CN113249460A - Primer group for gene group detection of anxiety patient medication guidance, related application and corresponding kit - Google Patents

Abstract

The invention provides a primer group for detecting a gene group for the medication guidance of anxiety patients, a related application and a corresponding kit, wherein the primer group comprises primers for detecting variation related to each gene in the gene group, and the gene group comprises the following genes: CYP2C9, CYP2C19, CYP3A4, CYP3A5, NAT2, ABCB1, SCN1A, UGT1A4, and POLG.

Description

Primer group for gene group detection of anxiety patient medication guidance, related application and corresponding kit

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a primer group for gene group detection of medication guidance of anxiety patients, application of the primer group in preparation of a corresponding kit and the corresponding kit.

Background

The mental health problem has become a serious public health problem and a prominent social problem in China. In recent years, the prevalence rate of mental diseases in China is increased year by year, and data shows that the number of patients with various mental diseases in China is more than 1 hundred million, and the number of patients with serious mental disorders is over 1800 ten thousand. Mental diseases are ranked first in the social burden of diseases in China, and are expected to reach 1/4 by 2020. The drug therapy is the main clinical means of the mental diseases at present, but the drug curative effect and the adverse reaction show huge individual differences. Research shows that genetic factors are important factors influencing drug response of mental diseases, and drug selection and dosage adjustment can be guided by detecting genes related to drug curative effect and adverse reaction through pharmacogenomics, so that the purpose of individual treatment is achieved.

However, genes and corresponding marker sites selected by some detection products in the current market are not suitable for Chinese people, and have no detection significance in Chinese people; meanwhile, some sites suitable for Chinese people are not listed, so that the problems of missed detection or inaccurate medication guidance occur.

Therefore, the variation of related genes can be accurately detected, and the purpose of individual treatment can be achieved.

In addition, for the detection of the marker locus, a fluorescence quantitative PCR method is commonly used at present, and the fluorescence quantitative PCR method is simple to operate, but has low flux, consumes time and labor; the Sanger assay as gold standard is both time and cost intensive.

Disclosure of Invention

The invention provides a primer group for gene group detection of anxiety disorder patient medication guidance, application thereof in preparation of a corresponding kit, a corresponding kit and a using method.

The invention provides a primer group for detecting a gene group for medication guidance of an anxiety patient, which is characterized in that: the primer group comprises primers for detecting variation related to each gene in a gene group, wherein the gene group comprises the following genes: CYP2C9, CYP2C19, CYP3A4, CYP3A5, NAT2, ABCB1, SCN1A, UGT1A4, and POLG.

The primer set provided by the invention also has the following characteristics: wherein, the genes CYP2C9, CYP2C19, CYP3A4, CYP3A5 and NAT2 are drug metabolism genes; the genes ABCB1, SCN1A and UGT1A4 are drug efficacy genes; the gene POLG is a drug toxicity gene.

The primer set provided by the invention also has the following characteristics: wherein, the drug targeted by the drug metabolism gene is selected from phenytoin, clobazam, diazepam, bravaracetam, alprazolam, clonazepam, midazolam, triazolam, zolpidem, zopiclone, buspirone, carbamazepine and perampanel; the drug curative effect gene is aimed at a drug selected from phenobarbital, carbamazepine, phenytoin and lamotrigine; the drug targeted by the drug toxicity gene is selected from valproic acid.

The primer set provided by the invention also has the following characteristics: wherein, the SNP sites related to each gene in the gene group are shown in Table 2.

The present invention also provides a primer set, comprising: and the amplification primer pairs are used for detecting each SNP locus related to each gene, and the nucleotide series of the amplification primers included in each amplification primer pair is SEQ ID NO.1-SEQ ID NO. 38.

The primer set provided by the present invention is also characterized by further comprising: and (3) corresponding extension primers required for detecting each SNP site, wherein the nucleotide series of each extension primer is shown as SEQ ID NO.39-SEQ ID NO. 57.

The invention also provides an application of the primer group in preparing a kit for the medication guidance of anxiety patients, which is characterized in that: wherein the primer group is the primer group.

The present invention also provides a kit for detecting a gene group for use in medication guidance for an anxiety patient, comprising: the primer set described above.

The kit provided by the invention is characterized by further comprising: and detecting reagents required for detecting the SNP sites based on a nucleic acid mass spectrometry method.

Action and Effect of the invention

The invention provides a primer group for detecting a gene group for drug guidance of an anxiety patient, application of the primer group in preparation of a corresponding kit and the corresponding kit, wherein the primer group comprises primers for detecting variation related to each gene in the gene group, and the gene group comprises the following genes: CYP2C9, CYP2C19, CYP3A4, CYP3A5, NAT2, ABCB1, SCN1A, UGT1A4 and POLG are verified to be more in line with the specific distribution frequency of Chinese population, and the corresponding variation of the gene group for the medication guidance of the anxiety patient can be accurately detected through a specific primer designed for the variation related to the specific gene, so that the method can be used for detecting the gene group, and can be used for the medication guidance of the anxiety patient to provide more accurate medication guidance.

Drawings

FIG. 1 is a schematic diagram of the thermal cycle in the elongation reaction according to example 1.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.

The methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

The following are the single nucleotide polymorphisms (SNP sites) referred to in the examples: mainly refers to DNA sequence polymorphism caused by single nucleotide variation on genome level, SNP is the most common variation form in human genome DNA sequence, and is estimated to account for a very small part of millions of SNPs, which have great influence on diseases and drug treatment.

Example 1

This example specifically describes a primer set and a kit, etc., which are useful for a gene group for drug administration guidance for anxiety patients.

The present invention provides a primer set for detecting anxiety patient medication-directed gene group, the primer set includes primers for detecting variation related to each gene in the gene group, wherein the gene group includes the following genes: CYP2C9, CYP2C19, CYP3A4, CYP3A5, NAT2, ABCB1, SCN1A, UGT1A4, and POLG.

Further, the functional effects of the drugs entering the human body are classified, and the genes CYP2C9, CYP2C19, CYP3A4, CYP3A5 and NAT2 in the genes are drug metabolism genes; the genes ABCB1, SCN1A and UGT1A4 are drug efficacy genes; HLA and POLG are drug toxicity genes.

In addition, in this embodiment, the drug targeted by the drug metabolism gene is selected from phenytoin, clobazam, diazepam, bwawacetam, alprazolam, clonazepam, midazolam, triazolam, zolpidem, zopiclone, buspirone, carbamazepine, and perampanel; the drug aiming at the drug curative effect gene is selected from phenobarbital, carbamazepine, phenytoin and lamotrigine; the drug against the drug toxicity gene is selected from valproic acid. The functional effects and drugs corresponding to the respective genes are shown in table 1.

Table 2 shows the SNP sites related to the medication guidance for anxiety patients for each gene included in the above gene group.

The descriptions in table 2, like 1, 2, 3, 4, 10, 17, 35, respectively, refer to the names of the corresponding variants in the industry, wherein all types are wild-type, i.e., 1, without mutations.

After the enzyme activity is reduced, the drug metabolism is slowed down, the concentration of the drug in the body can be increased, and the drug dosage is correspondingly adjusted according to the influence of the gene on the enzyme activity.

"25889 _25890 insA" indicates the insertion of an A at position 25889 to 25890.

In order to detect such variations, the primer set in this example includes amplification primers corresponding to the individual SNP variations for specific amplification, and the nucleotide sequence of each amplification primer pair is shown in table 3.

The primer set also includes extension primers corresponding to the individual SNP variations, and the nucleotide sequences of the extension primers are shown in Table 4.

The primer set provided by this embodiment can be used for preparing a kit for detecting a gene group for an anxiety patient medication instruction, and accordingly, the kit also includes the primer set, and in this embodiment, the kit further includes a detection reagent for detecting SNP variation based on a nucleic acid mass spectrometry method.

In this embodiment, the method of using the kit is as follows:

first, PCR reaction

1. Preparing 1. mu.M PCR primer mixture for each amplification primer (see Table 3) containing forward (forward F) and reverse (reverse R) primers for each SNP site in the multiplex reaction;

2. diluting the extracted DNA sample to 10 ng/mu L, and centrifuging;

3. the PCR mix was prepared according to the following table (Table 5) without adding the DNA sample and the control thereto:

4. 3uL of the PCR mixture prepared above was added to each well of a 96-well plate, and then a DNA sample (volume: 2. mu.L; concentration: 10 ng/. mu.L) was added to each well, and a negative control NTC (ribozyme-free water) was prepared;

5. the plates were sealed with a membrane, vortexed and centrifuged (4000rpm for 5 seconds);

6. the 96-well plate was placed on a PCR instrument for the following thermal cycling:

95 ℃ for 2 minutes

45 cycles:

95 ℃ for 30 seconds

60 ℃ for 30 seconds

60 seconds at 72 DEG C

5 minutes at 72 DEG C

Keeping the temperature at 4 DEG C

II, SAP reaction

1. The SAP mixtures were prepared according to the following table (table 6).

2. Mu.l of the above SAP mixture was added to each well after PCR reaction (total volume after addition: 7. mu.L).

3. The plates were sealed with a membrane, vortexed and centrifuged (4000rpm for 5 seconds).

4. Place the plate on a PCR instrument for the following procedures:

40 minutes at 37 DEG C

5 minutes at 85 DEG C

Keeping the temperature at 4 DEG C

Extension reaction

1. Preparing an extension primer mixture according to a linear adjustment method, namely, preparing a 500 mu M system by dry powder of extension primers (shown in table 4) in each tube, mixing extension primers with different volumes into a new tube according to a linear primer regression table, and adding water to a target volume to obtain a finished product, namely the extension primer working mixture.

2. The extensional mixed solution was prepared in accordance with the following table (Table 7).

3. Mu.l of the above extension mixture was added to each well after the SAP reaction and mixed (total volume after addition: 9. mu.L).

4. The plates were sealed with a membrane, vortexed and centrifuged (4000rpm for 5 seconds).

5. The 96-well plate was placed on a PCR instrument for thermal cycling as shown in FIG. 1.

Fourth, sample plate editing

1. And opening the software, double-clicking a type icon, clicking an Assay Editor icon, inputting a password "darwin", returning and entering an Assay Editor software interface.

2. Creating a folder, right-clicking at asset Projec/asset Group/Plex/asset to select Project administeror, entering Project name in name bar, clicking Add

3. Importing the Assay, selecting an Import asset Group Designer format … at the right key of the corresponding project, checking only asset Group, clicking Browse, selecting an Assay Group file, and clicking Import

4. And (3) creating a Plate, double-clicking a type icon, then clicking a Plate Editor icon, inputting a password "darwin", returning and entering a Plate Editor software interface. Right-clicking on the button on the Customer, Project, Plate, selects New Customer, inputs Customer ID, and clicks OK. Right clicking on corresponding Project, selecting New Project, inputting Project ID, and clicking OK. Right-clicking at the corresponding Project, selecting New Plate, entering Plate ID, selecting 96Wells, and clicking OK.

5. And selecting the Assay tab, selecting the sample hole of the experiment, finding the specific well of the experiment Assay in the left-side Assay column, pressing the right button, and clicking Add.

6. Selecting a sample tab, importing a sample name group: selecting Add New Sample Group at the corresponding Customer/project right button, selecting Sample Group file, filling in Sample ID, and clicking OK. Single import sample name: and selecting the sample hole of the experiment, finding the name of the experiment sample in the left sample column, right clicking Add to Add a single sample name. Batch import of sample names: the sample name import direction is set in the right Properties column, the experimental sample name Group is found in the left sample column, and the right click is applied Samples from Group "xxx".

7. And setting a plate and clicking save.

8. Double-clicking the Chip Linker button, inputting the password "darwin", returning, entering the Chip Linker software interface

9. The method comprises the steps of selecting a set plate on the left side, selecting IPLEX, Genotype + area on the right side, inputting a dispenser format (Nanodienser 96to 96-6-Pin point 96 Chip, Single Pin 96to 96-Single Pin point 96 Chip), inputting an extreme Name, Chip number Chip Barcode, clicking Add, and clicking Create.

Desalting CPM sample, spotting to chip and mass spectrometer to obtain data

1. 30ul of water was added to each well of the sample plate with the sample and centrifuged. The board is placed in the machine with the chips in the corresponding positions.

2. Clicking on the edited chip before In SpectroACQUIRE selection, clicking on Run Setup to check Turn Off HV After Analysis, Filter processed chips, dependent sample on chips, transfer chips to analyzer, Analysis chips, MTP cool14, chip heat 30, (if calibrator is used, Use Calibration Wells must be checked), clicking on Start Autorun, and starting.

3. And viewing the data result, double-clicking the Typer icon, then clicking the Typer Analyzer icon, inputting the password "darwin", returning, and entering a Typer Analyzer software interface.

Example 2

This example samples some anxiety cases (Table 8) by one-generation sequencing and the primer set, corresponding kit and corresponding method provided in example 1, and detects the variation as shown in Table 2, and the detection results are shown in Table 9.

In Table 9, genotyping indicates the A/G SNP alleles as examples:

a: indicating that the detected sample is AA homozygous;

AG: representing that the detected sample is AG heterozygote;

g: the detection sample is GG homozygous type

In table 9, the results are illustrated in grades as follows:

a. consiservive: the reliability of the obtained result is high (the instrument automatically judges and reads the typing result);

b. Moderate: the reliability of the obtained result is expressed to be medium (the automatic interpretation and typing result of the instrument);

aggregate: the reliability of the obtained result is low (the instrument automatically judges and reads the typing result);

user Call: manual interpretation (machine automatic interpretation without typing results);

# N/A: indicating that no test results were obtained.

The above results show that:

(1) as can be seen from table 9, of the 95 detection results, 90 with high reliability of the results account for approximately 95%, and 0 with low reliability.

(2) Table 9 the genotyping results obtained using the method of example 1 were consistent with the genotyping results obtained by first-generation sequencing, with 100% consistency.

It can be seen that the reliability of the detection result of the variation of the SNP sites is higher than 95% by using the primer set, the corresponding kit and the method of example 1 for detection, and the detection result of the variation of the SNP sites is verified by using the first-generation sequencing method as the detection gold standard, which shows that the primer set, the corresponding kit and the using method of example 1 can accurately detect the variation as shown in table 2, so that the primer set, the corresponding kit and the using method can be accurately used for the genome detection of the drug administration guidance of the anxiety patients as shown in table 1, and can more accurately provide the drug administration guidance for the anxiety patients.

Moreover, since the Massarray platform is a non-fluorescent detection platform that accurately measures PCR amplicons using mass spectrometry. The mass spectrometry and the end-point PCR are combined to realize multiple detection, the number of gene detection sites related to the invention is large, and the primer group designed by the invention can select a nucleic acid mass spectrometry Massarray platform with low medium flux, low cost and good flexibility to detect SNP sites of most genes, thereby being more beneficial to practical application.

Examples effects and effects

The primer set for the gene group detection of the medication guidance of the anxiety neurosis patients, the application thereof in preparing the corresponding kit and the corresponding kit provided in the embodiment 1 can be seen from the embodiment 2 that the variation shown in the table 2 can be accurately detected, so the primer set can be accurately used for the gene group detection of the medication guidance of the anxiety neurosis patients shown in the table 1, and the medication guidance can be more accurately provided for the anxiety neurosis patients.

In addition, the use method of the kit provided in embodiment 1 can select a nucleic acid mass spectrometry Massarray platform with low cost and good flexibility to perform SNP site detection of most genes through the primer set designed by the invention, and is more beneficial to practical application.

Claims (10)

1. A primer set for gene group detection of medication guidance of anxiety patients, which is characterized in that:

the primer group comprises primers for detecting variation related to each gene in the gene group,

wherein the gene group comprises the following genes: CYP2C9, CYP2C19, CYP3A4, CYP3A5, NAT2, ABCB1, SCN1A, UGT1A4, and POLG.

2. The primer set according to claim 1, wherein:

wherein, the genes CYP2C9, CYP2C19, CYP3A4, CYP3A5 and NAT2 are drug metabolism genes;

the genes ABCB1, SCN1A and UGT1A4 are drug efficacy genes;

the gene POLG is a drug toxicity gene.

3. The primer set according to claim 2, wherein:

wherein, the drug targeted by the drug metabolism gene is selected from phenytoin, clobazam, diazepam, bravaracetam, alprazolam, clonazepam, midazolam, triazolam, zolpidem, zopiclone, buspirone, carbamazepine and perampanel;

the drug targeted by the drug efficacy gene is selected from phenobarbital, carbamazepine, phenytoin and lamotrigine;

the drug targeted by the drug toxicity gene is valproic acid.

4. The primer set according to any one of claims 1 to 3, wherein:

wherein each SNP site involved in each of the genes in the gene group is as set forth in Table 2.

5. The primer set according to claim 4, comprising:

and the amplification primer pairs are used for detecting the SNP sites related to the genes respectively, and the nucleotide series of the amplification primers included in the amplification primer pairs is SEQ ID NO.1-SEQ ID NO. 38.

6. The primer set according to claim 5, further comprising:

and (3) detecting each SNP site by using corresponding extension primer, wherein the nucleotide series of each extension primer is SEQ ID NO.39-SEQ ID NO. 57.

7. The application of a primer group in preparing a kit for the medication guidance of anxiety patients is characterized in that:

wherein the primer set is the primer set according to any one of claims 1 to 6.

8. A kit for detecting a gene group for drug administration guidance of an anxiety patient, comprising:

the primer set of any one of claims 1 to 6.

9. The kit of claim 8, further comprising:

and detecting reagents required for detecting the SNP sites based on a nucleic acid mass spectrometry method.

10. The method of using the kit of any one of claims 8 to 9, wherein:

and detecting the SNP sites based on a nucleic acid mass spectrometry analysis method.

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