CN117402953A - Kit for detecting polymorphism of rheumatoid drug-related genes and application of kit - Google Patents
Kit for detecting polymorphism of rheumatoid drug-related genes and application of kit Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6858—Allele-specific amplification
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention discloses a kit for detecting polymorphism of genes related to rheumatoid drug administration and application thereof, comprising PCR mixed solution 1-4 and sample treatment solution for releasing genomic DNA of a sample to be detected; the PCR mixed solution 1 comprises a wild primer probe group for detecting the c.677C > T and c.1298A > C loci of the MTHFR gene and the c.80A > G loci of the SLC19A1 gene; the PCR mixed solution 2 comprises a mutant primer probe group for detecting c.677C > T and c.1298A > C loci of the MTHFR gene and c.80A > G loci of the SLC19A1 gene; the PCR mixed solution 3 comprises a wild type primer probe group for detecting the c.3435T > C locus of the ABCB1 gene and the c.675T > C locus of the ATIC gene; the PCR mixture 4 comprises a mutant primer probe group for detecting the c.3435T > C locus of the ABCB1 gene and the c.675T > C locus of the ATIC gene. The kit supports direct blood expansion, has the advantages of simple operation, stable system, strong specificity, high sensitivity and high flux, greatly reduces the cost and is widely applicable.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a kit for detecting polymorphism of rheumatoid drug-related genes and application thereof.
Background
Rheumatoid arthritis (rheumatoid arthritis, RA) is an autoimmune disease, mainly erosive and symmetrical polyarthritis, and if the autoimmune disease cannot be effectively controlled in time, not only is disability and teratogenesis caused, but also other organs such as heart, lung, pleura and the like of a patient are involved, and the daily life of the patient is seriously affected. Methotrexate (MTX) is a first-line drug for clinical treatment of RA, and has therapeutic effects by inhibiting folic acid, adenosine and nucleotide synthesis pathways generated from the head, and is the most recommended antirheumatic drug for improving the illness state of the college of rheumatology at home and abroad. Because of the individual differences, only 45% -65% of patients are effective in MTX treatment, and 30% of patients stop taking drugs because of poor MTX treatment or toxic and side effects, and common MTX side effects are clinically manifested by gastrointestinal reactions, bone marrow suppression, drug hepatitis, kidney damage, alopecia, dermatitis and the like. Individual variability of MTX may be caused by a number of factors, including genetic variation of transporters and metabolic enzymes, in addition to environmental factors (e.g., smoking, occupational exposure), infection, gene polymorphism is an important factor. The pharmacogenomics study shows that toxic and side effects of patients after taking MTX drugs are related to the polymorphic distribution of c.677C > T (rs 1801133) and c.1298A > C (rs 1801131) of MTHFR genes, c.3435T > C (rs 1045642) of ABCB1 genes, c.80A > G (rs 1051266) of SLC19A1 genes and c.6755T > C (rs 4673993) of ATIC genes.
Methylene tetrahydrofolate reductase (MTHFR) is a key enzyme for methotrexate to exert pharmacological effects, and the most common two single nucleotide polymorphisms c.677C > T and c.1298A > C are associated with toxicity during methotrexate treatment, skin and mucositis risk caused by methotrexate, with occurrence rates of 45.2% and 18.6% in Chinese populations, respectively. ABCB1c.3435T > C, SLC19A1c.80A > G are genes responsible for methotrexate drug delivery, and transporter gene polymorphisms are associated with the risk of hepatotoxicity of methotrexate. ATIC c.675T > C wild type resulted in poor efficacy of the drug after treatment with methotrexate in rheumatoid arthritis patients. Therefore, detection of SNP loci of related genes has important value and significance for more reasonable clinical use of MTX.
The existing methods for detecting gene polymorphism by molecular biology have various methods, for example, chinese patent No. CN105886607B discloses an MTHFR gene detection method and a kit, and the method adopts a sequencing method to detect the MTHFR gene polymorphism site, and the gene sequencing method is a gold standard for detecting SNP or mutation, has the advantages of high throughput and multiple sites, but also has the defects of complicated steps, long test period, high cost, low efficiency and the like, and is unfavorable for clinical large-area popularization. Other detection technologies such as detection compositions, kits and applications thereof for guiding the chemotherapy of the flood cancer species disclosed in Chinese patent document No. CN115232873B are continuously developed, the SNP locus is detected by adopting a time-of-flight mass spectrometry detection technology, and the operation process of the technology involves PCR amplification reaction, enzymolysis reaction, extension reaction, water supplementing and mass spectrometry detection, so that the operation steps are complicated and the detection sensitivity is low; in addition, the chinese patent of document number CN114369597a discloses a universal probe detection chip and application thereof, and relates to a gene chip detection technology, which requires first PCR amplification, then hybridizing a PCR product containing a target SNP site with a mutation probe and a wild probe, and determining the genotype of the sample by comparing the hybridization signal intensities of the two probes. The method has the advantages of complicated operation process, difficulty in realizing batch and automation, high chip requirement, high instrument cost and incapability of detecting unknown mutation.
Therefore, it is necessary to develop a detection scheme that is simple to operate, highly specific, highly sensitive, highly reliable and low in cost.
Disclosure of Invention
The invention aims at: aiming at the problems, a kit for detecting the polymorphism of the genes related to rheumatoid drug administration and application thereof are provided to solve the defects in the prior art.
The technical scheme adopted by the invention is as follows: a kit for detection of a polymorphism in a rheumatoid drug-related gene, comprising: a PCR mixed solution 1, a PCR mixed solution 2, a PCR mixed solution 3, a PCR mixed solution 4 and a sample treatment solution for releasing genome DNA of a sample to be tested;
the PCR mixed solution 1 comprises a wild primer probe group for detecting the c.677C > T and c.1298A > C loci of the MTHFR gene and the c.80A > G loci of the SLC19A1 gene;
the PCR mixed solution 2 comprises a mutant primer probe group for detecting c.677C > T and c.1298A > C loci of MTHFR genes and c.80A > G loci of SLC19A1 genes;
the PCR mixed solution 3 comprises a wild type primer probe group for detecting the c.3435T > C locus of the ABCB1 gene and the c.6755T > C locus of the ATIC gene;
the PCR mixed solution 4 comprises a mutant primer probe group for detecting the c.3435T > C locus of the ABCB1 gene and the c.6755T > C locus of the ATIC gene.
Further, the PCR mixture 1, the PCR mixture 2, the PCR mixture 3, and the PCR mixture 4 further include: taq enzyme, tris-HCl, mgCl 2 dNTPs and buffers.
Further, the PCR mixture 1, the PCR mixture 2, the PCR mixture 3, and the PCR mixture 4 further include: universal primers and/or universal probes for detecting the reference gene RPPH in human genomic DNA.
Further, the primer sequences of the PCR mixture 1, the PCR mixture 2, the PCR mixture 3 and the PCR mixture 4 are shown in the table 1; wherein:
the wild type primer probe group for detecting the MTHFR gene c.677C > T comprises a wild type primer with a sequence shown as SEQ ID NO.1, a public primer with a sequence shown as SEQ ID NO.3 and a probe with a sequence shown as SEQ ID NO. 4;
the mutant primer probe group for detecting the MTHFR gene c.677C > T locus comprises a mutant primer with a sequence shown as SEQ ID NO.2, a public primer with a sequence shown as SEQ ID NO.3 and a probe with a sequence shown as SEQ ID NO. 4;
the wild type primer probe group for detecting the C site of the MTHFR gene c.1298A > comprises a wild type primer with a sequence shown as SEQ ID NO.5, a public primer with a sequence shown as SEQ ID NO.7 and a probe with a sequence shown as SEQ ID NO. 8;
The mutant primer probe group for detecting the C site of the MTHFR gene c.1298A > comprises a mutant primer with a sequence shown as SEQ ID NO.6, a public primer with a sequence shown as SEQ ID NO.7 and a probe with a sequence shown as SEQ ID NO. 8;
the wild type primer probe group for detecting the SLC19A1 gene c.80A > G locus comprises a wild type primer with a sequence shown as SEQ ID NO.9, a public primer with a sequence shown as SEQ ID NO.11 and a probe with a sequence shown as SEQ ID NO. 12;
the mutant primer probe group for detecting the SLC19A1 gene c.80A > G locus comprises a mutant primer with a sequence shown as SEQ ID NO.10, a public primer with a sequence shown as SEQ ID NO.11 and a probe with a sequence shown as SEQ ID NO. 12;
the wild type primer probe group for detecting the c.3435T > C locus of the ABCB1 gene comprises a wild type primer with a sequence shown as SEQ ID NO.13, a public primer with a sequence shown as SEQ ID NO.15 and a probe with a sequence shown as SEQ ID NO. 16;
the mutant primer probe group for detecting the c.3435T > C locus of the ABCB1 gene comprises a mutant primer with a sequence shown as SEQ ID NO.14, a public primer with a sequence shown as SEQ ID NO.15 and a probe with a sequence shown as SEQ ID NO. 16;
The wild type primer probe group for detecting the ATIC gene c.675T > C locus comprises a wild type primer with a sequence shown as SEQ ID NO.17, a public primer with a sequence shown as SEQ ID NO.19 and a probe with a sequence shown as SEQ ID NO. 20;
the mutant primer probe group for detecting the c.675T > C locus of the ATIC gene comprises a mutant primer with a sequence shown as SEQ ID NO.18, a public primer with a sequence shown as SEQ ID NO.19 and a probe with a sequence shown as SEQ ID NO. 20.
TABLE 1 specific primer probe sequence listing
Further, the universal primers include primers having sequences shown in SEQ ID NOS.21-22 in Table 1; the universal probes include probes having the sequences shown as SEQ ID NO.23 in Table 1.
Further, the universal probe adopts a taqman probe, the 5 'end of the universal probe is connected with a fluorescence report group, and the 3' end of the universal probe is connected with a fluorescence quenching group; the fluorescent reporter group is selected from one fluorescent dye of FAM, TEXAS RED, HEX and CY 5; the fluorescence quenching group is selected from BHQ1 or BHQ2 fluorescent dye.
Further, the kit also comprises a positive control and a negative control, wherein the positive control comprises a DNA recombinant plasmid containing all target sequence polymorphic sites of the target gene; the negative control includes physiological saline.
Further, the sample processing liquid comprises SDS, formamide and HCl.
Further, the buffer is 10-by-10%, and the dosage of the 10-by-10% buffer is 5-10% v; the MgCl 2 The dosage of (2-3.25 v%; the dosage of dNTPs is 2-2.75 v%; the usage amount of the Tris-HCl is 1.2-1.8 v%.
Furthermore, the invention also comprises application of the kit in rheumatoid drug evaluation, wherein the kit evaluates the drug administration risk of the rheumatoid drug by detecting the polymorphism of the genes of human MTHFR, SLC19A1, ABCB1 and ATIC, and comprises the following steps:
A. releasing DNA of a sample to be detected by adopting a sample treatment liquid;
B. directly adding the DNA extract obtained in the step A into the PCR mixed solution 1, the PCR mixed solution 2, the PCR mixed solution 3 and the PCR mixed solution 4 for fully and uniformly mixing;
C. the fluorescent quantitative PCR amplification reaction procedure is:
pre-denaturation reaction: reacting for 3min at 95 ℃;
amplification reaction: 15s at 95 ℃ and 45s at 60 ℃ for 45 cycles;
D. judging the genotype of a sample to be tested;
E. and evaluating the medication risk of the rheumatoid medicament by the genotype of the sample to be tested.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. According to the kit, the preparation steps of a PCR amplification system are omitted by arranging the premixed PCR mixed solution 1, the premixed PCR mixed solution 2, the premixed PCR mixed solution 3 and the premixed PCR mixed solution 4, so that the operation is more convenient, the operation error is reduced, the use is convenient for a user, and the accuracy is high;
2. the kit can accurately detect the genome DNA as low as 0.5 ng/. Mu.L by designing the ARMS primer and the Taqman probe and matching with a specific PCR mixed solution system, has higher sensitivity, and can effectively avoid the condition of false positive or false negative by introducing an internal reference gene to monitor the whole process quality of the detection process;
3. compared with other detection methods in the market, the detection method of the kit greatly simplifies the operation intensity, can realize the result about 1 hour, and greatly reduces the cost; meanwhile, by setting two reaction systems, the detection result is easy to read, more patient gene information is provided for clinicians, more perfect personalized medicine reference is provided for the use of rheumatoid medicine methotrexate, and patients benefit.
Drawings
FIG. 1A is a diagram showing the detection results of different wild type primers in the embodiment 1 of the present invention for a sample to be detected carrying the CC genotype of the MTHFR gene and a sample to be detected carrying the TT genotype of the MTHFR gene, respectively;
FIG. 1B is a diagram showing the detection results of different mutant primers in the embodiment 1 of the present invention for a sample to be detected carrying the CC genotype of the MTHFR gene and a sample to be detected carrying the TT genotype of the MTHFR gene, respectively;
FIG. 2A is a diagram showing the detection results of different wild type primers in example 1 of the present invention for a sample to be detected carrying the AA genotype of the SLC19A1 gene and a sample to be detected carrying the GG genotype of the SLC19A1 gene, respectively;
FIG. 2B is a diagram showing the detection results of different mutant primers in the embodiment 1 of the present invention for a sample to be detected carrying the AA genotype of the SLC19A1 gene and a sample to be detected carrying the GG genotype of the SLC19A1 gene, respectively;
FIG. 3A is a diagram showing the detection results of different wild type primers in example 1 of the present invention for a sample to be detected carrying the AA genotype of MTHFRc.1298 gene and a sample to be detected carrying the CC genotype of MTHFRc.1298 gene, respectively;
FIG. 3B is a diagram showing the detection results of different mutant primers in the embodiment 1 of the present invention for a sample to be detected carrying the AA genotype of MTHFRc.1298 gene and a sample to be detected carrying the CC genotype of MTHFRc.1298 gene, respectively;
FIG. 4A is a diagram showing the detection results of different wild type primers in example 1 of the present invention for a sample to be detected carrying the TT genotype of the ABCB1 gene and a sample to be detected carrying the CC genotype of the ABCB1 gene, respectively;
FIG. 4B is a diagram showing the detection results of different mutant primers in the embodiment 1 of the present invention for a sample to be detected carrying the TT genotype of the ABCB1 gene and a sample to be detected carrying the CC genotype of the ABCB1 gene, respectively;
FIG. 5A is a diagram showing the detection results of different wild type primers in example 1 of the present invention for a sample to be tested carrying the TT genotype of ATIC gene and a sample to be tested carrying the CC genotype of ATIC gene, respectively;
FIG. 5B is a diagram showing the detection results of different mutant primers in the embodiment 1 of the present invention for a sample to be detected carrying the TT genotype of ATIC gene and a sample to be detected carrying the CC genotype of ATIC gene, respectively;
FIG. 6A is a graph showing the amplification results of a wild-type reaction system of PCR mixture 1 according to example 2 of the present invention using different PCR reaction formulations;
FIG. 6B is a graph showing the amplification results of a mutant reaction system of PCR mixture 2 according to example 2 of the present invention using different PCR reaction formulations;
FIG. 6C is a graph showing the amplification results of a wild-type reaction system of PCR mixture 3 according to example 2 of the present invention using different formulations of PCR reaction solutions;
FIG. 6D is a graph showing the amplification results of a mutant reaction system of PCR mixture 4 according to example 2 of the present invention using different formulations of PCR reaction solutions;
FIG. 7A is a graph showing the amplification result of a wild-type reaction system of the PCR mixture 1 in combination in example 2 of the present invention;
FIG. 7B is a graph showing the amplification results of a mutant reaction system in which PCR mixture 2 was used in combination in example 2 of the present invention;
FIG. 7C is a graph showing the amplification result of a wild-type reaction system in which PCR mixture 3 was used in combination in example 2 of the present invention;
FIG. 7D is a graph showing the amplification results of a wild-type reaction system in which PCR mixture 4 was used in combination in example 2 of the present invention;
FIG. 8A is a graph showing the amplification result of a wild-type reaction system of the PCR mixture 1 in combination II in example 2 of the present invention;
FIG. 8B is a graph showing the amplification results of a mutant reaction system of the PCR mixture 2 in combination II in example 2 of the present invention;
FIG. 8C is a graph showing the amplification result of the wild-type reaction system of the PCR mixture 3 in combination II in example 2 of the present invention;
FIG. 8D is a graph showing the amplification result of the wild-type reaction system of the PCR mixture 4 in combination II in example 2 of the present invention;
FIG. 9A is a graph showing the amplification result of a wild-type reaction system of PCR mixture 1 in combination III in example 2 of the present invention;
FIG. 9B is a graph showing the amplification results of a mutant reaction system of the PCR mixture 2 in combination III in example 2 of the present invention;
FIG. 9C is a graph showing the amplification result of the wild-type reaction system of the PCR mixture 3 in combination III in example 2 of the present invention;
FIG. 9D is a graph showing the amplification result of the wild-type reaction system of PCR mixture 4 in combination III in example 2 of the present invention;
FIG. 10A is a diagram showing the detection result of sample one to be detected using PCR mixture 1 according to embodiment 3 of the present invention;
FIG. 10B is a diagram showing the detection result of sample one to be detected using PCR mixture 2 according to embodiment 3 of the present invention;
FIG. 10C is a diagram showing the detection result of sample one to be detected using PCR mixture 3 according to embodiment 3 of the present invention;
FIG. 10D is a diagram showing the detection result of sample one to be detected using PCR mixture 4 according to embodiment 3 of the present invention;
FIG. 11A is a diagram showing the detection result of sample two to be detected by using PCR mixture 1 according to embodiment 3 of the present invention;
FIG. 11B is a diagram showing the detection result of sample two to be detected by using PCR mixture 2 according to embodiment 3 of the present invention;
FIG. 11C is a diagram showing the detection result of sample two to be detected using PCR mixture 3 according to embodiment 3 of the present invention;
FIG. 11D is a diagram showing the detection result of sample two to be detected using PCR mixture 4 according to embodiment 3 of the present invention;
FIG. 12A is a diagram showing the detection result of sample three to be detected by using the PCR mixture 1 according to the embodiment 3 of the present invention;
FIG. 12B is a diagram showing the detection result of sample three to be detected by using PCR mixture 2 according to embodiment 3 of the present invention;
FIG. 12C is a diagram showing the detection result of sample three to be detected by using PCR mixture 3 according to embodiment 3 of the present invention;
FIG. 12D is a diagram showing the detection result of sample three to be detected by using the PCR mixture 4 according to the embodiment 3 of the present invention;
FIG. 13A is a diagram showing the detection result of the kit of example 4 in the first reaction system for a sample to be detected with known concentration and genotype;
FIG. 13B is a graph showing the detection results of the kit of example 4 of the present invention on a sample to be detected of known concentration and genotype in a second reaction system.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
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.
The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving," are inclusive or open-ended and do not exclude additional unrecited elements or method steps.
The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
The terms "about" and "substantially" in this invention mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid, or analogue units thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be a nucleic acid that denatures one strand of double-stranded DNA. Alternatively, the single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA.
The features and capabilities of the present invention are described in further detail below in connection with examples.
Example 1
The specific sites and regions of the related genes are selected, and the primers c.677C > T, c.1298A > C (SNP ID: rs 1801133), c.80A > G (SNP ID: rs 1051266), c.3435T > C (SNP ID: rs 1045642), c.6755T > C (SNP ID: 4673993) of the MTR gene, c.677C > T, c.1298A > C, SLC A1 gene and c.3728T > C of the AR5 gene are respectively designed by using the Primer 3.0 software of ABI company according to the information of the reference sequence (NM_ 005957.5:) of the MTHFR gene, the reference sequence (NM_ 194255.4:) of the SLC19A1 gene, the reference sequence (NM_ 001348944.2:), the reference sequence of the ATIC gene, and the c.677C > T polymorphism (SNP ID: rs 1801133), c.1298A > C (SNP ID: rs 1801131), c.80A > G polymorphism (SNP ID: rs 1051266), c.3435T > C (SNP ID: rs 1045642) and c.6755T > C of the c.6755T > C gene of the MTR gene are respectively designed by using the Primer 3.0 software of ABI. In order to monitor the effectiveness of a reaction system, an internal reference primer and a probe are added into the detection system, a section of sequence of human conserved gene RPPH (the GeneBank reference sequence number is NG_ 009291.1) is selected, and each specific primer can only be combined with a DNA template of a corresponding genotype and amplified according to the allele-specific PCR principle for PCR amplification and fluorescence detection.
Specifically, taking an MTHFRc.677C > T site as an example (other sites are similar), simultaneously designing a plurality of wild type primers and a plurality of mutant type primers, respectively adopting the designed plurality of wild type primers to respectively detect a sample to be detected carrying the CC genotype of the MTHFR gene and a sample to be detected carrying the TT genotype of the MTHFR gene under the same PCR condition, wherein the detection results are shown in figure 1A; and respectively detecting the sample to be detected carrying the CC genotype of the MTHFR gene and the sample to be detected carrying the TT genotype of the MTHFR gene by adopting a plurality of designed mutant primers, wherein the detection results are shown in figure 1B. Combining the CT value of the amplification curve, the morphology of the amplification curve and the fluorescence signal value, WF3 and MF3 can be derived as the most preferred primers, and the specificity is high. Similarly, with reference to FIGS. 2A-2B, WR3 and MR3 are the most preferred primers for the SLC19A1c.80A > G site; referring to FIGS. 3A-3B, WF2 and MF3 are the most preferred primers for the MTHFRc.1298A > C site; referring to FIGS. 4A-4B for the ABCB1c.3435T > C site, WF3 and MF3 are the most preferred primers; referring to FIGS. 5A-5B, WR2 and MR2 are the most preferred primers for the ATICc.675T > C site. As shown in table 1:
table 1 shows the sequence listing of different primers designed
The final set of screened optimal primer probes is shown in Table 2 below:
TABLE 2 specific primer probe sequence listing
Furthermore, the probe adopts a Taqman fluorescent probe; the 5 '-end of the probe sequence is marked with a fluorescence report group, and the 3' -end is marked with a fluorescence quenching group. Specifically, a probe for detecting MTHFR c.677C > T gene locus is marked by FAM fluorescent dye, and the 3' end is marked by BHQ1 or BHQ2 fluorescent dye; the probe for detecting the MTHFR c.1298A > C gene locus is marked by TEXAS RED fluorescent dye, and the 3' end is marked by BHQ1 or BHQ2 fluorescent dye; the probe for detecting SLC19A1c.80A > G gene locus is marked by HEX fluorescent dye, and the 3' end is marked by BHQ1 or BHQ2 fluorescent dye; the probe for detecting the ABCB1c.3435T > C gene locus is marked by FAM fluorescent dye, and the 3' end is marked by BHQ1 or BHQ2 fluorescent dye; the probe for detecting ATIC c.675T > C gene locus is marked by HEX fluorescent dye, and the 3' end is marked by BHQ1 or BHQ2 fluorescent dye; the probe for detecting the RPPH gene locus is marked by CY5 fluorescent dye, and the 3' -end is marked by BHQ1 or BHQ2 fluorescent dye.
Example 2
Polymerase Chain Reaction (PCR) is a method for amplifying and detecting specific target genetic material, and is used for various purposes such as DNA diagnosis, DNA replication, detection of viruses and bacteria, quantification and measurement of target DNA concentration, and the like. In general, the PCR reaction is carried out by preparing a PCR reaction solution on site, and specifically, uniformly mixing a plurality of components such as an enzyme, buffer, dNTPs, a primer probe and the like to carry out PCR amplification immediately. The operation is not only easy to cause the situation of adding mistakes or omission, but also can cause errors of PCR experimental results due to different experimental skills or precision of operators, thereby prolonging the preparation time of the PCR reaction liquid and bringing the risk of pollution to the reagent in the continuous taking process. Therefore, the embodiment provides a kit for detecting polymorphism of human MTHFR, SLC19A1, ABCB1 and ATIC genes, which comprises a PCR mixed solution 1 and a PCR mixed solution 2 for detecting C sites of MTHFR genes c.677C > T and c.1298A > C sites and C.80A > G sites of SLC19A1 genes; PCR mixture 3 and PCR mixture 4 for detecting the c.3435T > C site of the ABCB1 gene and the c.675T > C site of the ATIC gene, and sample processing liquid for releasing genomic DNA of a sample to be tested.
Wherein the PCR mixture 1 comprises detection of MTHFR gene c.677C>T、c.1298A>C site, SLC19A1 gene c.80A>Wild primer probe group of G locus, primer probe group for detecting internal reference gene, taq enzyme, tris-HCl and MgCl 2 dNTPs and buffers.
PCR mixture 2 included detection of MTHFR gene c.677C>T、c.1298A>C site, SLC19A1 gene c.80A>G site mutant primer probe group, primer probe group for detecting reference gene, taq enzyme, tris-HCl and MgCl 2 dNTPs and buffers.
PCR mixture 3 includes detection of ABCB1 gene c.3435T>C site, ATIC gene c.675T>Wild primer probe group of C locus, primer probe group for detecting internal reference gene, taq enzyme, tris-HCl and MgCl 2 、dNTPsAnd Buffer.
PCR mixture 4 included detection of ABCB1 Gene c.3435T>C site, ATIC gene c.675T>C site mutant primer probe group, primer probe group for detecting internal reference gene, taq enzyme, tris-HCl and MgCl 2 dNTPs and buffers.
Stabilizers and protectors conventional in the art may be added to the PCR mixture 1-4, thereby extending the shelf life. The components such as the primer probe, the enzyme, the dNTPs and the buffer solution required by the PCR reaction system are mixed together, so that the preparation time of each component is obviously shortened, and when the kit is used, a sample can be detected by being mixed with the PCR mixed solution, the kit can be taken and used at any time, the experimental error and experimental pollution caused by repeated taking and mixing are obviously reduced, and the stringent requirements on detection time limit, operation proficiency and the like and the overall detection difficulty are reduced.
In order to facilitate the interpretation of the results, the detection kit provided in this embodiment sets a wild-type reaction system (i.e., a first reaction system) and a mutant reaction system (i.e., a second reaction system) for the mutation site of the target gene, respectively. Specifically, the PCR mixed solution 1 and the PCR mixed solution 3 correspond to a first reaction system and are used for detecting whether a sample to be detected contains wild type gene loci or not; the PCR mixed solution 2 and the PCR mixed solution 4 correspond to a second reaction system and are used for detecting whether the sample to be detected contains mutant gene loci.
The embodiment provides a detection kit, wherein the addition amount of a sample to be detected is 2 mu L, the volumes of a PCR mixed solution 1, a PCR mixed solution 2, a PCR mixed solution 3 and a PCR mixed solution 4 are 18 mu L, and the same PCR reaction solution formula is adopted, so that the types and the volume amounts of only primers and probes are different. Specifically, the usage specification of the primer probe is 100 mu M, and the added volume of the primer SEQ ID NO.1 and the primer SEQ ID NO.3 in the PCR mixed solution 1 is 0.08 mu L; the added volume of the primer SEQ ID NO.5 and the primer SEQ ID NO.7 is 0.04 mu L; the added volume of the primer SEQ ID NO.9 and the primer SEQ ID NO.11 is 0.09 mu L; the added volumes of the primers SEQ ID NO. 21-22 are 0.03 mu L, and the added volumes of the probes SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12 and SEQ ID NO.23 are 0.03 mu L;
In the PCR mixed solution 2, the added volume of the primer SEQ ID NO.2 and the primer SEQ ID NO.3 is 0.09 mu L; the added volume of the primer SEQ ID NO.6 and the primer SEQ ID NO.7 is 0.04 mu L; the added volume of the primer SEQ ID NO.10 and the primer SEQ ID NO.11 is 0.06 mu L; the added volume of the primers SEQ ID NO. 21-22 is 0.03 mu L; the addition volume of the probes SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.12 is 0.03 mu L;
in the PCR mixed solution 3, the added volume of the primer SEQ ID NO.13 and the primer SEQ ID NO.15 is 0.04 mu L; the added volume of the primer SEQ ID NO.17 and the primer SEQ ID NO.19 is 0.05 mu L; the added volume of the primers SEQ ID NO. 21-22 is 0.03 mu L; the addition volume of the probes SEQ ID NO.15, SEQ ID NO.20 and SEQ ID NO.23 is 0.03 mu L;
in the PCR mixed solution 4, the added volume of the primer SEQ ID NO.14 and the primer SEQ ID NO.15 is 0.05 mu L; the added volume of the primer SEQ ID NO.18 and the primer SEQ ID NO.19 is 0.05 mu L; the added volume of the primers SEQ ID NO. 21-22 is 0.03 mu L; the addition volume of the probes SEQ ID NO.16, SEQ ID NO.20 and SEQ ID NO.23 is 0.03 mu L;
the amounts of Taq enzyme added to each of the PCR mixture 1, the PCR mixture 2, the PCR mixture 3 and the PCR mixture 4 were 0.3. Mu.L.
In the process of detecting the gene polymorphism, besides the influence of irresistible factors such as environmental factors, equipment performance and the like, the specificity of a reaction system directly influences the detection accuracy and the practicability of the kit, so the inventor optimizes and improves the reaction system. Specifically, three PCR reaction liquid formulations are designed as shown in tables 3-5, and amplification detection is carried out on whole blood samples (MTHFRc.677 CT, MTHFRc.1298 AA, SLC19A1c.80 AG, ABCB1c.3435 CC, ATICc.675TC) with known genotypes under the same PCR conditions.
TABLE 3 PCR reaction solution formulation one
| Component (A) | Concentration of mother liquor | 1T test addition (μL) |
| Buffer 10 x (no glycerol) | — | 1 |
| Magnesium chloride | 50mM | 0.4 |
| dNTP | 10mM | 0.4 |
| Tris | PH＝7.53 | 0.25 |
TABLE 4 PCR reaction solution formulation II
| Component (A) | Concentration of mother liquor | 1T test addition (μL) |
| Buffer 10 x (no glycerol) | — | 1.65 |
| Magnesium chloride | 50mM | 0.65 |
| dNTP | 10mM | 0.55 |
| Tris | PH＝7.53 | 0.36 |
TABLE 5 PCR reaction solution formulation III
FIG. 6A is a graph showing the amplification results of a wild-type reaction system of a PCR mixture 1 using different PCR reaction liquid formulations, FIG. 6B is a graph showing the amplification results of a mutant reaction system of a PCR mixture 2 using different PCR reaction liquid formulations, FIG. 6C is a graph showing the amplification results of a wild-type reaction system of a PCR mixture 3 using different PCR reaction liquid formulations, and FIG. 6D is a graph showing the amplification results of a wild-type reaction system of a PCR mixture 4 using different PCR reaction liquid formulations, and it is found that the amplification effect of the PCR reaction liquid formulation III on the detection sample is improved in combination with the fluorescence signal intensity, the curve morphology and the Ct value, so that the determination of the formulation III as the optimal PCR reaction liquid is made.
In addition, in order to reduce the mutual interference between the primer probes of different mutation sites of different genes and coordinate amplification efficiency, the detection kit provided in this embodiment performs amplification detection by mixing a plurality of pairs of primer probes in the same premix, taking a whole blood sample (mthfrc.677 ct, mthfrc.1298aa, sli 19a1c.80aa, abcb1c.3435tt, aticc.675tc) of a known genotype as an example, for the purpose of combining 1-4 different gene sites of a PCR mixture:
Combining:
(1) Placing a wild primer probe composition for detecting the C site of the MTHFR gene c.1298A > and the C site of the ABCB1 gene c.3435T > and the C site of the ATIC gene c.675T > in the PCR mixed solution 1, wherein the amplification result corresponds to the amplification result of FIG. 7A; placing a mutant primer probe composition for detecting the C.1298A > C locus of the MTHFR gene, the C.3435T > C locus of the ABCB1 gene and the C.675T > C locus of the ATIC gene in the PCR mixed solution 2, wherein the amplification result corresponds to the diagram 7B; the fluorescence intensity value and the amplification curve form of the combination show that the fluorescence heights and the amplification efficiencies of the MTHFR gene c.677C > T locus and the ATIC gene c.675T > C locus are relatively low.
(2) Placing a wild primer probe composition for detecting SLC19A1 gene c.80A > G locus and MTHFR gene c.677C > T locus in the PCR mixed solution 3, wherein the amplification result corresponds to FIG. 7C; the PCR mixed solution 4 is placed with a mutant primer probe composition for detecting SLC19A1 gene c.80A > G locus and MTHFR gene c.677C > T locus, and the amplification result corresponds to FIG. 7D; the results showed that the fluorescence height and amplification efficiency of the two-site amplification curve were both excellent, but the target gene settings in PCR mixes 1 and 2 required a recombination test.
And (2) combining two:
(1) Placing a wild primer probe composition for detecting the C.1298A > C locus of the MTHFR gene, the C.677C > T locus of the MTHFR gene and the C.675T > C locus of the ATIC gene in the PCR mixed solution 1, wherein the amplification result corresponds to FIG. 8A; placing a mutant primer probe composition for detecting the C.1298A > C locus of the MTHFR gene, the C.677C > T locus of the MTHFR gene and the C.675T > C locus of the ATIC gene in the PCR mixed solution 2, wherein the amplification result corresponds to FIG. 8B; the combined fluorescence intensity value and the amplification curve form show that the fluorescence height of the c.675T > C locus of the ATIC gene is relatively low, namely the amplification efficiency is poor.
(2) Placing a wild primer probe composition for detecting SLC19A1 gene c.80A > G locus and ABCB1 gene c.3435T > C locus in the PCR mixed solution 3, wherein the amplification result corresponds to FIG. 8C; the PCR mixed solution 4 is placed with a mutant primer probe composition for detecting SLC19A1 gene c.80A > G locus and ABCB1 gene c.3435T > C locus, and the amplification result corresponds to FIG. 8D; the results show that the fluorescence height and the amplification efficiency of the two-site amplification curve are excellent, and the amplification is particularly aimed at the amplification of the SLC19A1 gene c.80A > G site.
And (3) combining three:
the primer probe composition for detecting the SLC19A1 gene c.80A > G locus in the PCR mixed liquid 3 and 4 and the primer probe composition for detecting the ATIC gene c.675T > C locus in the PCR mixed liquid 1 and 2 are exchanged by the comprehensive comparison combination II and the combination I, namely:
(1) Placing a wild primer probe composition for detecting the C.677C > T locus, the C.1298A > C locus and the C.80A > G locus of the SLC19A1 gene in the PCR mixed solution 1, wherein the amplification result corresponds to FIG. 9A; placing a mutant primer probe composition for detecting the C.677C > T locus, the C.1298A > C locus and the C.80A > G locus of the SLC19A1 gene in the PCR mixed solution 2, wherein the amplification result corresponds to FIG. 9B; the binding fluorescence intensity value and the amplification curve form show that the amplification efficiency of the three target genes is good.
(2) The wild primer probe composition for detecting the c.3435T > C locus of the ABCB1 gene and the c.675T > C locus of the ATIC gene is placed in the PCR mixed solution 3, and the amplification result corresponds to the graph 9C; the PCR mixed solution 4 is placed with a mutant primer probe composition for detecting the c.3435T > C, ATIC gene c.6755T > C locus, and the amplification result corresponds to the figure 9D; the results show that the fluorescence height and amplification efficiency of the two-site amplification curve are good, so that the combination III is determined to be the optimal target gene site combination.
The internal composition of the test kit provided in this example is shown in table 6 below:
TABLE 6 internal composition of kit
| Component name | Main component | Number of specifications |
| PCR mixture 1 | Primer probe, enzyme, tris-HCl and MgCl 2 、dNTPs、buffer | 18μL×20 |
| PCR mixture 2 | Primer probe, enzyme, tris-HCl and MgCl 2 、dNTPs、buffer | 18μL×20 |
| PCR mixture 3 | Primer probe, enzyme, tris-HCl and MgCl 2 、dNTPs、buffer | 18μL×20 |
| PCR mixture 4 | Primer probe, enzyme, tris-HCl and MgCl 2 、dNTPs、buffer | 18μL×20 |
| Positive control | DNA recombinant plasmid containing all target sequence polymorphic sites of target gene | 60μL×1 |
| Negative control | Sodium chloride | 60μL×1 |
| Sample processing liquid | SDS, formamide, HCl | 290μL×20 |
Specifically, the enzyme is Taq DNA polymerase; in addition, in order to simplify the nucleic acid extraction step of the sample to be detected, the detection kit provided by the invention is provided with the sample treatment liquid containing SDS, formamide and HCl, wherein the SDS can generate better synergistic effect with strong alkali, so that the protein and the nucleic acid are fully separated, the whole blood sample can be directly diluted and cracked, the genome DNA in the sample to be detected is released, and an experimenter can extract the DNA of the sample to be detected by processing the sample to be detected in one step, so that the detection efficiency is further improved.
The storage conditions of the kit are as follows: storing at-20+/-5 ℃ in a dark place;
the kit is suitable for the instrument: full automatic PCR analysis System Fascan48E, independently developed by the company Siamion technologies Co.
Example 3
The embodiment provides the method for using the kit provided in the embodiment 2, which specifically includes the following steps:
step 1: releasing DNA of a sample to be tested, taking 10 mu L of uniformly mixed whole blood sample, respectively adding the 10 mu L of uniformly mixed whole blood sample into the sample treatment liquid, and carrying out serial number vortex mixing for later use;
step 2: preparing a reagent, calculating the reaction test number [ the number of samples to be tested+positive control (1) +negative control (1) ], taking out the detection kit from the refrigerator, melting and shaking the component at room temperature, mixing uniformly, and centrifuging at 2000rpm for 10 seconds;
step 3: adding samples, respectively taking 2uL of treated samples to be tested, negative control and positive control, adding the treated samples to the reaction tubes, covering a tube cover, uniformly mixing by vortex for 3s, and transferring the reaction tubes to a detection area after centrifuging at 6000rpm for 5 s;
step 4: PCR amplification and fluorescence detection, placing each reaction tube on a fluorescence PCR amplification instrument according to a certain sequence, setting the reaction system to be 20 mu L, and carrying out PCR amplification according to the following procedures:
pre-denaturation reaction: reacting for 3min at 95 ℃;
amplification reaction: 15s at 95 ℃ and 45s at 60 ℃ for 45 cycles;
Detection fluorescence selection: detection genes (FAM channel, TEXAS RED channel, HEX channel), internal standard genes (CY 5 channel);
step 5: data analysis
1) Kit validity determination
Positive control: FAM, TEXAS RED and HEX channels Ct value is less than or equal to 36.5, and the amplification curve has obvious exponential growth phase;
negative control: each channel has no amplification, and Ct is more than 36.5 or has no Ct value;
if the conditions are not met, the experiment is regarded as invalid, and instruments, reagents, amplification conditions, experimental operation and the like should be checked;
2) Sample validity determination
Reference gene: in all sample detection, the Ct value of a CY5 channel is less than or equal to 36.5, and an amplification curve has obvious exponential growth phase; if this condition is not met, the sample is considered an invalid sample.
Under the conditions of the PCR reaction system and the PCR amplification program, on the premise that the internal reference gene signals form a normal amplification S-shaped curve, observing whether a target detection fluorescent signal in a specific PCR reaction system forms an amplification S-shaped curve, if so, judging that the DNA sample to be detected is positive for the polymorphism type represented by the specific reaction system, and judging the genotype of the sample to be detected according to the difference (delta Ct) between the Ct value of the mutant reaction system and the Ct value of the wild reaction system, wherein the judging mode of the specific genotype detection result is shown in a table 7.
TABLE 7 determination method for genotype test results
The invention is based on the designed ARMS specific primer, and the wild type or mutant type of each site is specifically amplified, and the results shown in FIGS. 10A-10D are PCR detection results of a reaction system 1 and a reaction system 2, wherein FIG. 10A and FIG. 10C represent the system 1 (i.e., a wild type reaction system), and FIG. 10B and FIG. 10D represent the system 2 (i.e., a mutant type reaction system). Specifically, as for the whole blood sample I, as shown in FIGS. 10A to 10D, the fluorescent signals of the internal standard genes in the reaction system I and the reaction system II are qualified, and the genotype of MTHFRc.677, the genotype of MTHFRc.1298, the genotype of SLC19A1c.80, the genotype of ABCB1c.3435, the genotype of ATICc.675, and the genotype of CC can be determined to be CT according to the genotype determination method of Table 7.
As shown in FIGS. 11A-11D, the second whole blood sample was identified as MTHFRc.677 genotype as CC, MTHFRc.1298 genotype as CC, SLC19A1c.80 genotype as AA, ABCB1c.3435 genotype as TC, and ATICc.675 genotype as TC.
As shown in FIGS. 12A-12D, the third whole blood sample was assayed for MTHFRc.677 genotype at TT, MTHFRc.1298 genotype at AA, SLC19A1c.80 genotype at GG, ABCB1c.3435 genotype at TT, and ATICc.6755T > C genotype at TT.
Example 4
For each site, 5 clinical samples of known genotypes were tested, and the DNA concentrations of the samples to be tested were diluted to 1 ng/. Mu.L, 0.75 ng/. Mu.L, 0.5 ng/. Mu.L, 0.25 ng/. Mu.L, and amplified using the test kit of example 2.
Analysis results: 1-0.5 ng/. Mu.L can be correctly interpreted, and Ct value is less than or equal to 32 (one of the detection standards of the kit), 2 samples 0.25 ng/. Mu.L in MTHFR c.677C > T can be correctly interpreted, and 4 samples 0.25 ng/. Mu.L in Ct >33,MTHFR c.1298A>C can be correctly interpreted, but Ct value is more than 33; SLC19A1 c.80A > G3 samples 0.25 ng/. Mu.L can be correctly interpreted but Ct >33.5; ABCB1 c.3435T > C has 2 samples 0.25 ng/. Mu.L correctly interpretable but Ct >33.5; ATIC c.675T > C there were 2 samples 0.25 ng/. Mu.L that could be correctly interpreted but Ct >34. 5 samples carrying ATIC c.675CC genotype and ABCB1c.3435CC genotype at the concentration of 0.5 ng/. Mu.L are repeatedly detected, and the detection results can be normally typed and accord with the detection standard, so that the kit provided by the invention can accurately detect the genotype of the sample with the genomic DNA concentration of 0.5 ng/. Mu.L, and the sensitivity is quite high. The results of the detection graphs in which the sample concentrations of ATIC c.675CC and ABCB1c.3435CC were 0.5 ng/. Mu.L are shown in FIGS. 13A-13B (the detection results of other sites are consistent).
Example 5
In this example, 25 whole blood samples were used as amplified samples, and the test results are shown in table 8, using the kit provided in example 2 and the test method provided in example 3, compared with the sequencing method:
table 8 results of accuracy testing
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As can be seen from Table 8, all the above 25 typing detection results of the detection samples are verified by DNA sequencing, and the coincidence rate of the sequencing result and the detection result of the detection kit reaches 100%, so that the reliability and accuracy of the detection result of the detection kit can be proved.
Example 6: correlation between gene detection result and methotrexate administration risk assessment
Pharmacogenomic studies have shown that the distribution of MTHFR, ABCB1, SLC19A1, ATIC gene polymorphisms is associated with the toxic side effects of methotrexate drug use following chemotherapy in patients. The mutation of the 667 and 1298 genes of the MTHFR gene reduces MTHFR activity and increases the risk of medication toxicity. ABCB1, SLC19A1 are genes responsible for methotrexate drug delivery, and transporter gene polymorphisms are associated with methotrexate toxicity. ATIC c.675T > C wild type resulted in poor efficacy of the drug after treatment with methotrexate in rheumatoid arthritis patients. Thus, different variants affect the therapeutic effect of the drug, and through extensive literature reading, the present example screens out the relationship between different genotypes of individuals and the risk of methotrexate administration, as shown in graph 9.
TABLE 9MTHFR, SLC19A1, ABCB1, ATIC genotype and methotrexate administration risk correlation Table
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The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A kit for detecting polymorphism of a rheumatoid drug-related gene, comprising: a PCR mixed solution 1, a PCR mixed solution 2, a PCR mixed solution 3, a PCR mixed solution 4 and a sample treatment solution for releasing genome DNA of a sample to be tested;
the PCR mixed solution 1 comprises a wild primer probe group for detecting the c.677C > T and c.1298A > C loci of the MTHFR gene and the c.80A > G loci of the SLC19A1 gene;
the PCR mixed solution 2 comprises a mutant primer probe group for detecting c.677C > T and c.1298A > C loci of MTHFR genes and c.80A > G loci of SLC19A1 genes;
the PCR mixed solution 3 comprises a wild type primer probe group for detecting the c.3435T > C locus of the ABCB1 gene and the c.6755T > C locus of the ATIC gene;
the PCR mixed solution 4 comprises a mutant primer probe group for detecting the c.3435T > C locus of the ABCB1 gene and the c.6755T > C locus of the ATIC gene.
2. The kit of claim 1, wherein the PCR mixture 1, the PCR mixture 2, the PCR mixture 3, and the PCR mixture 4 further comprise: taq enzyme, tris-HCl, mgCl 2 dNTPs and buffers.
3. The kit of claim 2, wherein the PCR mixture 1, PCR mixture 2, PCR mixture 3, and PCR mixture 4 further comprise: universal primers and/or universal probes for detecting the reference gene RPPH in human genomic DNA.
4. The kit of claim 3, wherein the primer sequences of PCR mixture 1, PCR mixture 2, PCR mixture 3 and PCR mixture 4 are shown in Table 1; wherein:
the wild type primer probe group for detecting the MTHFR gene c.677C > T comprises a wild type primer with a sequence shown as SEQ ID NO.1, a public primer with a sequence shown as SEQ ID NO.3 and a probe with a sequence shown as SEQ ID NO. 4;
the mutant primer probe group for detecting the MTHFR gene c.677C > T locus comprises a mutant primer with a sequence shown as SEQ ID NO.2, a public primer with a sequence shown as SEQ ID NO.3 and a probe with a sequence shown as SEQ ID NO. 4;
the wild type primer probe group for detecting the C site of the MTHFR gene c.1298A > comprises a wild type primer with a sequence shown as SEQ ID NO.5, a public primer with a sequence shown as SEQ ID NO.7 and a probe with a sequence shown as SEQ ID NO. 8;
The mutant primer probe group for detecting the C site of the MTHFR gene c.1298A > comprises a mutant primer with a sequence shown as SEQ ID NO.6, a public primer with a sequence shown as SEQ ID NO.7 and a probe with a sequence shown as SEQ ID NO. 8;
the wild type primer probe group for detecting the SLC19A1 gene c.80A > G locus comprises a wild type primer with a sequence shown as SEQ ID NO.9, a public primer with a sequence shown as SEQ ID NO.11 and a probe with a sequence shown as SEQ ID NO. 12;
the mutant primer probe group for detecting the SLC19A1 gene c.80A > G locus comprises a mutant primer with a sequence shown as SEQ ID NO.10, a public primer with a sequence shown as SEQ ID NO.11 and a probe with a sequence shown as SEQ ID NO. 12;
the wild type primer probe group for detecting the c.3435T > C locus of the ABCB1 gene comprises a wild type primer with a sequence shown as SEQ ID NO.13, a public primer with a sequence shown as SEQ ID NO.15 and a probe with a sequence shown as SEQ ID NO. 16;
the mutant primer probe group for detecting the c.3435T > C locus of the ABCB1 gene comprises a mutant primer with a sequence shown as SEQ ID NO.14, a public primer with a sequence shown as SEQ ID NO.15 and a probe with a sequence shown as SEQ ID NO. 16;
The wild type primer probe group for detecting the ATIC gene c.675T > C locus comprises a wild type primer with a sequence shown as SEQ ID NO.17, a public primer with a sequence shown as SEQ ID NO.19 and a probe with a sequence shown as SEQ ID NO. 20;
the mutant primer probe group for detecting the c.675T > C locus of the ATIC gene comprises a mutant primer with a sequence shown as SEQ ID NO.18, a public primer with a sequence shown as SEQ ID NO.19 and a probe with a sequence shown as SEQ ID NO. 20.
TABLE 1 specific primer probe sequence listing
5. The kit of claim 4, wherein the universal primers comprise primers having the sequences shown in SEQ ID NOS.21-22 in Table 1; the universal probes include probes having the sequences shown as SEQ ID NO.23 in Table 1.
6. The kit of claim 5, wherein the universal probe is a taqman probe, the 5 'end of the universal probe is connected with a fluorescent reporter group, and the 3' end of the universal probe is connected with a fluorescent quenching group; the fluorescent reporter group is selected from one fluorescent dye of FAM, TEXAS RED, HEX and CY 5; the fluorescence quenching group is selected from BHQ1 or BHQ2 fluorescent dye.
7. The kit of claim 1, further comprising a positive control comprising a recombinant DNA plasmid containing all target sequence polymorphic sites for a gene of interest and a negative control; the negative control includes physiological saline.
8. The kit of claim 1, wherein the sample processing fluid comprises SDS, formamide, HCl.
9. The kit of claim 2, wherein the buffer is 10 x buffer, and the amount of the 10 x buffer is 5v% to 10v%; the MgCl 2 The dosage of (2-3.25 v%; the dosage of dNTPs is 2-2.75 v%; the usage amount of the Tris-HCl is 1.2-1.8 v%.
10. Use of the kit according to any one of claims 1-9 for evaluating the risk of administration of a rheumatoid medicament by detecting the polymorphism of the human MTHFR, SLC19A1, ABCB1, ATIC genes, comprising the steps of:
A. releasing DNA of a sample to be detected by adopting a sample treatment liquid;
B. directly adding the DNA extract obtained in the step A into the PCR mixed solution 1, the PCR mixed solution 2, the PCR mixed solution 3 and the PCR mixed solution 4 for fully and uniformly mixing;
C. the fluorescent quantitative PCR amplification reaction procedure is:
pre-denaturation reaction: reacting for 3min at 95 ℃;
amplification reaction: 15s at 95 ℃ and 45s at 60 ℃ for 45 cycles;
D. judging the genotype of a sample to be tested;
E. and evaluating the medication risk of the rheumatoid medicament by the genotype of the sample to be tested.
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Inventor after: Yu Ruifang Inventor after: Wang Hong Inventor after: Li Hongdong Inventor after: Deng Bo Inventor after: Fan Liangbo Inventor after: Yang Jingjing Inventor after: Fu Xiaoni Inventor after: Miao Baogang Inventor after: Li Ming Inventor before: Yu Ruifang Inventor before: Wang Hong Inventor before: Li Hongdong Inventor before: Deng Bo Inventor before: Fan Liangbo Inventor before: Yang Jingjing Inventor before: Fu Xiaoni Inventor before: Miao Dangang Inventor before: Li Ming |