition sample containing EDTA anticoagulant for DNA extraction;
S2: premixing and split charging gene detection reagents to obtain PCR reaction liquids 1-8 aiming at different polymorphic sites respectively, and forming a PCR reaction system together;
S3: adding the DNA obtained in the step S1 into the PCR reaction system of the step S2, and carrying out PCR amplification after centrifugation;
s4: after the reaction is finished, carrying out genotype identification and interpretation;
wherein, the detection sites of the PCR reaction liquids 1-8 are respectively as follows:
。
12. the method of claim 11, wherein the conditions for the PCR amplification in step S3 are:
The conditions for the incubation of the UDG enzyme are: 37 ℃ for 2 minutes;
The conditions for the pre-denaturation are: 95 ℃ for 2 minutes;
the first phase consisted of 5 amplification cycles, provided that: denaturation: 95 ℃ for 15 seconds; annealing: 60 ℃ for 30 seconds; extension: 72 ℃,20 seconds;
The second stage consisted of 35 amplification cycles, provided that: denaturation: 95 ℃ for 15 seconds; annealing: setting fluorescence signal collection at 60 ℃ for 30 seconds; extension: 72℃for 30 seconds.
13. The method of claim 11, wherein the method of genotyping is: under the conditions of a PCR reaction system and a circulation program, FAM, VIC and CY5 fluorescence detection signals of each positive quality control product form a logarithmic amplification S-shaped curve; each negative quality control product has no amplification curve or Ct value of 0; the CY5 fluorescence detection signals of each sample to be detected should form a logarithmic amplification S-shaped curve; then observing FAM and VIC fluorescence detection signal amplification curves of the reaction tube of the sample to be detected, and if a logarithmic amplification S-shaped curve is formed, the sample to be detected contains corresponding base polymorphism sites; if the logarithmic amplification "S" type curve is not used or the Ct value is 0, there is no corresponding base polymorphism site.
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