CN108192966B - Complete set of primers for detecting drug metabolizing enzyme gene SNP locus and application thereof - Google Patents

Complete set of primers for detecting drug metabolizing enzyme gene SNP locus and application thereof Download PDF

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CN108192966B
CN108192966B CN201810143897.1A CN201810143897A CN108192966B CN 108192966 B CN108192966 B CN 108192966B CN 201810143897 A CN201810143897 A CN 201810143897A CN 108192966 B CN108192966 B CN 108192966B
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张影
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Abstract

The invention discloses a primer set for detecting drug metabolizing enzyme gene SNP sites and application thereof, wherein the primer set comprises amplification primer pairs and extension primers of 9 SNP sites of 7 drug metabolizing enzyme related genes of human genome DNA. A pair of multiplex PCR amplification primers and a single base extension primer are respectively designed at each site, and the SNP site typing of a sample to be detected is detected by adopting a MALDI-TOF MS (matrix assisted laser desorption ionization time of flight mass spectrometry) method. The invention establishes a drug metabolizing enzyme gene detection method based on gene molecule typing, which can finish a plurality of SNP typing in the same reaction at the same time, is accurate, efficient and low in cost, and the covered genes and loci are related to metabolizing enzyme genes related to 8 types of common drugs for children, thus being the most comprehensive coverage in drug gene detection products for children at present.

Description

Complete set of primers for detecting drug metabolizing enzyme gene SNP locus and application thereof
Technical Field
The invention relates to the technical field of gene detection, in particular to a set of primers for detecting drug metabolizing enzyme gene SNP sites and application thereof.
Background
In China, 3500 kinds of medicine preparations exist, and only 60 kinds of medicine preparations are specially used for children. In the only over 60 kinds of children's exclusive medicines, there are many cases of wrong use. Because of improper medication, about 30000 children fall into the silent world every year in China, and damages to liver and kidney functions, nervous systems and the like are more difficult to count. In china, the drug intoxication event in children is rising year by year, reaching 73% in 2014. 1 of 8 children has adverse reaction due to improper medication, which causes damage to liver and kidney function and nervous system.
The safety of medication is mainly related to the metabolic process of the medicine. If the metabolism speed of the medicine in the body is reduced, the medicine can be accumulated in the body, and thus adverse reactions occur. The metabolism rate of the drug in vivo is mainly related to drug metabolizing enzyme genes, and the metabolism rate of the drug in vivo changes in individuals with drug metabolizing enzyme genes, which is the main cause of adverse drug reactions.
The enzyme participating in drug metabolism has the highest activity of liver microsomal enzyme, and mainly catalyzes the metabolism of exogenous substances such as drugs, so the enzyme is also called drug metabolism enzyme, and is called drug metabolism enzyme for short. The main subtypes are 7 important subtypes in CYP1, CYP2 and CYP 3: CYP1a2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3a4, CYP3a 5. The genetic genes of individuals differ, resulting in a change in their enzymatic activity, which is phenotypically manifested by different individuals' ability to metabolize the same drug. According to the speed of metabolism of the medicine in vivo, the medicine can be divided into: 1. a rapid metabolic type; 2. an intermediate metabolic type; 3. a slow metabolic type; 4. ultra-slow metabolic type. If the medicine belongs to a slow metabolism type, the drug metabolism rate is obviously slower than that of the common people, and the medicine is avoided or other effective medicines are replaced so as to reduce the risk of drug accumulation poisoning caused by poor metabolism of the medicine.
In the research related to drug metabolism enzyme gene detection, a patent (CN200810208219.5) discloses a drug metabolism related site detection method, and a patent (CN201610214241.5) discloses a drug metabolism enzyme related gene SNP fluorescent labeling compound amplification kit, wherein 5 SNP sites of 4 related genes are respectively selected in the two patents, the detected genes are incomplete, and all key enzyme genes and single nucleotide polymorphism sites related to drug metabolism are not covered; in addition, the methods generally adopted in the above two patents and the previous patent are all fluorescence quantitative PCR methods, which are suitable for detecting about 5 known SNP sites, and when the sites are increased, the operation is complex and the time consumption is long.
Disclosure of Invention
Aiming at the technical problems in the related technology, the invention provides a set of primers for detecting drug metabolizing enzyme gene SNP sites and application thereof, and the MALDI-TOF MS method is used, so that 30 SNP types can be completed in the same reaction at the same time, the detection efficiency is improved, and the cost is reduced; through detecting related drug enzyme genes of 8 types of common drugs (antipyretic analgesic drugs, respiratory drugs, endocrine drugs, nervous drugs, digestive tract drugs, cardiovascular drugs, anti-infection drugs and anti-allergy drugs) for children, the metabolic capability of children to different drugs is known, and a scientific reference basis is provided for reasonable drug administration.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
the set of primers for detecting SNP sites of drug metabolizing enzyme genes comprises PCR amplification primer pairs of 9 SNP sites of 7 drug metabolizing enzyme related genes of human genome DNA and single-base extension primers.
Further, the 7 genes related to the drug metabolizing enzyme of the human genome DNA are 7 important subtypes of CYP1, CYP2, CYP 3: CYP1a2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3a4, CYP3a 5.
Further, the SNP sites are as follows: the site of rs762551 on CYP1A2, the site of rs1057910 on CYP2C9, the sites of rs4244285 and rs4986893 on CYP2C19, the site of rs1065852 on CYP2D6, the sites of rs3813867 and rs2031920 on CYP2E1, the site of rs28371759 on CYP3A4, and the site of rs776746 on CYP3A 5.
Further, the set of primers for detecting SNP sites of the drug metabolizing enzyme gene comprises:
the PCR amplification primer sequence of the rs28371759 locus is a nucleotide sequence shown in SEQ ID NO1 and SEQ ID NO2, and the single base extension primer sequence is a nucleotide sequence shown in SEQ ID NO 19;
the PCR amplification primer sequence of the rs4986893 site is a nucleotide sequence shown by SEQ ID NO3 and SEQ ID NO4, and the single base extension primer sequence is a nucleotide sequence shown by SEQ ID NO 20;
the PCR amplification primer sequence of the rs1057910 locus is the nucleotide sequence shown in SEQ ID NO5 and SEQ ID NO6, and the single base extension primer sequence is the nucleotide sequence shown in SEQ ID NO 21;
the PCR amplification primer sequence of the rs1065852 site is the nucleotide sequence shown by SEQ ID NO7 and SEQ ID NO8, and the single base extension primer sequence is the nucleotide sequence shown by SEQ ID NO 22;
the PCR amplification primer sequence of the rs3813867 site is the nucleotide sequence shown by SEQ ID NO9 and SEQ ID NO10, and the single-base extension primer sequence is the nucleotide sequence shown by SEQ ID NO 23;
the PCR amplification primer sequence of the rs776746 site is the nucleotide sequence shown in SEQ ID NO11 and SEQ ID NO12, and the single base extension primer sequence is the nucleotide sequence shown in SEQ ID NO 24;
the PCR amplification primer sequence of the rs762551 site is the nucleotide sequence shown in SEQ ID NO13 and SEQ ID NO14, and the single-base extension primer sequence is the nucleotide sequence shown in SEQ ID NO 25;
the PCR amplification primer sequence of the rs4244285 locus is the nucleotide sequence shown in SEQ ID NO15 and SEQ ID NO16, and the single base extension primer sequence is the nucleotide sequence shown in SEQ ID NO 26;
the PCR amplification primer sequence of the rs2031920 site is the nucleotide sequence shown by SEQ ID NO17 and SEQ ID NO18, and the single-base extension primer sequence is the nucleotide sequence shown by SEQ ID NO 27.
A gene detection kit comprising the set of primers for detecting SNP sites of a drug metabolizing enzyme gene according to any one of claims 1 to 4.
Further, the drug metabolizing enzyme gene comprises CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A 5.
Further, the SNP loci of the drug metabolizing enzyme genes comprise an rs762551 locus, an rs1057910 locus, an rs4244285 locus, an rs4986893 locus, an rs1065852 locus, an rs3813867 locus, an rs2031920 locus, an rs28371759 locus and an rs776746 locus.
Further, the set of primers consists of the amplification primer pair for each SNP site according to claim 7 and a single-base extension primer.
Furthermore, the gene detection result of the kit shows that 8 major medicaments commonly used by children comprise antipyretic and analgesic medicaments, respiratory medicaments, endocrine medicaments, nervous systems, digestive tract systems, cardiovascular and cerebrovascular systems, anti-infection medicaments and anti-allergy medicaments.
Furthermore, the kit gene detection takes human genome DNA as a sample, a complete set of primers are subjected to multiple PCR amplification and then a matrix-assisted laser desorption ionization time-of-flight mass spectrometry is adopted to detect SNP locus typing of the sample, and the gene detection result of the multiple PCR amplification product of the complete set of primers shows the drug metabolism capability of the sample.
Furthermore, the metabolic capability of children to different drugs is known by detecting the related drug enzyme genes of 8 major drugs commonly used by children, and scientific reference basis is provided for reasonable drug administration.
The invention has the beneficial effects that:
1. the detected genes and sites are updated, and the detection genes are more comprehensive than those of other patents;
2. the existing common detection technology method is improved, so that the operation is simpler and more convenient, and the cost and the time are saved;
3. improve the current situation of unreasonable medication of children and accurately administer the medicine according to the individual metabolic capability.
Detailed Description
For the convenience of understanding the above technical solutions of the present invention, the following detailed descriptions of the above technical solutions of the present invention are provided by way of specific embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
EXAMPLE 1 establishment of the protocol
S1 designing a technical route: determining main components of common medicaments for children (Table 1), and determining SNP typing results of genes and sites related to medicament metabolism and interpretation of medicament metabolism rate (Table 2);
s2 reaction system is established: designing a multiplex PCR amplification primer (table 3) and an extension primer (table 4), and adjusting the concentration of reaction components and reaction conditions;
s2 analysis of the sample: DNA extraction, multiple PCR amplification, flight time mass spectrometry detection, result analysis and medication suggestion.
TABLE 1 common drugs for children and their main ingredients
Figure GDA0003124417970000061
TABLE 2 typing of related SNP sites and drug metabolism rates thereof
Figure GDA0003124417970000062
TABLE 3 multiplex PCR amplification primer sequences
Figure GDA0003124417970000071
TABLE 4 Single-base extension primer sequences
Serial number SNP site Single base extension primer
19 rs28371759 CAGCTCTGTCCGATC
20 rs4986893 TGGCCTTACCTGGAT
21 rs1057910 TGGGGAGAAGGTCAA
22 rs1065852 CTGGGCTGCACGCTAC
23 rs3813867 TTCTTGGTTCAGGAGAG
24 rs776746 AGAGCTCTTTTGTCTTTCA
25 rs762551 AAGGGTGAGCTCTGTGGGC
26 rs4244285 CAGTAATTTGTTATGGGTTCC
27 rs2031920 TTAATTCATAGGTTGCAATTTT
Example 2 sample extraction and analysis
In order to verify the detection accuracy and effectiveness of the primer set for detecting the drug metabolizing enzyme gene, 10 samples are selected for gene detection, and the process is as follows:
A. saliva DNA extraction: saliva DNA extraction is carried out by utilizing a Kangji century oral swab genome DNA extraction kit. A certain amount of absolute ethyl alcohol is added into GW1 and GW2 reagents. Taking 500 mu L of saliva sample from a sampling tube, adding 300 mu L of GR, 20 mu L of protease K and 300 mu L of GL, shaking and mixing uniformly, shaking and incubating for 15min at 56 ℃, adding 300 mu L of absolute ethyl alcohol, shaking and mixing uniformly; adding 750 μ L of the solution into an adsorption column of a collection tube, centrifuging at 12000rpm for 1min, and discarding the solution; adding 400 μ L GW1 into adsorption column, centrifuging at 12000rpm for 1min, and discarding solution; adding 400 μ L GW2 into adsorption column, centrifuging at 12000rpm for 1min, and discarding solution; centrifuging at 12000rpm for 2min, standing and air drying; placing the adsorption column in a new 1.5mL centrifuge tube, suspending and adding 40 μ L GE, standing for 5min, centrifuging at 12000rpm for 1min, collecting DNA solution, inspecting quality, and storing at 4 deg.C.
B, PCR amplification: and D, respectively adding the DNA of the sample to be detected extracted in the step A into a 384-pore plate, and performing multiplex PCR amplification. To each reaction well was added a reaction system for multiplex PCR amplification (5. mu.L) which:
the DNA as a template was 1. mu.L,
the primer mix is 1 mu L, and the primer mix is a primer containing a primer base and a primer base,
10×Buffer 0.5μL
MgCl2(25mM)0.4μL，
dNTP(25mM)0.1μL，
ddH2O 1.8μL，
hotstar (5U/. mu.L) was made up to 5. mu.L.
After the reaction system is prepared, a PCR sealing film is used for sealing to prevent the sample from evaporating, and the mixture is vibrated, uniformly mixed and centrifuged; the sealed 384-well plate is placed on an ABI9700PCR instrument for reaction, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min (95 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 1min) for 45 cycles; keeping at 72 deg.C for 5min and 4 deg.C; obtaining PCR amplification products, and centrifuging for later use.
SAP digestion: and (3) completing the SNP locus typing result of the sample to be tested by using a reagent matched with the Sequenom platform and the operation steps. The PCR amplification product obtained in step B was added to each reaction well according to SAP digested reaction system (2. mu.L):
SAP×Buffer 0.17μL，
SAP Enzyme(1U/μL)0.3μL，
ddH2O 1.53μL；
after the reaction system is prepared, a PCR sealing film is used for sealing to prevent the sample from evaporating, and the mixture is vibrated, uniformly mixed and centrifuged; the sealed 384-well plate is placed on an ABI9700PCR instrument for reaction, and the reaction conditions are as follows: keeping at 37 deg.C for 40min, 85 deg.C for 5min, and 4 deg.C; obtaining the PCR product after alkaline phosphorylase treatment, and centrifuging for later use.
D. Single base extension reaction: and (3) completing the SNP locus typing result of the sample to be tested by using a reagent matched with the Sequenom platform and the operation steps. And D, adding the PCR product treated by the alkaline phosphorylase obtained in the step C into each corresponding reaction hole according to a single-base extension reaction system (2 mu L), wherein the reaction system comprises the following steps:
the extension primer mix is 0.94. mu.L,
Gold×Buffer 0.2μL，
Extension mix 0.2μL，
Iplex Enzyme 0.041μL，
ddH2O 0.619μL；
after the reaction system is prepared, a PCR sealing film is used for sealing to prevent the sample from evaporating, and the mixture is vibrated, uniformly mixed and centrifuged; the sealed 384-well plate is placed on an ABI9700PCR instrument for reaction, and the reaction conditions are as follows: 30s at 94 ℃, 5s at 94 ℃ ({ 5s at 94 ℃ (5 s at 52 ℃, 5s at 80 ℃) with 5 internal circulations }40 external circulations, 3min at 72 ℃, and keeping at 4 ℃; obtaining single base extension products, and centrifuging for later use.
E. Resin purification: and (3) completing the SNP locus typing result of the sample to be tested by using a reagent matched with the Sequenom platform and the operation steps. D, adding 16 mu L ddH into each well of 384-well plates of the single-base extension products obtained in the step D after gently tearing off the sealing film2O; clean A4 paper was placed on the 6MG 384 plate with a small spoonTaking a proper amount of purified resin; repeatedly flattening the purified resin by a plastic plate, and compacting to ensure that the resin content in each hole is uniform; pressing the 384 plate upside down on the 6MG 384 plate, exchanging the two plates, pressing the 6MG plate on the 6MG plate, knocking the back of the 6MG plate to make the resin fall into the 384-hole plate filled with the single-base extension product; after the sealing film is sealed, the mixture is turned upside down at 15rpm for 30min and fully purified.
F. Chip spotting: centrifuging the 384-well plate in the step E, starting a MassARRAY Nanodispenser RS1000 spotting instrument, and transferring the extension product after resin purification to a 384-well SpectroCHIP chip; and (3) carrying out MALDI-TOF analysis on the spotted chip, and typing the detection result by using TYPER4.0 software and outputting the result.
Example 3 analysis of Gene test results
The gene detection results (Table 5) of 9 SNP sites of 10 samples in example 2 were analyzed, and a rational medication recommendation was given in combination with the typing of SNP sites related to drug metabolizing enzyme genes and the drug metabolism rates (Table 2).
TABLE 5 genotype of 9 SNP sites of drug metabolism-related genes
Site of the body 1 2 3 4 5 6 7 8 9 10
rs1065852 A AG AG AG A G AG AG A AG
rs762551 A A AC C AC AC AC A A A
rs1057910 A A A A A A A A A A
rs4244285 G G G AG AG G AG AG G G
rs4986893 AG G G G G G AG G G G
rs28371759 A A A A A A A A A A
rs776746 C C CT C CT C C T CT C
rs3813867 G G CG G G G CG G G G
rs2031920 C C CT CT T C C C C CT
As can be seen from Table 5, the genotypes of the 9 loci were all correctly detected, with a detection rate of 100%. The amplification primer and the extension primer designed by the invention can detect 9 SNP sites in the same reaction, and are used for developing a drug metabolizing enzyme gene detection kit and guiding safe medication. In the aspect of safe medication for children, the gene detection of the invention can understand the metabolic capability of children to different drugs and provide scientific reference basis for reasonable medication. The genes and loci covered by the invention are related to 8 types of medicaments related to children medicaments, and are the most comprehensive in gene detection products for children safety medicaments.
Example 4 drug metabolism relationships and drug administration suggestions of human drug-metabolizing enzyme genes
The activities of drug metabolizing enzymes in human population generally show the phenomena of fast metabolizing type (EM), intermediate metabolizing type (IM) and slow metabolizing type (PM) distribution.
CYP2D6 is responsible for the metabolism of various drugs such as anticonvulsants, sedatives, anti-inflammatory drugs, endocrine drugs and the like.
CYP1a2 accounts for 13% of the total liver P450 oxidase content and is involved in the metabolism of many drugs, steroid hormones and pain-causing substances. The mutation at this site is the most functional and most frequent mutation, and individuals carrying the C allele have reduced rates of metabolism of caffeine, phenacetin, theophylline, and acetaminophen.
CYP2C9 is involved in the metabolism of 6% of drugs in current clinical medication. Their catalytic action on drug metabolism is usually distributed in two states, i.e. fast and slow metabolizers. The dosage should be different between the fast and slow metabolizers.
CYP2C19 x 2 causes splicing deletion, CYP2C19 x 3 is a stop codon mutation, and the enzymatic activity of the homozygous individual is only 4-6% of that of the wild type homozygous genotype individual. CYP2C9 genetic polymorphism causes variation of enzyme activity, thereby causing phenomena of drug metabolism ethnicity and individual difference. 75-85% of the eastern population are caused by CYP2C19 x 2, and about 20-25% are caused by CYP2C19 x 3.
CYP3A4 is a main cytochrome P450 enzyme in human liver, accounts for about 25% of total CYP450 enzyme in adult liver, is mainly distributed in liver cells and liver bile duct epithelium, is the most liver drug enzyme in liver, participates in about 90% of drugs metabolized in intestinal tract, and is metabolized by CYP3A4 enzyme in about 50% of drugs clinically.
The SNP of the CYP3A5 can cause abnormal splicing of CYP3A5 mRNA, and cause premature shearing of CYP3A5 protein by a stop codon, so that the CYP3A5 x 3 homozygous individual loses the enzymatic activity, and the expression and the activity of the CYP3A5 protein in the liver and the intestinal tract are obviously reduced. CYP3A5 is involved in the metabolism of tacrolimus, midazolam, dapsone, cortisone, nifedipine and other medicines.
CYP2E1 has more substrates, wherein most of the substrates are precancerous substances and prototoxicants, and the substrates are medicines. Because the difference of the active individuals is obvious, the medicine has certain influence on the medicine treatment effect and adverse reaction of different individuals. CYP2E1 is a main component enzyme in the ethanol oxidation system, and can participate in the metabolism of more than 80 low molecular hydrophobic toxic substances, carcinogens and medicaments, so that the low molecular compounds are converted into metabolites with stronger chemical reaction than parent compounds. The CYP2E1 content accounts for 6.6% of the liver system of human body on average. Metabolizing mainly chlorzoxazone; also metabolize the fluorides of acetaminophen, dapsone, aniline, ethanol and general anesthetics.
Medication recommendations (examples): acetaminophen is a main component of antipyretic and analgesic medicines commonly used by children, has the function of central nervous antipyresis, and is a main component of children common medicines such as pediatric paracetamol, chlorphenamine maleate granules (pediatric kuaike), chlorphenamine phenomenol suspension (pediatric tylenol) and the like. The in vivo drug delivery enzymes of acetaminophen are CYP1A2 and CYP2E1, and the results of gene detection show that in Table 5 of example 3, the patients 3 and 4 have unfavorable variation on two genes, and are slow metabolizers of acetaminophen, and the adverse reaction of acetaminophen drugs is aggravated; the main metabolic enzyme of ibuprofen, which is the same antipyretic analgesic drug component, is CYP2C9, and the metabolic enzyme genes of the two drugs are normal, so that the antipyretic analgesic drug containing ibuprofen is preferred to be used for reducing the usage of acetaminophen when two subjects take the drug.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> Beijing Scale Yongda scientific and technological development Co., Ltd
<120> set of primers for detecting drug metabolizing enzyme gene SNP site and application thereof
<130> 2018.02
<160> 27
<170> PatentIn version 3.3
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acgttggatg cagctggata ccagaaagac 30
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acgttggatg gttcttaatt cataggttgc 30
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acgttggatg tccacaagtg atttggctgg 30
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<223> rs28371759 single-base extension primer
<400> 19
cagctctgtc cgatc 15
<210> 20
<211> 15
<212> DNA
<213> Artificial
<223> rs4986893 single base extension primer
<400> 20
tggccttacc tggat 15
<210> 21
<211> 15
<212> DNA
<213> Artificial
<223> rs1057910 single-base extension primer
<400> 21
tggggagaag gtcaa 15
<210> 22
<211> 16
<212> DNA
<213> Artificial
<223> rs1065852 single-base extension primer
<400> 22
ctgggctgca cgctac 16
<210> 23
<211> 17
<212> DNA
<213> Artificial
<223> rs3813867 Single-base extension primer
<400> 23
ttcttggttc aggagag 17
<210> 24
<211> 19
<212> DNA
<213> Artificial
<223> rs776746 single base extension primer
<400> 24
agagctcttt tgtctttca 19
<210> 25
<211> 19
<212> DNA
<213> Artificial
<223> rs762551 Single base extension primer
<400> 25
aagggtgagc tctgtgggc 19
<210> 26
<211> 21
<212> DNA
<213> Artificial
<223> rs4244285 single-base extension primer
<400> 26
cagtaatttg ttatgggttc c 21
<210> 27
<211> 22
<212> DNA
<213> Artificial
<223> rs2031920 single-base extension primer
<400> 27
ttaattcata ggttgcaatt tt 22

Claims (2)

1. The complete set of primers for detecting the SNP locus of the drug metabolizing enzyme gene is characterized in that: PCR amplification primer pairs and single base extension primers of 9 SNP sites of 7 drug metabolizing enzyme-related genes comprising human genome DNA; the 7 drug metabolizing enzyme related genes of the human genome DNA are 7 important subtypes in CYP1, CYP2 and CYP 3: CYP1a2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3a4, CYP3a 5; the 9 SNP loci are as follows: rs762551 site on CYP1A2, rs1057910 site on CYP2C9, rs4244285 and rs4986893 site on CYP2C19, rs1065852 site on CYP2D6, rs3813867 and rs2031920 site on CYP2E1, rs28371759 site on CYP3A4, and rs776746 site on CYP3A 5;
the PCR amplification primer sequence of the rs28371759 locus is SEQ ID NO: 1 and SEQ ID NO: 2, and the sequence of the single-base extension primer is shown as SEQ ID NO: 19;
the PCR amplification primer sequence of the rs4986893 site is SEQ ID NO: 3 and SEQ ID NO: 4, and the sequence of the single-base extension primer is shown as SEQ ID NO: 20;
the PCR amplification primer sequence of the rs1057910 locus is SEQ ID NO: 5 and SEQ ID NO: 6, and the sequence of the single-base extension primer is SEQ ID NO: 21;
the PCR amplification primer sequence of the rs1065852 site is SEQ ID NO: 7 and SEQ ID NO: 8, and the sequence of the single-base extension primer is shown as SEQ ID NO: 22;
the PCR amplification primer sequence of the rs3813867 site is SEQ ID NO: 9 and SEQ ID NO: 10, and the single-base extension primer sequence is SEQ ID NO: 23;
the PCR amplification primer sequence of the rs776746 site is SEQ ID NO: 11 and SEQ ID NO: 12, and the single-base extension primer sequence is SEQ ID NO: 24;
the PCR amplification primer sequence of the rs762551 site is SEQ ID NO: 13 and SEQ ID NO: 14, and the sequence of the single-base extension primer is shown as SEQ ID NO: 25;
the PCR amplification primer sequence of the rs4244285 locus is SEQ ID NO: 15 and SEQ ID NO: 16, and the sequence of the single-base extension primer is shown as SEQ ID NO: 26;
the PCR amplification primer sequence of the rs2031920 site is SEQ ID NO: 17 and SEQ ID NO: 18, and the sequence of the single-base extension primer is shown as SEQ ID NO: 27, or a nucleotide sequence as set forth in figure 27.
2. A gene detection kit comprising the set of primers for detecting SNP sites of a drug metabolizing enzyme gene according to claim 1.
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