CN113528637A - Primer group for amplifying anti-infective drug gene SNP locus, detection primer group and application thereof - Google Patents
Primer group for amplifying anti-infective drug gene SNP locus, detection primer group and application thereof Download PDFInfo
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Abstract
The invention provides a primer group for amplifying anti-infective drug gene SNP sites, a detection primer group and application thereof, belonging to the technical field of gene detection. The primer group for amplifying the anti-infective drug gene SNP locus and the single base extension primer provided by the invention form a detection primer group which can be used for detecting the anti-infective drug gene SNP locus polymorphism. The detection primer group can simultaneously detect 14 SNP loci of 8 medication related genes related to 13 drugs and 6 drug active ingredients, so that the detection is more efficient, and the genotype of the SNP loci of the detected related genes of each anti-infective drug lays a research foundation for subsequently and effectively guiding individualized administration and safe medication.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer group for amplifying anti-infective drug gene SNP sites, a detection primer group and application thereof.
Background
Anti-infective agents refer to various drugs used to treat infections caused by pathogens (bacteria, viruses, fungi, chlamydia, mycoplasma, spirochetes, etc.). In the case of anti-infective drugs, the drug response produced by the same drug dose for different individuals varies greatly, and causes of such variation include intrinsic factors and extrinsic factors, wherein the main factor of intrinsic factors is the difference in the genetic material, i.e., genotype, of the individual.
Common anti-infective agents include voriconazole, erythromycin, rifampicin, isoniazid, oseltamivir, ribavirin and the like. Most individual differences of voriconazole can be explained by its CYP2C19 gene polymorphism, CYP2C19 is a member of cytochrome P450 family, CYP2C19 gene has multiple alleles, wherein allele 1 is normal metabolic type, allele 2, 3, 4, 5, 6, 8 is slow metabolic type, and allele 17 is ultra fast metabolic type. Therefore, patients of different genotypes should be treated with different doses of voriconazole. Erythromycin is one of macrolide antibiotics, has similar antibacterial spectrum to penicillin, has strong antibacterial effect on gram-positive bacteria such as staphylococcus aureus and hemolytic streptococcus, and also has certain effect on gram-negative bacteria. Clinical studies show that patients with glucose-6-phosphate dehydrogenase deficiency (G6PD gene mutation) can cause malignant toxic and side effects such as hemolysis by using erythromycin. Pharmacogenomic studies also show that the ABCC2 genetic polymorphism is related to the erythromycin metabolic rate. Rifampicin is mainly used in therapeutic drugs for brucellosis and sensitive mycobacterial infections. It can cause nausea, emesis, anorexia, diarrhea, gastralgia, abdominal distention, etc., and various anaphylaxis. Clinical researches suggest that the genetic polymorphism of the NAT1/NAT2 gene can influence the metabolism of isoniazid in vivo, thereby causing toxic and side effects. Isoniazid is mainly used in the progressive stage, dissolution and dissemination stage and absorption and transformation stage of various types of pulmonary tuberculosis, and can also be used in tuberculous meningitis and other extrapulmonary tuberculosis. In addition, isoniazid also has certain curative effect on dysentery, whooping cough, hordeolum, etc. Adverse reactions include gastrointestinal symptoms (such as anorexia, nausea, emesis, abdominal pain, constipation, etc.), blood system symptoms (anemia, leukopenia, eosinophilia, blood phlegm, hemoptysis, epistaxis, eyeground hemorrhage, etc.), and liver damage; allergies (rashes or others), endocrine disorders (gynecomastia in men, lactation, irregular menstruation, impotence, etc.). Clinical studies suggest that slow acetylation is more likely to cause reactions in the blood system, endocrine system, nervous system and mental system when isoniazid is administered in large doses or for a long time; the fast acetylation is easy to cause liver damage, and the expression product of NAT1/NAT2 gene can participate in the acetylation of isoniazid in vivo. Oseltamivir (trade name "tamiflu") is an antiviral drug that inhibits neuraminidase, and can be used for the treatment of influenza a and b; meanwhile, the medicine can also be used as preventive medicine for people with high risk of influenza complications, such as pregnant women, children less than 5 years old, old people more than 65 years old, chronic disease patients, severe obesity patients, and the like. Relevant pharmacokinetic studies show that the rs200707504 site polymorphism in the CES1 gene can influence the metabolic rate of oseltamivir in vivo, thereby influencing the treatment effect. Ribavirin has broad-spectrum antiviral effect, and has inhibitory effect on various viruses such as hepatitis C, respiratory syncytial virus, influenza virus, herpes simplex virus, etc.; it can be used for preventing and treating influenza (caused by influenza virus A and B), adenovirus pneumonia, hepatitis A, herpes, measles, etc. Large dose application (including nasal drop) can cause heart damage, and for patients with respiratory diseases, such as chronic obstructive pulmonary disease or asthma, can cause dyspnea, chest pain, etc. Clinical studies have shown that multiple gene site polymorphisms in the IFNL3/IFNL4 gene have an effect on the therapeutic efficacy of hepatitis C.
The traditional detection method of the polymorphism of the gene locus of the targeted drug comprises first-generation sequencing, fluorescent quantitative PCR and high-throughput sequencing. However, these detection methods have some drawbacks: the first generation sequencing detection flux is low, only one SNP site can be detected at one time, 14 SNP sites need to be sequenced for 14 generations, the detection time efficiency is low, and the cost is too high. The fluorescent quantitative PCR detection can detect 3-5 SNP sites at most once, 4-5 reactions are needed for detecting 14 SNP sites, multiple PCR needs to be designed independently, the cost of probes used by the fluorescent quantitative PCR is high, and a relatively expensive fluorescent quantitative PCR instrument is also needed. Although the high-throughput sequencing technology can realize simultaneous detection of multiple sites, the requirement on the minimum sample number of one experiment is high, and the experiment cost and the analysis time are also remarkably high.
Disclosure of Invention
In view of the above, the present invention aims to provide a primer set for amplifying anti-infective drug gene SNP sites, which can simultaneously amplify 14 SNP sites of 8 drug-related genes related to 13 drugs and 6 drug active ingredients, so as to efficiently perform detection.
The invention also aims to provide a primer group for detecting the SNP locus polymorphism of the anti-infective drug gene and application thereof, which not only can realize the detection of a plurality of SNP loci of a plurality of anti-infective drug target genes and have high detection flux, but also can simultaneously detect a plurality of gene loci and reduce the detection cost.
The invention provides a primer group for amplifying anti-infective drug gene SNP sites, which comprises primers with nucleotide sequences shown in a sequence table SEQ ID NO. 1-SEQ ID NO. 28.
The invention provides a primer group for detecting anti-infective drug gene SNP site polymorphism, which comprises the primer group for amplifying anti-infective drug gene SNP site and a single base extension primer with a nucleotide sequence shown in a sequence table SEQ ID NO. 29-SEQ ID NO. 42.
The invention provides an application of the primer group or the primer group in detecting the SNP locus polymorphism of the anti-infective drug gene.
Preferably, the anti-infective drug comprises voriconazole, veovan, pinapril, lifukang, memantine, erythromycin estolate, erythromycin ethylsuccinate, rifampicin, isoniazid, oseltamivir phosphate capsule, ribavirin oral liquid, ribavirin dispersible tablet and/or ribavirin injection.
Preferably, the effective pharmaceutical ingredients of the anti-infective drug comprise voriconazole, erythromycin, rifampicin, isoniazid, oseltamivir and/or ribavirin.
Preferably, the anti-infective drug genes include CYP2C19, G6PD, ABCC2, NAT1, NAT2, CES1, IFNL3, and/or IFNL 41.
Preferably, the SNP site of the anti-infective drug gene comprises rs4244285, rs4986893, rs12248560, rs137852327, rs5030869, rs72554665, rs72554664, rs717620, rs1801280, rs1799930, rs200707504, rs12979860, rs8099917 and/or rs 2228570.
The invention provides a method for detecting SNP locus polymorphism of anti-infective drug genes, which comprises the following steps:
1) extracting the genome DNA of a sample to be detected;
2) performing multiple PCR amplification by using the primer group by using the extracted DNA as a template to obtain multiple PCR amplification products;
3) purifying the multiple PCR amplification product, and performing multiple extension reaction under the action of single-base extension primers in the primer group to obtain an extension product;
4) and sequencing the extension product to obtain the genotype of the anti-infective drug gene SNP locus.
Preferably, the reaction system for multiplex PCR amplification in step 2) is 10 μ l: 1 ul 1 × HotStarTaq buffer, 0.6 ul 25mM Mg2+0.2. mu.l of 10mM dNTP, 0.15. mu.l of 1U HotStarTaqpolymerase, 1. mu.l of DNA template and 1. mu.l of multiplex PCR primer mixture, and 10. mu.l of ultrapure water was added; the multiple PCR primer mixed solution comprises a primer group for amplifying the anti-infective drug gene SNP locus, wherein the concentration of each primer in the primer group is 1-3 mu M;
the reaction procedure of the multiplex PCR amplification in the step 2): 5min at 95 ℃; 20s at 94 ℃, 40s at 65 ℃, 1.5min at 72 ℃ and 11 cycles; 20 cycles of 94 ℃ for 20s, 59 ℃ for 30s, and 72 ℃ for 1.5 min; 72 ℃ for 2 min.
Preferably, the reaction system of the multiple extension reaction in step 3) is 10 μ l: mu.l of the SNaPshot Multiplex Kit, 2. mu.l of the purified Multiplex PCR amplification product, 1. mu.l of the extension primer mixture and 2. mu.l of ultrapure water; the concentration of each single-base extension primer in the extension primer mixture is 1-3 mu M;
reaction procedure for the multiplex extension reaction in step 3): 1min at 96 ℃; 96 ℃ for 10s, 55 ℃ for 5s, 60 ℃ for 30s, 28 cycles.
The primer group for amplifying the anti-infective drug gene SNP locus provided by the invention comprises primers with nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO. 28 in a sequence table. The primer group can simultaneously amplify DNA fragments of 14 SNP loci of 8 medication related genes related to 13 medicaments and 6 medicament active ingredients, and realize the multiplex PCR amplification of the SNP loci of various anti-infective medicament genes. The primer group provided by the invention not only can effectively improve the detection efficiency, but also can greatly reduce the detection cost.
The primer group for detecting the SNP locus polymorphism of the anti-infective drug gene comprises the primer group for amplifying the SNP locus of the anti-infective drug gene and a single-base extension primer with a nucleotide sequence shown in a sequence table SEQ ID NO. 29-SEQ ID NO. 42. The primer group has the following advantages: 1) the medicine contains more active ingredients of common anti-infective medicines, including 13 medicines and 6 active ingredients; 2) the number of covered genes and SNP sites is large, and 8 genes and 14 SNP sites are included; 3) the confidence level of the gene sites is high, and according to the data of a pharmacogenetic knowledge base (pharmGKB), 14 SNP sites related in the detection method have 9 confidence levels of 1 level, 2 confidence levels of 2 levels, 23 confidence levels and 14 levels; 4) the gene locus design and optimization are carried out aiming at the own gene database of Chinese people, and the effectiveness is high; 5) the method has high sensitivity, can be realized only by 1ng of DNA, and is widely suitable for trace DNA samples such as blood cards, oral cavity testers and the like; 6) the detection efficiency is high, 384 samples can be detected in one experiment, and the whole experiment process is completed within 6 hours; 7) the flux is high, and the detection of a plurality of sites can be realized only once by the SNaPshot SNP typing technology.
Drawings
FIG. 1 is a map showing the results of detection of all target SNP sites in a sample according to an embodiment of the present invention.
Detailed Description
The invention provides a primer group for amplifying anti-infective drug gene SNP sites, which comprises primers with nucleotide sequences shown in a sequence table SEQ ID NO. 1-SEQ ID NO. 28. The invention also provides a primer group for detecting the SNP locus polymorphism of the anti-infective drug gene, which comprises the primer group for amplifying the SNP locus of the anti-infective drug gene and a single-base extension primer with a nucleotide sequence shown in a sequence table SEQ ID NO. 29-SEQ ID NO. 42. The sequences of the PCR amplification primer set and the single-base extension primer for SNP sites are shown in Table 1.
TABLE 1PCR amplification primer sequence and Single-base extension primer sequence of SNP site of interest of the present invention
The invention provides an application of the primer group or the primer group in detecting the SNP locus polymorphism of the anti-infective drug gene.
In the present invention, the type of the anti-infective drug, the drug active ingredient, the drug targeting gene, and the relationship of the SNP site in the gene are shown in table 2.
TABLE 2 correlation between genes related to anti-infective drugs and SNP sites
The invention provides a method for detecting SNP locus polymorphism of anti-infective drug genes, which comprises the following steps:
1) extracting the genome DNA of a sample to be detected;
2) performing multiple PCR amplification by using the primer group by using the extracted DNA as a template to obtain multiple PCR amplification products;
3) purifying the multiple PCR amplification product, and performing multiple extension reaction under the action of single-base extension primers in the primer group to obtain an extension product;
4) and sequencing the extension product to obtain the genotype of the anti-infective drug gene SNP locus.
The invention extracts the genome DNA of a sample to be detected.
The method for extracting DNA is not particularly limited in the present invention, and a method for extracting DNA well known in the art, for example, a kit method, may be used. The sample to be tested is preferably saliva. The saliva is preferably collected in the form of a oropharyngeal swab.
After the DNA is extracted, the invention takes the extracted DNA as a template and uses the primer group to carry out multiple PCR amplification to obtain multiple PCR amplification products.
In the present invention, the reaction system for multiplex PCR amplification is preferably 10. mu.l: 1 ul 1 × HotStarTaq buffer, 0.6 ul 25mM Mg2+0.2. mu.l of 10mM dNTP, 0.15. mu.l of 1U HotStarTaqpolymerase, 1. mu.l of DNA template and 1. mu.l of multiplex PCR primer mixture, and 10. mu.l of ultrapure water was added; the multiple PCR primer mixed solution comprises the primer group, the concentration of each primer in the primer group is 1-3 mu M, and the concentration of the amplification primer corresponding to each SNP locus is shown in Table 3.
TABLE 3 concentration of amplification primers corresponding to each SNP site of the present invention
| SNP_ID | rs200707504 | rs4986893 | rs1801280 | rs8099917 | rs1799930 |
| Single primer concentration (μ M) | 1～2 | 1～2 | 2～3 | 1～3 | 1～2 |
| SNP_ID | rs72554664 | rs4244285 | rs5030869 | rs12979860 | rs717620 |
| Single primer concentration (μ M) | 2～3 | 1～3 | 1～3 | 1～2 | 1～2 |
| SNP_ID | rs2228570 | rs12248560 | rs72554665 | rs137852327 | - |
| Single primer concentration (μ M) | 1～2 | 1～3 | 1～2 | 1～2 | - |
The reaction procedure for the multiplex PCR amplification is preferably as follows: 5min at 95 ℃; 20s at 94 ℃, 40s at 65 ℃, 1.5min at 72 ℃ and 11 cycles; 20 cycles of 94 ℃ for 20s, 59 ℃ for 30s, and 72 ℃ for 1.5 min; 72 ℃ for 2 min. The apparatus for the multiplex PCR amplification reaction of the present invention is not particularly limited, and a PCR apparatus known in the art may be used.
After obtaining the multiple PCR amplification product, the multiple PCR amplification product is purified and then is subjected to multiple extension reaction under the action of the single base extension primer in the primer group to obtain an extension product.
In the present invention, the purification method preferably comprises adding 5U SAP enzyme and 2U Exonuclease I enzyme per 10. mu.l of the system of the multiple PCR amplification products for incubation. The incubation condition is preferably incubation for 30min at 36-38 ℃. After said incubation also preferably comprises inactivating the enzyme; the enzyme inactivation condition is preferably that the enzyme is inactivated at 72-78 ℃ for 12-17 min, and more preferably at 75 ℃ for 15 min.
In the present invention, the reaction system of the multiplex extension reaction is 10 μ l: mu.l of the SNaPshot Multiplex Kit, 2. mu.l of the purified Multiplex PCR amplification product, 1. mu.l of the extension primer mixture and 2. mu.l of ultrapure water; the concentration of each single-base extension primer in the extension primer mixture is 1-3 mu M, and the concentration of primers corresponding to different SNP sites is preferably shown in Table 4.
TABLE 4 concentration of each extension primer corresponding to each SNP site in the extension primer mixture
Reaction procedure for the multiplex extension reaction: 1min at 96 ℃; 96 ℃ for 10s, 55 ℃ for 5s, 60 ℃ for 30s, 28 cycles.
In the present invention, the extension product also preferably includes purification. The purification was performed with SAP enzyme. The purified system preferably adds 1U of SAP enzyme per 10. mu.l of extension product. The purification conditions are preferably a 30min incubation at 37 ℃ followed by 15min inactivation at 75 ℃.
Obtaining a purified extension product, and sequencing the purified extension product to obtain the genotype of the anti-infective drug gene SNP locus.
In the invention, the purified extension product is prepared into a sequencing solution; the sequencing solution is preferably prepared by mixing 0.3. mu.l Liz120 SIZE STANDARD and 9. mu.l Hi-Di per 1. mu.l of purified extension product. The sequencing solution is preferably denatured at 95 ℃ for 5min prior to on-machine sequencing. The sequencing is preferably performed using an ABI3730XL sequencer. After sequencing, the sequencing results are preferably analyzed by GeneMapper4.1(applied biosystems Co., Ltd., USA) to obtain the genotype of each SNP site.
In the invention, the obtained SNP genotype of each gene lays a foundation for further using anti-infective drugs according to the specific genotype guidance.
The primer set for amplifying the anti-infective drug gene SNP site, the detection primer set and the use thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. DNA extraction from saliva/throat swab or the like
10 pieces of saliva collected from different patients or swabs wiped across the cheek were transferred into 2ml centrifuge tubes, and the swab sections were cut from the rod with scissors and 40. mu.l of buffer GA was added. Add 20. mu.l of Proteinase K solution, vortex for 30s and mix, stand at 56 ℃ for 60min, vortex for several times every 15 min. Add 400. mu.l buffer GB, mix well by inversion, stand at 70 ℃ for 10min, at which time the solution should be clear, centrifuge briefly to remove droplets on the inner wall of the tube cover, then squeeze to remove the swab, transfer as much lysate as possible to a new centrifuge tube. Add 200. mu.l of absolute ethanol, mix well by inversion, centrifuge briefly to remove droplets on the inner wall of the tube cap. Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CR2 (adsorption column CR2 is put into a collection tube), centrifuging at 12000rpm for 30s, pouring off the waste liquid in the collection tube, and putting adsorption column CR2 back into the collection tube. Add 500. mu.l buffer GD to the adsorption column CR2, centrifuge at 12000rpm for 30s, discard the tube and replace the column CR2 back in the tube. 600 μ l of the rinsing solution PW 2 was added to the adsorption column CR2, centrifuged at 12000rpm for 30s, the waste liquid in the collection tube was discarded, and the adsorption column CR2 was returned to the collection tube. Repeating the previous step. Centrifuging at 12000rpm for 2min, and discarding waste liquid. The adsorption column CR2 was left at room temperature for several minutes to allow the residual rinse solution from the adsorption material to dry out completely. Transferring the adsorption column CR2 into a clean centrifuge tube, suspending and dripping 30 μ l of elution buffer TB into the middle position of the adsorption membrane, standing at room temperature for 3min, centrifuging at 12000rpm for 2min, collecting DNA solution, inspecting quality, and storing at 4 deg.C.
2. Multiplex PCR amplification reaction
2.1 preparation of PCR reaction System
A10. mu.l reaction containing 1 × HotStarTaqbuffer, 3.0mM Mg2+0.3mM dNTP, 1U HotStarTaqpolymerase (Qiagen Inc.), 1. mu.l of sample DNA and 1. mu.l of multiplex PCR amplification primer mixture were supplemented with ultrapure water to 10. mu.l.
2.2 the sequences of the primers in the mixture of primers for multiplex PCR amplification are shown in SEQ ID NO. 1-SEQ ID NO. 28, and the concentrations of the primers in the mixture of primers for multiplex PCR amplification are shown in Table 3.
TABLE 3 concentration of amplification primers corresponding to each SNP site
Note: each primer (upstream and downstream primers) was diluted to 100. mu.M and added to a primMix of 100. mu.L final volume in Table 3, with the upstream and downstream primers mixed together.
2.3 multiplex PCR amplification reaction procedure
3. Multiplex PCR amplification product purification
To 10. mu.l of the multiplex PCR amplification product, 5U of SAP enzyme and 2U of Exonuclease I enzyme were added, and incubated at 37 ℃ for 30 minutes, followed by inactivation at 75 ℃ for 15 minutes.
Wherein two enzymes SAP and Exonuclease I were purchased from NEB (New England Biolabs (Beijing) LTD.), the concentration of SAP was 1U/. mu.L, the concentration of Exonuclease I was 20U/. mu.L, the addition amount was SAP 5. mu.L, and Exon I was 0.1. mu.L.
SnaPshot multiplex single base extension reaction
4.1 extension reaction
A10. mu.l extension reaction system included 5. mu.l of the SNaPshot Multiplex Kit (ABI), 2. mu.l of the purified Multiplex PCR product, 1. mu.l of the extension primer mixture, and 2. mu.l of ultrapure water.
4.2 Each extension primer in the extension primer mixture is shown in SEQ ID NO. 29-SEQ ID NO. 42| and the concentration of each extension primer is shown in Table 4.
TABLE 4 concentration of extension primers corresponding to different SNP sites
| SNP_ID | rs200707504 | rs4986893 | rs1801280 | rs8099917 | rs1799930 |
| Single primer concentration (μ M) | 1 | 1 | 2 | 1 | 1 |
| SNP_ID | rs72554664 | rs4244285 | rs5030869 | rs12979860 | rs717620 |
| Single primer concentration (μ M) | 2 | 1 | 3 | 1 | 1 |
| SNP_ID | rs2228570 | rs12248560 | rs72554665 | rs137852327 | - |
| Single primer concentration (μ M) | 1 | 1 | 1 | 1 | - |
Note: each extension primer was diluted to 100. mu.M and added to a primer Mix in the amount indicated in the table to a final volume of 100. mu.L, with both the upstream and downstream primers mixed together.
4.3 reaction sequence
5. Purification of extension products
Mu.l of the extension product was added with 1U of SAP enzyme, incubated at 37 ℃ for 30 minutes and then inactivated at 75 ℃ for 15 minutes.
6. ABI3730XL sequencer on extension products
Mu.l of the purified extension product was mixed with 0.3. mu.l of Liz120 SIZE STANDARD and 9. mu.l of Hi-Di, denatured at 95 ℃ for 5 minutes and applied to an ABI3730XL sequencer.
7. The raw data collected on the ABI3730XL sequencer was analyzed by genemappers 4.1(applied biosystems co., ltd., USA) to obtain specific genotype information for the targeted SNP site.
Meanwhile, in the experiment, the same samples are subjected to Sanger sequencing to obtain the genotype of the target SNP locus.
The sequencing results of the detection method of the invention and Sanger sequencing are shown in Table 5.
TABLE 5 genotyping results for SNPs in genes obtained by two assays
TABLE 5 genotyping results (continuance) for SNP obtained by two detection methods
The result shows that the detection results of 10 random samples are completely consistent with the result of the Sanger sequencing method, and the result accuracy of the detection system disclosed by the invention is 100%. FIG. 1 is a map showing the results of detection of all target SNP sites in a single sample. As can be seen from FIG. 1, the resulting peaks of each genotype are clearly proportioned, separated from each other and non-intersecting with each other, indicating that the primer of the present invention has good specificity and repeatability.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<213> Artificial Sequence (Artificial Sequence)
<400> 3
acgttggatg gactgtaagt ggtttctcag 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
acgttggatg aacatcagga ttgtaagcac 30
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acgttggatg gacccagcat cgacaatgta 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acgttggatg caaatacagc actggcatgg 30
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acgttggatg actgtataca gcatggttcc 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
acgttggatg caatttgtca ctgttcctcc 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
acgttggatg aagatgttgg agacgtctgc 30
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acgttggatg cctgccaaag aagaaacacc 30
<210> 11
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
acgttggatg tctccagctc aatctggtgc 30
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
acgttggatg tcagcgacga gctccgtga 29
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
acgttggatg tccatcgatt cttggtgttc 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
acgttggatg gcaataattt tcccactatc 30
<210> 15
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
acgttggatg caaagggtac ctggacgac 29
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
acgttggatg cattctccac atagaggacg 30
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
acgttggatg tcgtgcctgt cgtgtactga 30
<210> 18
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
acgttggatg agcgcggagt gcaattcaac 30
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
acgttggatg agcatgattc ctggactgcg 30
<210> 20
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acgttggatg cctgttccac tttctttgat g 31
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
acgttggatg acgttccggt caaagtctcc 30
<210> 22
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
acgttggatg tggcctgctt gctgttctta 30
<210> 23
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
acgttggatg caaatttgtg tcttctgttc 30
<210> 24
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
acgttggatg ggatttgagc tgaggtcttc 30
<210> 25
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
acgttggatg tgcatacctg tgggctatgg 30
<210> 26
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
acgttggatg agcagtgggg tgaaaatacg 30
<210> 27
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
acgttggatg attgttggcc tgcacctctg 30
<210> 28
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
acgttggatg caactcaaca cccaaggagc 30
<210> 29
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tctcctcccg ctgac 15
<210> 30
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ctggccttac ctggat 16
<210> 31
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
acgtaattcc tgccgtca 18
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
catggttcca atttgggtga 20
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gactcaaaat cttcaattgt t 21
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gggaggcagt ggggtgaaaa ta 22
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gttaagtaat ttgttatggg ttcc 24
<210> 36
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cccgcgggtc caccacc 17
<210> 37
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tgagctcccc gaaggcg 17
<210> 38
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ccttgactgc gtctggaac 19
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
ctctggccgc cattgcctcc 20
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ttgtgtcttc tgttctcaaa g 21
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cctcacctca gcgacgagct cc 22
<210> 42
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ggcgctgaga tgcatttcaa ca 22
Claims (10)
1. A primer group for amplifying anti-infective drug gene SNP sites is characterized by comprising primers with nucleotide sequences shown as SEQ ID NO. 1-28 in a sequence table.
2. A primer set for detecting anti-infective drug gene SNP site polymorphism, which is characterized by comprising the primer set for amplifying anti-infective drug gene SNP site as claimed in claim 1 and a single-base extension primer with a nucleotide sequence shown as SEQ ID NO. 29-42 of the sequence table.
3. Use of the primer set for amplifying an anti-infective gene SNP site according to claim 1 or the primer set for detecting an anti-infective gene SNP site polymorphism according to claim 2 for detecting an anti-infective gene SNP site polymorphism.
4. The use of claim 3, wherein the anti-infective agent comprises voriconazole, veovan, pinacol, lifukang, milnacin, etodol, erythromycin ethylsuccinate, rifampin, isoniazid, oseltamivir phosphate capsule, ribavirin oral liquid, ribavirin dispersible tablet, and/or ribavirin injection.
5. The use of claim 3, wherein the pharmaceutically active ingredient of the anti-infective agent comprises voriconazole, erythromycin, rifampicin, isoniazid, oseltamivir and/or ribavirin.
6. The use of claim 3, wherein the anti-infective agent genes comprise CYP2C19, G6PD, ABCC2, NAT1, NAT2, CES1, IFNL3, and/or IFNL 41.
7. The use as claimed in any one of claims 3 to 6, wherein the SNP site of the antiinfective gene comprises rs4244285, rs4986893, rs12248560, rs137852327, rs5030869, rs72554665, rs72554664, rs717620, rs1801280, rs1799930, rs200707504, rs12979860, rs8099917 and/or rs 2228570.
8. A method for detecting SNP locus polymorphism of anti-infective drug genes is characterized by comprising the following steps:
1) extracting the genome DNA of a sample to be detected;
2) performing multiplex PCR amplification by using the extracted DNA as a template and the primer set according to claim 1 to obtain a multiplex PCR amplification product;
3) purifying the multiple PCR amplification product, and performing multiple extension reaction under the action of single-base extension primers in the primer group of claim 2 to obtain an extension product;
4) and sequencing the extension product to obtain the genotype of the anti-infective drug gene SNP locus.
9. The detection method according to claim 8, wherein the reaction system of the multiplex PCR amplification in step 2) is 10 μ l: 1 ul 1 × HotStarTaqbuffer, 0.6 ul 25mM Mg2+0.2. mu.l of 10mM dNTP, 0.15. mu.l of 1U HotStarTaq polymerase, 1. mu.l of DNA template and 1. mu.l of multiplex PCR primer mixture, and 10. mu.l of ultrapure water was added; the multiplex PCR primer mixture comprises the primer group of claim 1, wherein the concentration of each primer in the primer group is 1-3 μ M;
the reaction procedure of the multiplex PCR amplification in the step 2): 5min at 95 ℃; 20s at 94 ℃, 40s at 65 ℃, 1.5min at 72 ℃ and 11 cycles; 20 cycles of 94 ℃ for 20s, 59 ℃ for 30s, and 72 ℃ for 1.5 min; 72 ℃ for 2 min.
10. The detection method according to claim 8 or 9, wherein the reaction system of the multiplex extension reaction in step 3) is 10 μ l: mu.l of the SNaPshot Multiplex Kit, 2. mu.l of the purified Multiplex PCR amplification product, 1. mu.l of the extension primer mixture and 2. mu.l of ultrapure water; the concentration of each single-base extension primer in the extension primer mixture is 1-3 mu M;
reaction procedure for the multiplex extension reaction in step 3): 1min at 96 ℃; 96 ℃ for 10s, 55 ℃ for 5s, 60 ℃ for 30s, 28 cycles.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946759A (en) * | 2015-06-15 | 2015-09-30 | 广州金域医学检验中心有限公司 | CYP2C19*17 gene polymorphism SNaPshot detection method and application |
| CN108192966A (en) * | 2018-02-12 | 2018-06-22 | 北京天平永达科技发展有限公司 | For detecting the primer set of drug metabolic enzyme gene SNP site and its application |
| CN109536605A (en) * | 2019-01-31 | 2019-03-29 | 深圳市第二人民医院 | The PCR primer combination and application of statins adverse reaction genotype polymorphism |
| CN111304320A (en) * | 2020-04-17 | 2020-06-19 | 浙江迪谱诊断技术有限公司 | Primer sequence and kit for detecting safe medication gene of children |
| CN111304321A (en) * | 2020-04-17 | 2020-06-19 | 浙江迪谱诊断技术有限公司 | Primer combination sequence and kit for detecting child safety medication related gene mutation site |
| WO2021004010A1 (en) * | 2019-07-11 | 2021-01-14 | 南京先声医学检验有限公司 | Primer set, application, product and method for detecting snp loci related to medication for mental and neurological diseases |
-
2021
- 2021-02-22 CN CN202110199126.6A patent/CN113528637B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946759A (en) * | 2015-06-15 | 2015-09-30 | 广州金域医学检验中心有限公司 | CYP2C19*17 gene polymorphism SNaPshot detection method and application |
| CN108192966A (en) * | 2018-02-12 | 2018-06-22 | 北京天平永达科技发展有限公司 | For detecting the primer set of drug metabolic enzyme gene SNP site and its application |
| CN109536605A (en) * | 2019-01-31 | 2019-03-29 | 深圳市第二人民医院 | The PCR primer combination and application of statins adverse reaction genotype polymorphism |
| WO2021004010A1 (en) * | 2019-07-11 | 2021-01-14 | 南京先声医学检验有限公司 | Primer set, application, product and method for detecting snp loci related to medication for mental and neurological diseases |
| CN111304320A (en) * | 2020-04-17 | 2020-06-19 | 浙江迪谱诊断技术有限公司 | Primer sequence and kit for detecting safe medication gene of children |
| CN111304321A (en) * | 2020-04-17 | 2020-06-19 | 浙江迪谱诊断技术有限公司 | Primer combination sequence and kit for detecting child safety medication related gene mutation site |
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