CN112680510B - Primer pair and kit for detecting gene polymorphism related to etanercept drug administration - Google Patents
Primer pair and kit for detecting gene polymorphism related to etanercept drug administration Download PDFInfo
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Abstract
The invention relates to a primer pair and a kit for detecting gene polymorphism related to etanercept drug administration, and belongs to the technical field of in-vitro nucleic acid detection. The primer pair comprises amplification primers respectively aiming at SLCO1C1rs3794271, PTPRC rs10919563, STAT4rs7574865, HLA-Ers1264457, TRAF1rs3761847 and KLRD1rs2302489 alleles and GAPDH internal reference genes; the kit comprises a primer solution comprising the amplification primer. The kit provided by the invention has high sensitivity which can reach one ten thousandth, and the minimum detection limit is only 1-2 copies, so that the kit is particularly suitable for detecting low-content mutation samples; compared with a sequencing method, the detection result of the invention can be observed in real time, the product does not need gel electrophoresis detection, and the completely closed tube operation is performed, thereby effectively reducing the risk of PCR product pollution; in addition, the method has the advantages of high detection speed and suitability for high-flux sample detection.
Description
Technical Field
The invention relates to the technical field of in-vitro nucleic acid detection, in particular to a primer pair and a kit for detecting gene polymorphism related to etanercept drug.
Background
Rheumatic diseases refer to diseases involving joints and surrounding soft tissues, including muscles, ligaments, bursa and fascia, in which joint lesions are often accompanied by swelling and movement disorders in addition to pain, and some patients may also have impaired joint and visceral function, severely affecting the quality of life of the patient. The etiology and pathogenesis of rheumatic diseases are complicated, but autoimmune diseases are mainly pathogenesis, and inflammatory reaction is mainly pathological basis. Tumor Necrosis Factor (TNF) -alpha is taken as a pro-inflammatory factor, plays an important role in the pathogenesis of rheumatic diseases, and a TNF-alpha inhibitor can inhibit the cytokine to play an anti-inflammatory and immunosuppressive role, so that the advent of the TNF-alpha inhibitor brings the light to the treatment of a plurality of refractory rheumatic diseases.
Etanercept (Etanercept) is a TNF- α inhibitor that is currently very widely used worldwide, and has been used clinically abroad for more than ten years. Etanercept is a humanized TNF-alpha receptor-antibody fusion protein, which is formed by combining the extracellular segments of 2 type ii TNF-alpha receptors p75 and the Fc segment of human IgG1, and has a relative molecular weight of 150kDa and a half-life period in the human body of (102±30) h, similar to the endogenous soluble receptor structure. The medicine is highly compatible with soluble TNF-alpha in extracellular fluid and TNF-alpha on the surface of cell membrane, and has effects of controlling inflammation and blocking disease progress by inhibiting TNF-alpha activity, and is an anti-rheumatism biological agent. Etanercept can also bind to TNF-beta, which has similar biological activity to TNF-alpha and plays a role in the immune function of the body, especially in the formation of lymphoid organs and in the development of inflammation, but the relationship between the inhibition of etanercept by TNF-beta and clinical efficacy is not clear. Etanercept was approved by the U.S. Food and Drug Administration (FDA) in 11 1998 for the treatment of Rheumatoid Arthritis (RA), psoriatic arthritis (PsA), ankylosing Spondylitis (AS), and Juvenile Idiopathic Arthritis (JIA).
In recent years, pharmacogenomic studies have found that etanercept has a correlation with polymorphisms at multiple gene sites, as shown in the following table:
with respect to the above table, a person skilled in the art generally (without performing creative efforts) will select a specific genetic locus according to the respective actual requirements when developing a genetic test program, for example, when using real-time fluorescent Quantitative PCR (QPCR), may select the locus of TNF rs1800629 (the locus has a high evidence-based level), and when using a second-generation sequencing platform, may select the locus for simultaneous detection (although no prior art for all relevant loci of etanercept is currently found). The QPCR technology platform is applicable to clinical rapid detection reports (applicable to patients with short hospitalization period such as etanercept), but has general flux and detection sensitivity; the second generation sequencing platform is suitable for clinical high-throughput long-period detection project report (suitable for tumor long-term inpatients), but is not suitable for clinical conventional drug gene detection, and is especially not suitable for etanercept such patients with extremely short inpatient period, and the clinical actual requirements can not be met in both economy and periodicity.
Therefore, the present inventors have remarkable advantages in terms of detection sensitivity and cost by using ARMS PCR technology platform (ARMS is also called Allele-Specific PCR, allle Specific PCR, AS-PCR) (the "gold standard" currently used for gene detection is a PCR direct sequencing method, but the direct sequencing method has a complicated operation procedure, low sensitivity, and can detect only more than 20 to 30% of mutants, etc., compared with the direct sequencing method, ARMS-TaqMan method has higher sensitivity and lower cost).
In addition, the inventor selects six gene loci of SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs2302489 through creative work (by referring to documents, sorting the evidence-based grades of all gene loci and sorting the mutation frequency and other data of Chinese people and combining the pharmaceutical economy factors), so that the detection of the combined gene loci is more suitable for accurate administration of etanercept in Chinese patient groups (CHB+CHS) when etanercept administration is carried out on Chinese rheumatic disease patient groups.
The kit adopts an ARMS combined TaqMan fluorescent probe method: designing an allele-specific PCR amplification primer, adding a specific fluorescent probe which is an oligonucleotide while adding a pair of primers during PCR amplification, and respectively marking a reporting fluorescent group and a quenching fluorescent group at two ends of the probe. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme is used for enzyme digestion degradation of the probe to separate the reporting fluorescent group from the quenching fluorescent group, so that a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescence molecule is formed for each amplified DNA chain, and the accumulation of the fluorescence signal and the formation of PCR products are completely synchronous.
In the prior published literature, only parting detection of SLCO1C1 and STAT4 genes is carried out (the application is not aimed at etanercept), but the requirement of clinical etanercept drug related genotyping cannot be met when the kit is used alone, six gene detection is needed to be used in combination, and a plurality of operation problems and system crossing problems exist in clinical practice.
Therefore, the redesign optimization of the six-gene primer related to etanercept drug administration and the stability of the corresponding PCR system have a larger improvement space; after a large number of creative designs and experimental screening, the six gene detection is combined into a whole, so that the PCR reaction system of the kit is optimized, and the product performance of the kit is obviously improved.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a primer pair and a kit for detecting gene polymorphism of etanercept drug-related SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs2302489, which have the advantages of accurate detection result, simple operation procedure, high sensitivity, strong specificity and high sequencing speed and effectively meet the clinical examination requirements; the ARMS is combined with QPCR technology.
The invention provides a primer pair for detecting polymorphism of etanercept drug-related genes, wherein polymorphic sites detected by the etanercept drug-related genes are SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs2302489 respectively, and the primer pair comprises:
amplification primers for the SLCO1C1 rs3794271 allele were as follows:
amplification of SLCO1C1 rs3794271 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO: as shown in fig. 1, 5'-GGCCAATCTATCAAGGTAGGGC-3',
a probe, as set forth in SEQ ID NO: 5'-CTCCAAGTAGAAATTTAGGGTCCTGGGAATTCATCC-3' as shown in 2;
amplifying SLCO1C1 rs3794271 wild upstream primer as shown in SEQ ID NO:3, shown in the following: 5'-TGCCCAAACCACAGAACACC-3';
amplifying SLCO1C1 rs3794271 mutant upstream primer as shown in SEQ ID NO:4, as follows: 5'-TGCCCAAACCACAGAACACT-3';
amplification primers for PTPRC rs10919563 allele were as follows:
amplification of PTPRC rs10919563 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO:5, 5'-AGCTGAGTCATGGGTATAAGGG-3',
a probe, as set forth in SEQ ID NO:6, 5'-TATATGCATTTTATAGCAATTACTATAATTATTTA-3';
Amplifying the PTPRC rs10919563 wild type upstream primer as shown in SEQ ID NO: shown in fig. 7, 5'-CCATTATAAGGACATTCACGTTTCAC-3';
amplifying the PTPRC rs10919563 mutant upstream primer as shown in SEQ ID NO:8, 5'-CCATTATAAGGACATTCACGTTTCAT-3';
amplification primers for STAT4 rs7574865 allele were as follows:
amplification STAT4 rs7574865 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO: as shown in fig. 9, 5'-TGAAGGTAGTGGTGTGGAT-3',
a probe, as set forth in SEQ ID NO:10, 5'-ACATTTTGGTCACCAACTTTTCATACTTTTACTGC-3';
amplifying STAT4 rs7574865 wild type upstream primer as shown in SEQ ID NO:11, 5'-CCACTGAAATAAGATAACCACTAGTC-3';
amplifying STAT4 rs7574865 mutant upstream primers as shown in SEQ ID NO:12, 5'-CCACTGAAATAAGATAACCACTAGTA-3';
amplification primers for HLA-E rs1264457 allele were as follows:
amplification of HLA-E rs1264457 wild type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO:13, 5'-GAGAGTCTCAGGCGCCTT-3',
a probe, as set forth in SEQ ID NO:14, 5'-TCGGGCCCCAGCTCGCAGCCAT-3';
amplification of HLA-E rs1264457 wild-type upstream primer as shown in SEQ ID NO:15, as shown in: 5'-GCGGAGGAAGCGACC-3';
Amplifying HLA-E rs1264457 mutant upstream primers as shown in SEQ ID NO:16, as shown in: 5'-GCGGAGGAAGCGACT-3';
amplification primers for the TRAF1 rs3761847 allele were as follows:
amplification of TRAF1 rs3761847 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO:17, 5'-GATGGCAATACCTGCTTCACAG-3',
a probe, as set forth in SEQ ID NO:18, 5'-CCTCAATACCACCCTCTCTACCTGCT-3';
amplification of TRAF1 rs3761847 wild-type upstream primer as shown in SEQ ID NO:19, 5'-GTCCCTTCTCTCCCCTGCA-3';
amplifying TRAF1 rs3761847 mutant upstream primer as shown in SEQ ID NO:20, 5'-GTCCCTTCTCTCCCCTGCG-3';
amplification primers for the KLRD1 rs2302489 allele were as follows:
amplification of KLRD1 rs2302489 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO:21, 5'-GTAGAGAAGGCACGATGTGTAC-3',
a probe, as set forth in SEQ ID NO:22, 5'-TTTGCTAAATTTCTTCATACTCAACTTTCAGATTC-3';
amplifying a KLRD1 rs2302489 wild type upstream primer as shown in SEQ ID NO:23, 5'-CATTTAAATACACAATTTTTCATTCTCGA-3';
amplifying the KLRD1 rs2302489 mutant upstream primer as shown in SEQ ID NO:24, 5'-CATTTAAATACACAATTTTTCATTCTCGT-3';
Amplification primers for the GAPDH reference gene were as follows:
an upstream primer for amplifying GAPDH gene, as shown in SEQ ID NO:25, 5'-ATCCTGGGCTACACTGAGCAC-3';
amplifying the downstream primer of GAPDH gene as shown in SEQ ID NO:26, 5'-CTCAGTGTAGCCCAGGATGCCCTT-3';
probes for amplifying the GAPDH gene, as set forth in SEQ ID NO:27, 5'-AGGTGGTCTCCTCTGACTTCAA-3'.
The probe labels of the primer pair described above include: SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 14. SEQ ID NO:18 and SEQ ID NO:22 is marked by a fluorescence reporter group (FAM); the 5' -end of the probe shown in SEQ ID NO. 27 is marked by adopting a fluorescence reporter group (JOE); SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 14. SEQ ID NO: 18. SEQ ID NO:22 and SEQ ID NO. 27 are labeled with a fluorescence quenching group (TAMRA).
The invention also provides a kit for detecting polymorphism of etanercept drug-related genes, wherein polymorphic sites detected by the etanercept drug-related genes are SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-Ers1264457, TRAF1 rs3761847 and KLRD1 rs2302489 respectively, and the kit comprises:
Primer liquid 1, wherein the primer liquid 1 contains a nucleotide sequence shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 2, wherein the primer liquid 2 contains a nucleotide sequence shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 3, wherein the primer liquid 3 contains a nucleotide sequence shown as SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 4, wherein the primer liquid 4 contains a nucleotide sequence shown in SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 5, wherein the primer liquid 5 contains a nucleotide sequence shown as SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 6, wherein the primer liquid 6 contains a nucleotide sequence shown in SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 7, wherein the primer liquid 7 contains a nucleotide sequence shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
Primer liquid 8, wherein the primer liquid 8 contains a nucleotide sequence shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 9, wherein the primer liquid 9 contains a nucleotide sequence shown in SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 10, wherein the primer liquid 10 contains a nucleotide sequence shown in SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 11, wherein the primer liquid 11 contains a nucleotide sequence shown in SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 12, wherein the primer liquid 12 contains a nucleotide sequence shown in SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe; .
In a preferred embodiment of the kit for detecting gene polymorphism related to etanercept administration provided by the present invention, the kit further comprises: the positive control is a nucleotide sequence inserted with SEQ ID NO:1 and SEQ ID NO:3 amplification product, inserted with SEQ ID NO:1 and SEQ ID NO:4 amplification product, inserted with SEQ ID NO:5 and SEQ ID NO:7 amplification product, inserted with SEQ ID NO:5 and SEQ ID NO:8 amplification product, inserted with SEQ ID NO:9 and SEQ ID NO:11, inserted with SEQ ID NO:9 and SEQ ID NO:12 amplification product, inserted with SEQ ID NO:13 and SEQ ID NO:15 amplification product, inserted with SEQ ID NO:13 and SEQ ID NO:16 amplification product, inserted with SEQ ID NO:17 and SEQ ID NO:19, inserted with SEQ ID NO:17 and SEQ ID NO:20 amplification product, inserted with SEQ ID NO:21 and SEQ ID NO:23 amplification product, inserted with SEQ ID NO:21 and SEQ ID NO:24 amplification product, 13 plasmids inserted with SEQ ID NO. 25 and SEQ ID NO. 26 amplification product;
Wherein the plasmid vector is pMD18-T plasmid.
In a preferred embodiment of the kit for detecting gene polymorphism related to etanercept drug delivery provided by the invention, the quantitative ratio of the SLCO1C1 rs3794271 wild homozygote plasmid, the SLCO1C1 rs3794271 mutant homozygote plasmid, the PTPRC rs10919563 wild homozygote plasmid, the PTPRC rs10919563 mutant homozygote plasmid, the STAT4 rs7574865 wild homozygote plasmid, the STAT4 rs7574865 mutant homozygote plasmid, the HLA-E rs1264457 wild homozygote plasmid, the HLA-E rs1264457 mutant homozygote plasmid, the TRAF1 rs3761847 wild homozygote plasmid, the TRAF1 rs3761847 mutant homozygote plasmid, the KLRD1 rs2302489 wild homozygote plasmid, the KLRD1 rs2302489 mutant homozygote plasmid and the internal reference GAPDH plasmid in the positive control plasmid mixture is 1:1:1:1:1:1:1:1:1:1:1:1:2.
in a preferred embodiment of the kit for detecting gene polymorphism related to etanercept administration provided by the present invention, the kit further comprises a blank, and the blank is sterilized purified water.
Compared with the prior art, the primer pair and the kit for detecting the gene polymorphism related to etanercept drug have the following beneficial effects:
1. The primer pair with high sensitivity and good specificity and the kit thereof are designed and optimized by combining ARMS with QPCR technology, so that the kit has the advantages of accurate quality, high sensitivity and strong specificity when detecting SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs2302489 gene polymorphism; in addition, the method has the advantages of simple sample treatment, simple sequencing step, high sequencing speed, one-time on-machine reaction completion in one hour, direct detection site fluorescence curve giving and visual result;
2. the primer pair with high sensitivity and good specificity and the kit thereof are designed and optimized by combining ARMS with QPCR technology, so that when the kit detects SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs2302489 gene polymorphism, the kit can monitor the reaction progress in real time, has short reaction time, can be used for fluorescent quantitative PCR instrument by simple treatment of PCR products, is easy to operate and high-throughput sample detection, has higher sensitivity than a gold standard method, namely a capillary electrophoresis sequencing method, and is more suitable for mutation analysis;
3. by arranging the blank reference substance and the positive reference substance in the kit, the kit can better ensure the accuracy of detection results when detecting SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs2302489 gene polymorphism.
Drawings
FIG. 1 is a graph of fluorescent amplification of a clinical sample SLCO1C1 rs3794271 wild type;
FIG. 2 is a graph of fluorescence amplification of a clinical sample SLCO1C1 rs3794271 mutant heterozygote;
FIG. 3 is a graph of fluorescent amplification of a clinical sample SLCO1C1 rs3794271 mutant homozygosity;
FIG. 4 is a fluorescent amplification plot of a clinical sample SLCO1C1 rs3794271 positive control;
FIG. 5 is a graph of fluorescence amplification of a clinical sample SLCO1C1 rs3794271 blank;
FIG. 6 is a graph of fluorescent amplification of a clinical sample PTPRC rs10919563 wild type;
FIG. 7 is a graph of fluorescence amplification of a clinical sample PTPRC rs10919563 mutant heterozygotes;
FIG. 8 is a graph of fluorescence amplification of a clinical sample PTPRC rs10919563 mutant homozygosity;
FIG. 9 is a graph of fluorescence amplification of a positive control of PTPRC rs10919563 of a clinical sample;
FIG. 10 is a graph of fluorescence amplification of a clinical sample PTPRC rs10919563 blank;
FIG. 11 is a graph of fluorescent amplification of wild-type STAT4 rs7574865 in clinical samples;
FIG. 12 is a fluorescent amplification plot of a clinical sample STAT4 rs7574865 mutant heterozygote;
FIG. 13 is a graph of fluorescence amplification of a clinical sample STAT4 rs7574865 mutant homozygosity;
FIG. 14 is a fluorescent amplification plot of a clinical sample STAT4 rs7574865 positive control;
FIG. 15 is a fluorescent amplification plot of a clinical sample STAT4 rs7574865 blank;
FIG. 16 is a graph of fluorescent amplification of wild type HLA-E rs1264457 as a clinical sample;
FIG. 17 is a graph of fluorescence amplification of HLA-E rs1264457 mutant heterozygotes of clinical samples;
FIG. 18 is a graph of fluorescence amplification of HLA-E rs1264457 mutant homozygosity for clinical samples;
FIG. 19 is a fluorescent amplification plot of a clinical sample HLA-E rs1264457 positive control;
FIG. 20 is a fluorescent amplification plot of a clinical sample HLA-E rs1264457 blank;
FIG. 21 is a graph of fluorescence amplification of a wild type TRAF1 rs3761847 sample;
FIG. 22 is a graph of fluorescence amplification of a heterozygous TRAF1 rs3761847 mutation;
FIG. 23 is a graph of fluorescence amplification of a homozygous version of the TRAF1 rs3761847 mutation in a clinical sample;
FIG. 24 is a graph of fluorescence amplification of a TRAF1 rs3761847 positive control for clinical samples;
FIG. 25 is a graph of fluorescence amplification of a TRAF1 rs3761847 blank for clinical samples;
FIG. 26 is a graph of fluorescent amplification of wild type KLRD1 rs2302489 of the clinical samples;
FIG. 27 is a graph of fluorescence amplification of a clinical sample KLRD1 rs2302489 mutant heterozygote;
FIG. 28 is a graph of fluorescence amplification of a clinical sample KLRD1 rs2302489 mutant homozygosity;
FIG. 29 is a fluorescent amplification plot of a positive control of clinical sample KLRD1 rs 2302489;
FIG. 30 is a graph of fluorescent amplification of a clinical sample KLRD1 rs2302489 blank control;
FIGS. 31 to 32 are graphs of fluorescence amplification for SLCO1C1 rs3794271 sets of designed primers; wherein the results of FIG. 31 are inaccurate, and only the results of FIG. 32 are truly reliable;
FIGS. 33 to 34 are graphs of fluorescence amplification for PTPRC rs10919563 sets of designed primers; wherein the results of FIG. 33 are inaccurate, only the results of FIG. 34 are truly reliable;
FIGS. 35 to 36 are graphs of fluorescence amplification of STAT4 rs7574865 sets of designed primers; wherein the results of fig. 35 are inaccurate, and only the results of fig. 36 are truly reliable;
FIGS. 37 to 38 are graphs of fluorescence amplification of HLA-E rs1264457 sets of designed primers; wherein the results of FIG. 37 are inaccurate, only the results of FIG. 38 are truly reliable;
FIGS. 39 to 40 are graphs of fluorescence amplification for TRAF1 rs3761847 sets of designed primers; wherein the results of FIG. 39 are inaccurate, only the results of FIG. 40 are truly reliable;
FIGS. 41-42 are graphs of fluorescence amplification for KLRD1 rs2302489 sets of designed primers; wherein the results of fig. 41 are inaccurate and only the results of fig. 42 are truly reliable.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1: preparation of kit (30 test/box)
1. Design and Synthesis of primers and probes
Specific mutation sites rs3794271, rs10919563, rs7574865, rs1264457, rs3761847 and rs2302489 are selected aiming at human SLCO1C1 gene, PTPRC gene, STAT4 gene, HLA-E gene, TRAF1 gene and KLRD1 gene, the selected primers and probes are designed in the mutation sites and nearby conserved regions, SNP (SNP retrieval of target gene sequence is carried out through an online NCBI website) is avoided in the Primer binding region, primer Blast is carried out through the online NCBI website, allele-specific PCR amplification primers are designed, specific amplification of the Primer pair is confirmed, and when the 3 '-terminal base of the PCR primers is mismatched with template DNA thereof, the amplification efficiency is drastically reduced, and PCR amplification signals can only appear when the 3' -base of the primers are paired with the template. The probe is positioned in a region between a pair of primers, and the binding region is prevented from SNP; wherein the amplification primer and the fluorescent probe are purified by PAGE and then HPLC, wherein the target probes SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 10, SEQ ID NO. 14, SEQ ID NO. 18 and SEQ ID NO. 22 are marked by a fluorescent reporter group (FAM), the 5 'end of the reference probe SEQ ID NO. 27 is marked by a fluorescent reporter group (JOE), and the 3' end is marked by a fluorescent quenching group (TAMRA).
TABLE 1 mutation sites and types
| Sequence number | Mutation site | Base change |
| 1 | SLCO1C1 rs3794271 | G>A |
| 2 | PTPRC rs10919563 | A>G |
| 3 | STAT4 rs7574865 | G>T |
| 4 | HLA-E rs1264457 | G>T |
| 5 | TRAF1 rs3761847 | A>G |
| 6 | KLRD1 rs2302489 | A>T |
The amplified sequences are shown in Table 2:
TABLE 2 specific amplification primers and primer sequences
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2. Control selection
The positive control is a nucleotide sequence inserted with SEQ ID NO:1 and SEQ ID NO:3 amplification product, inserted with SEQ ID NO:1 and SEQ ID NO:4 amplification product, inserted with SEQ ID NO:5 and SEQ ID NO:7 amplification product, inserted with SEQ ID NO:5 and SEQ ID NO:8 amplification product, inserted with SEQ ID NO:9 and SEQ ID NO:11, inserted with SEQ ID NO:9 and SEQ ID NO:12 amplification product, inserted with SEQ ID NO:13 and SEQ ID NO:15 amplification product, inserted with SEQ ID NO:13 and SEQ ID NO:16 amplification product, inserted with SEQ ID NO:17 and SEQ ID NO:19, inserted with SEQ ID NO:17 and SEQ ID NO:20 amplification product, inserted with SEQ ID NO:21 and SEQ ID NO:23 amplification product, inserted with SEQ ID NO:21 and SEQ ID NO:24 amplification product, 13 plasmids inserted with SEQ ID NO. 25 and SEQ ID NO. 26 amplification product; the number ratio of SLCO1C1 rs3794271 wild homozygote plasmid, SLCO1C1 rs3794271 mutant homozygote plasmid, PTPRC rs10919563 wild homozygote plasmid, PTPRC rs10919563 mutant homozygote plasmid, STAT4 rs7574865 wild homozygote plasmid, STAT4 rs7574865 mutant homozygote plasmid, HLA-E rs1264457 wild homozygote plasmid, HLA-E rs1264457 mutant homozygote plasmid, TRAF1 rs3761847 wild homozygote plasmid, TRAF1 rs3761847 mutant homozygote plasmid, KLRD1 rs2302489 wild homozygote plasmid, KLRD1 rs2302489 mutant homozygote plasmid and internal reference GAPDH plasmid in the plasmid mixture is 1:1:1:1:1:1:1:1:1:1:1:1:2. wherein the plasmid vector is pMD18-T plasmid.
The blank is sterilized purified water.
3.PCR premix composition
TABLE 3 PCR premix composition
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| 2×Fast TaqMan Mixture | 150 | 4500 |
| 50×Low ROX | 6 | 180 |
| Total amount of | 156 | 4680 |
4. Composition of primer liquid 1
TABLE 4 composition of primer liquid 1
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| SLCO1C1-WT-F(10uM) | 0.5 | 15 |
| SLCO1C1-R(10uM) | 0.5 | 15 |
| SLCO1C1-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
5. Composition of primer liquid 2
TABLE 5 composition of primer liquid 2
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| SLCO1C1-MT-F(10uM) | 0.5 | 15 |
| SLCO1C1-R(10uM) | 0.5 | 15 |
| SLCO1C1-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
6. Composition of primer liquid 3
TABLE 6 composition of primer liquid 3
7. Composition of primer liquid 4
TABLE 7 composition of primer solution 4
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| PTPRC-MT-F(10uM) | 0.5 | 15 |
| PTPRC-R(10uM) | 0.5 | 15 |
| PTPRC-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
8. Composition of primer liquid 5
TABLE 8 composition of primer solution 5
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| STAT4-WT-F(10uM) | 0.5 | 15 |
| STAT4-R(10uM) | 0.5 | 15 |
| STAT4-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
9. Composition of primer liquid 6
TABLE 9 composition of primer solution 6
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| STAT4-MT-F(10uM) | 0.5 | 15 |
| STAT4-R(10uM) | 0.5 | 15 |
| STAT4-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
10. Composition of primer liquid 7
TABLE 10 composition of primer solution 7
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| HLA-E-WT-F(10uM) | 0.5 | 15 |
| HLA-E-R(10uM) | 0.5 | 15 |
| HLA-E-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
11. Composition of primer liquid 8
TABLE 11 composition of primer solution 8
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12. Composition of primer liquid 9
TABLE 12 composition of primer solution 9
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| TRAF1-WT-F(10uM) | 0.5 | 15 |
| TRAF1-R(10uM) | 0.5 | 15 |
| TRAF1-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
13. Composition of primer liquid 10
TABLE 13 composition of primer solution 10
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| TRAF1-MT-F(10uM) | 0.5 | 15 |
| TRAF1-R(10uM) | 0.5 | 15 |
| TRAF1-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
14. Composition of primer liquid 11
TABLE 14 composition of primer solution 11
15. Composition of primer solution 12
TABLE 15 composition of primer solution 12
| Raw material name | Testing Single use amount (μL) | 30 test/box (mu L) |
| KLRD1-MT-F(10uM) | 0.5 | 15 |
| KLRD1-R(10uM) | 0.5 | 15 |
| KLRD1-P(10uM) | 0.5 | 15 |
| Ref2-F(10uM) | 0.5 | 15 |
| Ref2-R(10uM) | 0.5 | 15 |
| Ref2-P(10uM) | 0.5 | 15 |
| Total amount of | 3 | 90 |
16. Composition of positive control
TABLE 16 composition of positive control
| Raw material name | Volume (mu L) | 30 test/box (mu L) |
| 13 plasmid mixtures | 6 | 180 |
17. Composition of blank control
TABLE 17 composition of blank
| Raw material name | Volume (mu L) | 30 test/box (mu L) |
| Sterilizing purified water | 108 | 3240 |
Example 2: use of a kit
1. Sample detection
Preparing a system according to the template number: and (3) taking a PCR reaction tube, adding corresponding primer liquid, PCR premix liquid and sterilized purified water, adding sample DNA, sterilizing and purifying or positive reference substance as a template, and forming a PCR reaction system. PCR amplification was performed according to the PCR reaction procedure.
Each site has two reaction solutions of Wild (WT) and Mutation (MT), and 6 sites are 12 reaction solutions. The reaction solutions were prepared as follows:
TABLE 18 preparation of the reaction solutions
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The reaction procedure of the system is as follows:
TABLE 19 PCR reaction procedure
2. ABI7500 fluorescent quantitative PCR
Pressing the right start key starts ABI 7500. After the machine is started, a power indicator light at the left end of the machine is long-lighted. And opening the bin gate, putting the prepared reagent into the bin, and recording the self-placing position.
1) Double clicking on the "7500Software v2.0.5" icon opens the software. Clicking "OK" in the pop-up window goes into the program.
2) Clicking "New Experiment" and sequentially selecting "7500 (96 wells)", "Quantitain-standby Curve", "in the pop-up" Experiment Properties "interface"The Reagents "icon lightens it.
3) Clicking "Plate Setup", selecting "FAM" in the drop-down box in the "Reporter" column, selecting "TAMRA" in the drop-down box in the "quantiser" column, clicking "Add New Target", selecting "JOE" in the drop-down box in the "Reporter" column, and selecting "TAMRA" in the drop-down box in the "quantiser" column; repeatedly clicking the "Add New Sample" causes a sufficient number of boxes to pop up under the "Sample Name" and the unique number of each Sample is entered in these boxes.
4) Clicking "Assign Targets and Samples", selecting "ROX" in the drop-down selection box in the column "Passive Reference", selecting the hole site where the reagent is placed, confirming that it corresponds to the instrument, clicking "v" in the box in the column "Assign sample(s) to the selected wells", clicking "v" in the box in the column "Assign target(s) to the selected wells", and clicking the corresponding icon to lighten it: "U" (sample to be tested), "S" (positive control), "N" (blank control).
5) Setting reaction conditions: clicking "Run Method" into the reaction condition setting panel, formulating the required amplification program, setting 95 ℃ for 30 seconds in the Holding Stage column, moving the cursor to the Holding Stage column, setting 95 ℃ for 10 seconds in section 1 "Step 1", setting 60 ℃ for 30 seconds in Step 2", clicking the fluorescence acquisition icon in the section to lighten, inputting 50 in the Number of Cycles column, and inputting 25 in the Reaction Volume Per Well column.
6) After confirming no errors, clicking "START R …" to number the current PCR and store the number in a corresponding folder, clicking to confirm, and starting operation. There is a period of preheating after the start, and the "IN USE" indicator lights flash when the cycle is formally started.
7) After running, the bin gate is opened, the product is poured into the garbage can, and the instrument use record is filled.
3. Result judgment
After the reaction is completed, the baseline and the threshold are automatically adjusted. After setting, the Ct value of each sample can be obtained from the Ct of the View Well Table window by clicking the analysis button.
4. Quality control standard
Positive control: the FAM and JOE channels have obvious S-shaped amplification curves, and the curve Ct is less than or equal to 35;
blank control: the FAM and JOE channels have no obvious S-shaped amplification curve; or a curve Ct > 35;
All the conditions must be met in the same experiment, otherwise, the experimental result is invalid.
5. Reporting the results:
positive judgment value or reference section
(1) Positive results: has obvious S-shaped amplification curve with Ct less than or equal to 35
(2) Negative results: no obvious S-shaped amplification curve; or curve Ct > 35
[ analysis of detection results ]
The sample detection hole JOE channel is positive and wet, and the sample result is judged according to the following table to determine the genotype of the sample.
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Fig. 1 shows a wild type of SLCO1C1 rs3794271 in the clinical test results, fig. 2 shows a mutant heterozygous type of SLCO1C1 rs3794271 in the clinical test results, fig. 3 shows a mutant homozygous type of SLCO1C1 rs3794271 in the clinical test results, and fig. 4 and 5 show fluorescence graphs of a positive control and a blank control in the clinical test results, respectively. In FIG. 1, the FAM curve Ct in the SLCO1C1 (WT) reaction solution is less than or equal to 35, and the FAM in the SLCO1C1 (MT) reaction solution has no obvious S-type curve, and is judged to be the SLCO1C1 rs3794271 wild type. FIG. 2 shows that the FAM curve Ct in the SLCO1C1 (WT) reaction solution is not more than 35, and the FAM curve Ct in the SLCO1C1 (MT) reaction solution is not more than 35, and the SLCO1C1 rs3794271 heterozygous is determined. In FIG. 3, FAM in SLCO1C1 (WT) reaction solution has no obvious S-shaped curve, and FAM curve Ct in SLCO1C1 (MT) reaction solution is less than or equal to 35, and is judged to be SLCO1C1 rs3794271 mutant. In FIG. 4, the FAM curves of SLCO1C1 (WT) reaction solution and SLCO1C1 (MT) reaction solution are equal to or less than 35 Ct and the JOE curves are equal to or less than 35 Ct. FIG. 5 FAM of SLCO1C1 (WT) reaction solution and SLCO1C1 (MT) reaction solution has no obvious S-shaped curve, and JOE has no obvious S-shaped curve.
Fig. 6 shows the wild type of PTPRC rs10919563 in the clinical test result, fig. 7 shows the mutant heterozygous type of PTPRC rs10919563 in the clinical test result, fig. 8 shows the mutant homozygous type of PTPRC rs10919563 in the clinical test result, and fig. 9 and 10 show the fluorescence curves of the positive control and the blank control in the clinical test result, respectively. FIG. 6 shows that the curve Ct of FAM in PTPRC (WT) reaction solution is less than or equal to 35, and that FAM in PTPRC (MT) reaction solution has no obvious S-type curve, and it is determined that PTPRC rs10919563 is wild type. FIG. 7 shows that the FAM curve Ct in the PTPRC (WT) reaction solution is not more than 35, and the FAM curve Ct in the PTPRC (MT) reaction solution is not more than 35, and it is judged that PTPRC rs10919563 is heterozygous. FIG. 8 shows that the FAM in the PTPRC (WT) reaction solution has no obvious S-shaped curve, and the FAM curve Ct in the PTPRC (MT) reaction solution is less than or equal to 35, and is judged to be PTPRC rs10919563 mutant. FIG. 9 shows that the FAM curve of the PTPRC (WT) reaction solution and the PTPRC (MT) reaction solution have Ct of 35 or less and the JOE curve has Ct of 35 or less. FIG. 10 shows that the FAM and PTPRC (WT) reactions have no apparent S-shape and the JOE has no apparent S-shape.
Fig. 11 shows the wild type of STAT4 rs7574865 in the clinical test results, fig. 12 shows the mutant heterozygous type of STAT4 rs7574865 in the clinical test results, fig. 13 shows the mutant homozygous type of STAT4 rs7574865 in the clinical test results, and fig. 14 and 15 show the fluorescence graphs of the positive control and the blank control in the clinical test results, respectively. FIG. 11 shows that the FAM curve Ct in the STAT4 (WT) reaction solution is less than or equal to 35, and that the FAM in the STAT4 (MT) reaction solution has no obvious S-type curve, and is judged to be the wild type STAT4 rs 7574865. FIG. 12 shows that the FAM curve Ct in the STAT4 (WT) reaction solution is less than or equal to 35, and the FAM curve Ct in the STAT4 (MT) reaction solution is less than or equal to 35, and the result is judged to be STAT4 rs7574865 heterozygous. FIG. 13 shows that FAM in STAT4 (WT) reaction solution has no obvious S-type curve, and that FAM in STAT4 (MT) reaction solution has a Ct of 35 or less, and is judged to be a mutant form of STAT4 rs 7574865. FIG. 14 shows that the FAM curves of STAT4 (WT) reaction solution and STAT4 (MT) reaction solution are equal to or less than 35 in Ct and equal to or less than 35 in JOE curve. FIG. 15 FAM of STAT4 (WT) and STAT4 (MT) reactions showed no significant S-shape, nor JOE.
FIG. 16 shows the wild type HLA-E rs1264457 in the clinical test results, FIG. 17 shows the mutant heterozygous type HLA-E rs1264457 in the clinical test results, FIG. 18 shows the mutant homozygous type HLA-E rs1264457 in the clinical test results, and FIGS. 19 and 20 show the fluorescence profiles of the positive control and the blank control in the clinical test results, respectively. FIG. 16 shows that the curve Ct of FAM in HLA-E (WT) reaction solution is less than or equal to 35, and that FAM in HLA-E (MT) reaction solution has no obvious S-type curve, and is judged to be HLA-E rs1264457 wild type. FIG. 17 shows that the FAM curve Ct in the HLA-E (WT) reaction solution is less than or equal to 35, and the FAM curve Ct in the HLA-E (MT) reaction solution is less than or equal to 35, and the HLA-E rs1264457 heterozygous type is determined. FIG. 18 shows that FAM in HLA-E (WT) reaction solution has no obvious S-shaped curve, and that FAM in HLA-E (MT) reaction solution has a Ct of 35 or less, and is judged to be HLA-E rs1264457 mutant. FIG. 19 shows that the FAM curve of the HLA-E (WT) reaction solution and the HLA-E (MT) reaction solution have Ct of 35 or less and the JOE curve has Ct of 35 or less. FIG. 20 FAM and JOE in HLA-E (WT) and HLA-E (MT) reactions showed no obvious S-shape.
Fig. 21 shows the wild type of TRAF1 rs3761847 in the clinical test results, fig. 22 shows the mutant heterozygous type of TRAF1 rs3761847 in the clinical test results, fig. 23 shows the mutant homozygous type of TRAF1 rs3761847 in the clinical test results, and fig. 24 and fig. 25 show the fluorescence graphs of the positive control and the blank control in the clinical test results, respectively. FIG. 21 shows that the curve Ct of FAM in TRAF1 (WT) reaction solution is less than or equal to 35, and that FAM in TRAF1 (MT) reaction solution has no obvious S-shaped curve, and is judged to be TRAF1 rs3761847 wild type. FIG. 22 shows that the FAM curve Ct in the TRAF1 (WT) reaction solution is less than or equal to 35, and the FAM curve Ct in the TRAF1 (MT) reaction solution is less than or equal to 35, and the TRAF1 rs3761847 heterozygous type is determined. FIG. 23 shows that FAM in TRAF1 (WT) reaction solution has no obvious S-shaped curve, and that FAM in TRAF1 (MT) reaction solution has a Ct of 35 or less, and is determined to be TRAF1 rs3761847 mutant. FIG. 24 shows that the FAM curves of TRAF1 (WT) reaction solution and TRAF1 (MT) reaction solution are equal to or less than 35 in Ct, and the JOE curves are equal to or less than 35 in Ct. FIG. 25 FAM and JOE reactions showed no significant S-shape curves for TRAF1 (WT) and TRAF1 (MT) reactions.
Fig. 26 shows the wild type of KLRD1 rs2302489 in the clinical test results, fig. 27 shows the mutant heterozygous type of KLRD1 rs2302489 in the clinical test results, fig. 28 shows the mutant homozygous type of KLRD1 rs2302489 in the clinical test results, and fig. 29 and fig. 30 show the fluorescence profiles of the positive control and the blank control in the clinical test results, respectively. FIG. 26 shows that the FAM curve Ct in KLRD1 (WT) reaction solution is less than or equal to 35, and that the FAM in KLRD1 (MT) reaction solution has no obvious S-type curve, and it is judged that KLRD1 rs2302489 is wild type. FIG. 27 shows that the FAM curve Ct in the KLRD1 (WT) reaction solution is not more than 35, and that in the KLRD1 (MT) reaction solution is not more than 35, and that it is judged as KLRD1 rs2302489 heterozygous. FIG. 28 shows that the FAM in the KLRD1 (WT) reaction solution has no obvious S-shaped curve, and that the FAM curve Ct in the KLRD1 (MT) reaction solution is less than or equal to 35, and is judged to be a KLRD1 rs2302489 mutant type. FIG. 29 shows that the FAM curve of KLRD1 (WT) reaction solution and KLRD1 (MT) reaction solution have Ct.ltoreq.35 and the JOE curve has Ct.ltoreq.35. FIG. 30 shows that the FAM of KLRD1 (WT) reaction solution and KLRD1 (MT) reaction solution have no obvious S-shaped curve, and JOE has no obvious S-shaped curve.
FIGS. 31 to 32 are graphs of fluorescence amplification for SLCO1C1 rs3794271 sets of designed primers; wherein the results of fig. 31 are inaccurate and only the results of fig. 32 are truly reliable.
FIGS. 33 to 34 are graphs of fluorescence amplification for PTPRC rs10919563 sets of designed primers; wherein the results of FIG. 33 are inaccurate, only the results of FIG. 34 are truly reliable;
FIGS. 35 to 36 are graphs of fluorescence amplification of STAT4 rs7574865 sets of designed primers; wherein the results of fig. 35 are inaccurate, and only the results of fig. 36 are truly reliable;
FIGS. 37 to 38 are graphs of fluorescence amplification of HLA-E rs1264457 sets of designed primers; wherein the results of FIG. 37 are inaccurate, only the results of FIG. 38 are truly reliable;
FIGS. 39 to 40 are graphs of fluorescence amplification for TRAF1 rs3761847 sets of designed primers; wherein the results of FIG. 39 are inaccurate, only the results of FIG. 40 are truly reliable;
FIGS. 41-42 are graphs of fluorescence amplification for KLRD1 rs2302489 sets of designed primers; wherein the results of fig. 41 are inaccurate and only the results of fig. 42 are truly reliable.
Example 3: large sample validation of kits
1. The relevant components of the kit were prepared according to the preparation method shown in example 1 and stored at-20℃for further use.
2. 30 known whole blood samples are taken, DNA of the samples is extracted by using a 'nucleic acid extraction or purification reagent' (record number: xiang Chang mechanical device 20160167), the concentration of the DNA samples is detected by using a nucleic acid protein tester, and 30 samples A260/280 are all between 1.6 and 2.0.
3. DNA loading was performed and detected by an ABI 7500 fluorescent quantitative PCR apparatus according to the procedure shown in example 2.
4. According to the interpretation standard shown in the example 2, the results are interpreted and counted (the detection result coincidence rate is counted), and the sample coincidence rate is 100%; specific information of the detection results is shown in the following table:
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the primer pair and the kit for detecting the gene polymorphism related to etanercept drug use have the following beneficial effects:
1. the primer pair with high sensitivity and good specificity and the kit thereof are redesigned and optimized by utilizing ARMS combined with QPCR technology, so that the kit has the advantages of accurate quality, high sensitivity and strong specificity when detecting SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs2302489 gene polymorphism; in addition, the method has the advantages of simple sample treatment, simple sequencing step, high sequencing speed, one-time on-machine reaction completion in one hour, direct detection site fluorescence curve giving and visual result;
2. the primer pair with high sensitivity and good specificity and the kit thereof are redesigned and optimized by utilizing ARMS combined with QPCR technology, so that when the kit detects SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs2302489 gene polymorphism, the reaction progress can be monitored in real time, the reaction time is short, the PCR product can be simply processed, and the fluorescent quantitative PCR instrument can be used for detecting samples with simple operation and high flux, and compared with a gold standard method, namely, the sensitivity of a capillary electrophoresis sequencing method is higher, the kit is more suitable for mutation analysis;
3. By arranging the blank control and the positive control in the kit, the accuracy of detection results can be better ensured when the kit detects the gene polymorphism of VSLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-E rs1264457, TRAF1 rs3761847 and KLRD1 rs 2302489.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent processes or direct or indirect applications in other related arts using the present invention are included in the scope of the present invention.
Sequence listing
<110> Nanchang Haoshishi medical laboratory Co., ltd
<120> primer set and kit for detecting polymorphism of gene related to etanercept drug administration
<160> 27
<170> SIPOSequenceListing 1.0
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ggccaatcta tcaaggtagg gc 22
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<213> Artificial sequence (Artificial Sequence)
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ctccaagtag aaatttaggg tcctgggaat tcatcc 36
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<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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tgcccaaacc acagaacacc 20
<210> 4
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tgcccaaacc acagaacact 20
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
agctgagtca tgggtataag gg 22
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<213> Artificial sequence (Artificial Sequence)
<400> 6
tatatgcatt ttatagcaat tactataatt attta 35
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<213> Artificial sequence (Artificial Sequence)
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ccattataag gacattcacg tttcac 26
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<400> 8
ccattataag gacattcacg tttcat 26
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<400> 9
tgaaggtagt ggtgtggat 19
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<213> Artificial sequence (Artificial Sequence)
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acattttggt caccaacttt tcatactttt actgc 35
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<213> Artificial sequence (Artificial Sequence)
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ccactgaaat aagataacca ctagtc 26
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ccactgaaat aagataacca ctagta 26
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<213> Artificial sequence (Artificial Sequence)
<400> 14
tcgggcccca gctcgcagcc at 22
<210> 15
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gcggaggaag cgacc 15
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<213> Artificial sequence (Artificial Sequence)
<400> 16
gcggaggaag cgact 15
<210> 17
<211> 22
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<213> Artificial sequence (Artificial Sequence)
<400> 17
gatggcaata cctgcttcac ag 22
<210> 18
<211> 26
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<213> Artificial sequence (Artificial Sequence)
<400> 18
cctcaatacc accctctcta cctgct 26
<210> 19
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
gtcccttctc tcccctgca 19
<210> 20
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gtcccttctc tcccctgcg 19
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
gtagagaagg cacgatgtgt ac 22
<210> 22
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
tttgctaaat ttcttcatac tcaactttca gattc 35
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catttaaata cacaattttt cattctcga 29
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<213> Artificial sequence (Artificial Sequence)
<400> 24
catttaaata cacaattttt cattctcgt 29
<210> 25
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
atcctgggct acactgagca c 21
<210> 26
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ctcagtgtag cccaggatgc cctt 24
<210> 27
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aggtggtctc ctctgacttc aa 22
Claims (6)
1. A primer pair for detecting polymorphism of etanercept drug-related genes, wherein polymorphic sites detected by the etanercept drug-related genes are respectively SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-Ers1264457, TRAF1 rs3761847 and KLRD1 rs2302489, the primer pair comprising:
amplification primers for the SLCO1C1 rs3794271 allele were as follows:
Amplification of SLCO1C1 rs3794271 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO: as shown in fig. 1, 5'-GGCCAATCTATCAAGGTAGGGC-3',
a probe, as set forth in SEQ ID NO:2 'ctgcaagtaattaggtcctgggaatttcatcc-3';
amplifying SLCO1C1 rs3794271 wild upstream primer as shown in SEQ ID NO:3, shown in the following: 5'-TGCCCAAACCACAGAACACC-3';
amplifying SLCO1C1 rs3794271 mutant upstream primer as shown in SEQ ID NO:4, as follows: 5'-TGCCCAAACCACAGAACACT-3';
amplification primers for PTPRC rs10919563 allele were as follows:
amplification of PTPRC rs10919563 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO:5, 5'-AGCTGAGTCATGGGTATAAGGG-3',
a probe, as set forth in SEQ ID NO:6, 5'-TATATGCATTTTATAGCAATTACTATAATTATTTA-3';
amplifying the PTPRC rs10919563 wild type upstream primer as shown in SEQ ID NO: shown in fig. 7, 5'-CCATTATAAGGACATTCACGTTTCAC-3';
amplifying the PTPRC rs10919563 mutant upstream primer as shown in SEQ ID NO:8, 5'-CCATTATAAGGACATTCACGTTTCAT-3';
amplification primers for STAT4 rs7574865 allele were as follows:
amplification STAT4 rs7574865 wild-type and mutant universal downstream primers and probes:
A downstream primer as set forth in SEQ ID NO: as shown in fig. 9, 5'-TGAAGGTAGTGGTGTGGAT-3',
a probe, as set forth in SEQ ID NO:10, 5'-ACATTTTGGTCACCAACTTTTCATACTTTTACTGC-3';
amplifying STAT4 rs7574865 wild type upstream primer as shown in SEQ ID NO:11, 5'-CCACTGAAATAAGATAACCACTAGTC-3';
amplifying STAT4 rs7574865 mutant upstream primers as shown in SEQ ID NO:12, 5'-CCACTGAAATAAGATAACCACTAGTA-3';
amplification primers for HLA-Ers1264457 allele were as follows:
amplification of HLA-Ers1264457 wild-type and mutant Universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO:13, 5'-GAGAGTCTCAGGCGCCTT-3',
a probe, as set forth in SEQ ID NO:14, 5'-TCGGGCCCCAGCTCGCAGCCAT-3';
amplification of HLA-Ers1264457 wild-type upstream primers, as set forth in SEQ ID NO:15, as shown in: 5'-GCGGAGGAAGCGACC-3';
amplifying HLA-Ers1264457 mutant upstream primers as shown in SEQ ID NO:16, as shown in: 5'-GCGGAGGAAGCGACT-3';
amplification primers for the TRAF1 rs3761847 allele were as follows:
amplification of TRAF1 rs3761847 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO:17, 5'-GATGGCAATACCTGCTTCACAG-3',
A probe, as set forth in SEQ ID NO:18, 5'-CCTCAATACCACCCTCTCTACCTGCT-3';
amplification of TRAF1 rs3761847 wild-type upstream primer as shown in SEQ ID NO:19, 5'-GTCCCTTCTCTCCCCTGCA-3';
amplifying TRAF1 rs3761847 mutant upstream primer as shown in SEQ ID NO:20, 5'-GTCCCTTCTCTCCCCTGCG-3';
amplification primers for the KLRD1 rs2302489 allele were as follows:
amplification of KLRD1 rs2302489 wild-type and mutant universal downstream primers and probes:
a downstream primer as set forth in SEQ ID NO:21, 5'-GTAGAGAAGGCACGATGTGTAC-3',
a probe, as set forth in SEQ ID NO:22, 5'-TTTGCTAAATTTCTTCATACTCAACTTTCAGATTC-3';
amplifying a KLRD1 rs2302489 wild type upstream primer as shown in SEQ ID NO:23, 5'-CATTTAAATACACAATTTTTCATTCTCGA-3';
amplifying the KLRD1 rs2302489 mutant upstream primer as shown in SEQ ID NO:24, 5'-CATTTAAATACACAATTTTTCATTCTCGT-3';
amplification primers for the GAPDH reference gene were as follows:
an upstream primer for amplifying GAPDH gene, as shown in SEQ ID NO:25, 5'-ATCCTGGGCTACACTGAGCAC-3';
amplifying the downstream primer of GAPDH gene as shown in SEQ ID NO:26, 5'-CTCAGTGTAGCCCAGGATGCCCTT-3';
Probes for amplifying the GAPDH gene, as set forth in SEQ ID NO:27, 5'-AGGTGGTCTCCTCTGACTTCAA-3'.
2. The primer pair of claim 1, wherein the probe further comprises: SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 14. SEQ ID NO:18 and SEQ ID NO:22 is marked by a fluorescence reporter group FAM at the 5' end of the probe; the 5' -end of the probe shown in SEQ ID NO. 27 is marked by adopting a fluorescence reporter group JOE; SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 14. SEQ ID NO: 18. SEQ ID NO:22 and SEQ ID NO. 27, respectively, are labeled with a fluorescence quenching group TAMRA.
3. A kit for detecting polymorphism of etanercept drug-related genes, wherein polymorphic sites detected by the etanercept drug-related genes are SLCO1C1 rs3794271, PTPRC rs10919563, STAT4 rs7574865, HLA-Ers1264457, TRAF1 rs3761847 and KLRD1 rs2302489, respectively, the kit comprising:
primer liquid 1, wherein the primer liquid 1 contains a nucleotide sequence shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
Primer liquid 2, wherein the primer liquid 2 contains a nucleotide sequence shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 3, wherein the primer liquid 3 contains a nucleotide sequence shown as SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 4, wherein the primer liquid 4 contains a nucleotide sequence shown in SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 5, wherein the primer liquid 5 contains a nucleotide sequence shown as SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 6, wherein the primer liquid 6 contains a nucleotide sequence shown in SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 7, wherein the primer liquid 7 contains a nucleotide sequence shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 8, wherein the primer liquid 8 contains a nucleotide sequence shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
Primer liquid 9, wherein the primer liquid 9 contains a nucleotide sequence shown in SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 10, wherein the primer liquid 10 contains a nucleotide sequence shown in SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 11, wherein the primer liquid 11 contains a nucleotide sequence shown in SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe;
primer liquid 12, wherein the primer liquid 12 contains a nucleotide sequence shown in SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO:26 and a probe.
4. A kit according to claim 3, wherein the kit further comprises: a positive control, which is inserted with SEQ ID NO:1 and SEQ ID NO:3 amplification product, inserted with SEQ ID NO:1 and SEQ ID NO:4 amplification product, inserted with SEQ ID NO:5 and SEQ ID NO:7 amplification product, inserted with SEQ ID NO:5 and SEQ ID NO:8 amplification product, inserted with SEQ ID NO:9 and SEQ ID NO:11, inserted with SEQ ID NO:9 and SEQ ID NO:12 amplification product, inserted with SEQ ID NO:13 and SEQ ID NO:15 amplification product, inserted with SEQ ID NO:13 and SEQ ID NO:16 amplification product, inserted with SEQ ID NO:17 and SEQ ID NO:19, inserted with SEQ ID NO:17 and SEQ ID NO:20 amplification product, inserted with SEQ ID NO:21 and SEQ ID NO:23 amplification product, inserted with SEQ ID NO:21 and SEQ ID NO:24 amplification product, 13 plasmids inserted with SEQ ID NO. 25 and SEQ ID NO. 26 amplification product;
Wherein the plasmid vector is pMD18-T plasmid.
5. The kit of claim 4, wherein the positive control plasmid mixture comprises the following quantitative ratios of the SLCO1C1rs3794271 wild homozygote plasmid, the SLCO1C1rs3794271 mutant homozygote plasmid, the PTPRC rs10919563 wild homozygote plasmid, the PTPRC rs10919563 mutant homozygote plasmid, the STAT4 rs7574865 wild homozygote plasmid, the STAT4 rs7574865 mutant homozygote plasmid, the HLA-Ers1264457 wild homozygote plasmid, the HLA-Ers1264457 mutant homozygote plasmid, the TRAF 1rs 3761847 wild homozygote plasmid, the TRAF 1rs 3761847 mutant homozygote plasmid, the KLRD 1rs 2302489 wild homozygote plasmid, the KLRD 1rs 2302489 mutant homozygote plasmid, and the internal reference GAPDH plasmid: 1:1:1:1:1:1:1:1:1:1:1:2.
6. the kit of claim 3, further comprising a blank, wherein the blank is sterilized purified water.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009032316A2 (en) * | 2007-09-05 | 2009-03-12 | Celera Corporation | Genetic polymorphisms associated with rheumatoid arhritis, methods of detection and uses thereof |
| WO2014177746A1 (en) * | 2013-05-03 | 2014-11-06 | Fundació Hospital Universitari Vall D'hebron - Institut De Recerca | Prognosis of response to treatment with anti-tnf-alpha in patients with rheumatoid arthritis |
| WO2016024254A1 (en) * | 2014-08-15 | 2016-02-18 | Fundació Hospital Universitari Vall D’Hebron-Institut De Recerca | Therapy selection for psoriasis and psoriatic arthritis |
| CN110499364A (en) * | 2019-07-30 | 2019-11-26 | 北京凯昂医学诊断技术有限公司 | A kind of probe groups and its kit and application for detecting the full exon of extended pattern hereditary disease |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009032316A2 (en) * | 2007-09-05 | 2009-03-12 | Celera Corporation | Genetic polymorphisms associated with rheumatoid arhritis, methods of detection and uses thereof |
| WO2014177746A1 (en) * | 2013-05-03 | 2014-11-06 | Fundació Hospital Universitari Vall D'hebron - Institut De Recerca | Prognosis of response to treatment with anti-tnf-alpha in patients with rheumatoid arthritis |
| WO2016024254A1 (en) * | 2014-08-15 | 2016-02-18 | Fundació Hospital Universitari Vall D’Hebron-Institut De Recerca | Therapy selection for psoriasis and psoriatic arthritis |
| CN110499364A (en) * | 2019-07-30 | 2019-11-26 | 北京凯昂医学诊断技术有限公司 | A kind of probe groups and its kit and application for detecting the full exon of extended pattern hereditary disease |
Non-Patent Citations (1)
| Title |
|---|
| 类风湿关节炎患者TNF-α表达水平及其基因rs1800629位点多态性分析;郭燕;刘小君;许海涛;;中国骨与关节杂志(第07期);全文 * |
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