CN110643699B - Cyclosporin A metabolic gene detection kit and application method thereof - Google Patents
Cyclosporin A metabolic gene detection kit and application method thereof Download PDFInfo
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Abstract
The invention discloses a cyclosporin A metabolism gene detection kit, which comprises a primer pair shown in SEQ ID No.1-2 and a probe pair shown in SEQ ID No.3-4 for detecting site mutation of ABCB13435C > T gene, a primer pair shown in SEQ ID No.5-6 and a probe pair shown in SEQ ID No.7-8 for detecting site mutation of ABCB11236C > T gene. The kit can accurately judge the metabolism type of the cyclosporine A by a person to be detected, and can judge the metabolism degree of the cyclosporine A by combining data, thereby providing a more reliable reference basis for safe and effective clinical medication.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a gene detection kit for detecting cyclosporine A metabolism and an application method thereof.
Background
Drug metabolism (Drug metabolism) refers to a biological conversion process of a Drug in a body, and a Drug metabolic reaction refers to a biological conversion reaction of a Drug in a body, and a product of the biological conversion reaction is a metabolite. Studies have shown that drug metabolism in vivo is primarily controlled by metabolic enzymes. The oxidation and binding reaction in the drug metabolism process, the induction or inhibition of drug metabolizing enzymes, the species, organ tissues and sex differences of drug metabolism, the stereoselectivity differences of drug metabolism, the gene polymorphism of enzymes involved in drug metabolism and the like are all related to drug toxicity, thereby affecting the clinical medication safety. Therefore, the research on drug metabolism can effectively clarify the toxic and side effects possibly generated in clinical medication, and provide theoretical support for guiding clinical reasonable medication and preventing the toxic and side effects.
Cyclosporin A (CsA) is a cyclic polypeptide consisting of 11 amino acids, and is a strong and selective immunosuppressant separated from soil mould. Cyclosporin a is widely used in organ transplantation and treatment of immune diseases, and has been used in recent years for the treatment of refractory nephrotic syndrome and other kidney diseases. Compared with other immunosuppressive agents, cyclosporin a is characterized by acting selectively on T lymphocytes, and has no effect on cells of various lineages in bone marrow. However, renal toxicity is the most important problem in cyclosporin A therapy, and studies have found that cyclosporin A causes changes in the structure and function of glomerular stroma and renal vasculature, leading to renal stroma fibrosis, vascular hyalinization, glomerulosclerosis, and the like, which can occur even if the serum concentration of cyclosporin A is normal. The current clinical routine administration method is to give different patients the same dose of cyclosporin A and then to adjust the dose by real-time monitoring the blood level of the patients after administration. Although the clinically used method can realize individualized administration through tests, the clinical method usually needs to be tried after a certain period of time and a patient needs to take blood for several times, and the initial stage of transplantation of the patient is the most critical stage, so that the failure of safe and effective administration can greatly affect the treatment and possibly cause adverse reactions such as transplantation failure or hepatorenal toxicity. Therefore, it is very necessary to individually administer drugs according to individual differences among different patients.
P-pg is a product encoded by a multidrug resistance gene (MDR1), and P-gp is considered to be an important factor influencing the absorption and excretion of drugs in vivo. After being orally taken into a human body, the cyclosporin A is passively diffused into epithelial cells of a human small intestine, and the P-gp expressed in the epithelial cells of the small intestine continuously transports the cyclosporin A to an intestinal cavity so as to increase the proportion of the cyclosporin A discharged in a prototype form; meanwhile, circulation is formed between the absorption cells and the intestinal lumen, the cyclosporine A is delayed to enter human blood circulation, and the contact of the cyclosporine A and the intestinal CYP3A enzyme is increased, so that the metabolism of the cyclosporine A in the intestinal tract is obviously improved, and the drug absorption is reduced; in addition, the distribution of the cyclosporin A in target tissues can be influenced by P-gp in tissues and blood brain barrier, so that the rejection resistance and toxic reaction of the cyclosporin A are reduced; p-gp in the bile and renal tubules can affect the clearance of cyclosporin a and other substrates.
P-gp is encoded by ABCB1(ATP-binding cassette subset B member 1) gene, its cDNA is 4660bp in full length, contains 28 exons, and can be transcribed to obtain 4.5kb mRNA. The C3435T polymorphic site (rs1045642) of the ABCB1 gene can affect the folding structure of protein through codon change, so that the affinity of the protein to regulatory molecules or substrates is changed, and the metabolism of drugs is affected.
Patent document CN201610654232.8 discloses a biomarker for predicting drug-induced nephrotoxicity of a pediatric patient and an application thereof, and the description mentions that any one or more polymorphisms in CYP3a5 x 3, ABCB 12677G > T, ABCB13435C > T, PRKCB rs11074606, CypA-promoter (-11G/C), TGF- β 1codon25(Arg (25) > Pro) gene polymorphisms can predict the risk of renal toxicity of the pediatric patient caused by using cyclosporine a, and the prediction capability is stronger if 2 or more polymorphisms occur simultaneously; or CYP3A5 x 3, ABCB 12677G > T, ABCB13435C > T, PRKCB rs11074606, CypA-promoter (-11G/C) and TGF-beta 1codon25(Arg (25) > Pro) polymorphism can also predict the risk of using cyclosporine A to cause nephrotoxicity in children patients.
Patent document CN201510137264.6 discloses a set of primers for detecting polymorphisms of CYP3a4, CYP3a5 and MDR1 genes based on the ARMs-PCR method and a kit prepared therefrom. The polymorphism of human CYP3A4, CYP3A5 and MDR1 genes is typed through primer combination, and the primer combination is used for detecting the metabolic capability of related medicines of Chinese population.
In the prior art, sequencing or PCR-RFLP methods are mostly adopted for detecting the genotyping, and the two methods both need to detect samples such as blood, tissues and the like. The application utilizes saliva or oral epithelial cells as a sample to be detected, and has the characteristic of no wound compared with sample detection of blood, tissues and the like. In addition, the invention is based on a fluorescent quantitative PCR platform, and the result can be judged by detecting the change of fluorescence after the detection is finished, thereby the invention is free from the operation requirement and the instrument limitation of the conventional gel imaging and greatly reduces the detection cost.
Disclosure of Invention
The invention aims to provide a cyclosporine A metabolic gene detection kit and an application method of the kit. The kit determines the metabolic type of a person to be tested on cyclosporine A by detecting ABCB13435C > T gene locus mutation typing and ABCB11236C > T gene locus mutation typing and combining a specific algorithm, and guides clinical reasonable and effective medication.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention provides a cyclosporine A metabolic gene detection kit, which comprises a primer pair shown in SEQ ID No.1-2 and a probe pair shown in SEQ ID No.3-4 for detecting site mutation of ABCB13435C > T gene, a primer pair shown in SEQ ID No.5-6 and a probe pair shown in SEQ ID No.7-8 for detecting site mutation of ABCB11236C > T gene.
The sequence of SEQ ID No.1 is: 5'-AAGTTGATCTGTGAACTCTTGT-3'
The sequence of SEQ ID No.2 is: 5'-CTATAGGCCAGAGAGGCTGCCA-3'
The sequence of SEQ ID No.3 is: 5'-AGATCGTGAGGGCAG-3'
The sequence of SEQ ID No.4 is: 5'-AGATTGTGAGGGCAG-3'
The sequence of SEQ ID No.5 is: 5'-AAGAGTTTCTGATGTTTTCTTG-3'
The sequence of SEQ ID No.6 is: 5'-AGCCTCTGCATCAGCTGGACTG-3'
The sequence of SEQ ID No.7 is: 5'-AAGGGCCTGAACCTG-3'
The sequence of SEQ ID No.8 is: 5'-AAGGGTCTGAACCTG-3'
Preferably, the 5 'end of each group of probe pairs for detecting the gene mutation site is respectively marked with different fluorescent groups, and the 3' end is marked with MGB. The invention has no special limitation on the type of the fluorescent group, and the fluorescent group is commonly used in the field. In a preferred embodiment of the invention, the 5' ends of the probe pairs are labeled with FAM fluorophore and VIC fluorophore, respectively.
Preferably, the kit further comprises real-time qPCR MasterMix and ultrapure water.
In a second aspect, the invention provides an application method of a cyclosporine A metabolic gene detection kit, which comprises the steps of (1) determining a sample to be detected ABCB13435C>T and ABCB11236C>Carrying out T gene site mutation typing; (2) analyzing the result, the result being according to the formula P ═ es/(1+es) The value of P is calculated and,
wherein, S is 6.66+0.32 XM 1+0.18 XM 2-0.07 XM 3,
m1 is related to ABCB13435C > T gene locus mutation typing, wherein M1 is 1 when the typing is CT or TT, and M1 is 2 when the typing is CC;
m2 is related to ABCB11236C > T gene locus mutation typing, wherein M2 is 1 when the typing is CT or TT, and M2 is 2 when the typing is CC;
m3 is age.
When P <0.813, is of the cyclosporine a slow metabolic type;
when 0.813< P <0.852, it belongs to the cyclosporin a intermediate metabotropic pattern;
when P >0.852, it belongs to the cyclosporin A fast metabolic form.
In the present invention, the 2 gene site mutations are typed as shown in the following table:
TABLE 12 typing Table of site mutations of genes
In the invention, the sample to be tested is selected from saliva or oral epithelium, and the total free DNA extraction method of the sample to be tested is a conventional DNA extraction method.
Preferably, the application method of the kit comprises the following steps:
(1) taking total free DNA of a sample to be detected as a template, adding a primer pair of SEQ ID No.1-2 and a probe pair of SEQ ID No.3-4 to prepare PCR reaction liquid, transferring the prepared reaction liquid to a 96-well plate, and carrying out PCR reaction; after the reaction is finished, transferring the 96-well plate into a fluorescent quantitative PCR reader, reading the result and determining mutation typing of the ABCB13435C > T gene locus;
(2) taking total free DNA of a sample to be detected as a template, adding a primer pair of SEQ ID No.5-6 and a probe pair of SEQ ID No.7-8 to prepare PCR reaction liquid, transferring the prepared reaction liquid to a 96-well plate, and carrying out PCR reaction; after the reaction is finished, transferring the 96-well plate into a fluorescent quantitative PCR reader, reading the result and determining mutation typing of the ABCB11236C > T gene locus;
(3) according to the formula P ═ es/(1+es) The value of P is calculated and,
wherein, S is 6.66+0.32 XM 1+0.18 XM 2-0.07 XM 3, and the value of M1-3 is as shown above.
Preferably, the preparation method of the PCR reaction solution is as follows:
adding a primer pair, a probe pair and a DNA template to be detected into a reaction system, and then adding real-time qPCR MasterMix and ultrapure water to prepare a PCR reaction solution for PCR.
In a preferred embodiment of the present invention, the PCR reaction procedure is as follows: pre-denaturation at 95 deg.C for 10 min; 95 ℃, 15s, 60 ℃, 1min, 40 cycles; storing at 10 deg.C.
In a third aspect, the invention provides application of genetic locus mutation in preparing a product for detecting the metabolic type of cyclosporine A, wherein the genetic locus mutation comprises ABCB13435C > T genetic locus mutation and ABCB11236C > T genetic locus mutation.
In a fourth aspect, the invention provides a primer pair and a probe pair for preparing a product for detecting the metabolic type of cyclosporine A, which comprise a primer pair shown in SEQ ID No.1-2 and a probe pair shown in SEQ ID No.3-4 for detecting the site mutation of ABCB13435C > T gene, a primer pair shown in SEQ ID No.5-6 and a probe pair shown in SEQ ID No.7-8 for detecting the site mutation of ABCB11236C > T gene.
Preferably, the 5 'end of each group of probe pairs for detecting the gene mutation site is respectively marked with different fluorescent groups, and the 3' end is marked with MGB. The invention has no special limitation on the type of the fluorescent group, and the fluorescent group is commonly used in the field. In a preferred embodiment of the invention, the 5' ends of the probe pairs are labeled with FAM fluorophore and VIC fluorophore, respectively.
The inventive detection kit can divide the metabolism type of the object to be detected for the cyclosporin A into cyclosporin A slow metabolism type, middle metabolism type and fast metabolism type. Compared with the prior art, the metabolic degree of the person to be detected to the cyclosporine A can be further judged by utilizing a specific algorithm on the basis of judging the metabolic type. Therefore, the kit provided by the application has more accurate detection result and better specificity, and can provide more reliable reference data for safe and effective clinical medication.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of Cyclosporin A Metabolic Gene detection kit
A cyclosporin A metabolism gene detection kit, comprising:
primers and probes for detecting site mutation of ABCB13435C > T gene:
an upstream primer: 5'-AAGTTGATCTGTGAACTCTTGT-3'
A downstream primer: 5'-CTATAGGCCAGAGAGGCTGCCA-3'
1, probe 1: 5'-AGATCGTGAGGGCAG-3', 5 '-end labeled FAM, 3' -end linked MGB
And (3) probe 2: 5'-AGATTGTGAGGGCAG-3', 5 '-end mark VIC, 3' -end connection MGB
Primers and probes for detecting site mutation of ABCB11236C > T gene:
an upstream primer: 5'-AAGAGTTTCTGATGTTTTCTTG-3'
A downstream primer: 5'-AGCCTCTGCATCAGCTGGACTG-3'
1, probe 1: 5'-AAGGGCCTGAACCTG-3', 5 '-end labeled FAM, 3' -end linked MGB
And (3) probe 2: 5'-AAGGGTCTGAACCTG-3', 5 '-end mark VIC, 3' -end connection MGB
In addition, the kit also comprises real-time qPCR MasterMix and ultrapure water.
Specifically, the reaction system for detecting site mutation of ABCB13435C > T gene is shown in the following table:
TABLE 2 ABCB13435C > T gene locus mutation detection system
| Composition (I) | Volume of |
| qPCR MasterMix | 10μL |
| Upstream primer | 0.5μL |
| Downstream primer | 0.5μL |
| Probe 1 | 0.5μL |
| Probe 2 | 0.5μL |
| Ultrapure water | 8μL |
| In total | 20μL |
The reaction system for detecting the site mutation of ABCB11236C > T gene is shown in the following table:
TABLE 3 ABCB11236C > T Gene site mutation detection System
| Composition (I) | Volume of |
| qPCR MasterMix | 10μL |
| Upstream primer | 0.5μL |
| Downstream primer | 0.5μL |
| Probe 1 | 0.5μL |
| Probe 2 | 0.5μL |
| Ultrapure water | 8μL |
| In total | 20μL |
Example 2 detection of Cyclosporin A Metabolic type Using Cyclosporin A Metabolic Gene detection kit
(1) Collecting oral epithelial cells of human body (A, female, 37 years old), extracting total free DNA, and extracting total free DNA with QIAamp Circulating Nucleic Acid Kit of QIAGEN company, adjusting DNA concentration to 10-40 ng/. mu.L;
(2) determining the site mutation typing of ABCB13435C > T gene: adding 0.5 mu L of upstream primer (SEQ ID No.1), 0.5 mu L of downstream primer (SEQ ID No.2), 0.5 mu L of probe 1(SEQ ID No.3), 0.5 mu L of probe 2(SEQ ID No.4), 0.5 mu L, real-time qPCR MasterMix10 mu L and 8 mu L of ultrapure water into a reaction system, adding the DNA template prepared in the step (1) to prepare a PCR reaction solution, transferring the prepared reaction solution into a 96-well plate, and carrying out PCR reaction; after the reaction is finished, transferring the 96-well plate to a fluorescent quantitative PCR reader to read the result, and mutating the ABCB13435C > T gene locus of a person to be tested into CC typing;
(3) determining site mutation typing of ABCB11236C > T gene: adding 0.5 mu L of upstream primer (SEQ ID No.5), 0.5 mu L of downstream primer (SEQ ID No.6), 0.5 mu L of probe 1(SEQ ID No.7), 0.5 mu L of probe 2(SEQ ID No.8), 0.5 mu L, real-time qPCR MasterMix10 mu L and 8 mu L of ultrapure water into a reaction system, adding the DNA template prepared in the step (1) to prepare a PCR reaction solution, transferring the prepared reaction solution into a 96-well plate, and carrying out PCR reaction; after the reaction is finished, transferring the 96-well plate to a fluorescent quantitative PCR reader to read the result, and mutating the ABCB11236C > T gene locus of the person to be tested into CT typing;
(4) according to the formula P ═ es/(1+es) P value is calculated from S6.66 +0.32 × M1+0.18 × M2-0.07 × M3, where M1 is 2, M2 is 1, M3 is 37, S is 4.89, and P is 0.99, and thus, the subject is cyclosporine a fast metabolic type.
Example 3 detection of Cyclosporin A Metabolic type Using Cyclosporin A Metabolic Gene detection kit
(1) Human (B, female, 82 years old) oral epithelial cells were collected and total free DNA was extracted: the procedure is as in example 2;
(2) determining the site mutation typing of ABCB13435C > T gene: the method is the same as the embodiment 2, after the reaction is finished, the 96-well plate is transferred to a fluorescent quantitative PCR reader to read the result, and the mutation of the ABCB13435C > T gene locus of a person to be tested is CT typing;
(3) determining site mutation typing of ABCB11236C > T gene: the method is the same as the example 2, after the reaction is finished, the 96-well plate is transferred to a fluorescent quantitative PCR reader to read the result, and the ABCB11236C > T gene locus mutation of the person to be detected is CC typing;
(5) according to the formula P ═ es/(1+es) P value was calculated from S ═ 6.66+0.32 × M1+0.18 × M2-0.07 × M3, where M1 ═ 1, M2 ═ 2, M3 ═ 82, S ═ 1.6, and P ═ 0.83, and therefore, the subjects were cyclosporine a intermediate metabotropic.
Example 4 detection of Cyclosporin A Metabolic type Using Cyclosporin A Metabolic Gene detection kit
(1) Human (C, male, 84 years old) oral epithelial cells were collected and total free DNA was extracted: the procedure is as in example 2;
(2) determining the site mutation typing of ABCB13435C > T gene: the method is the same as the example 2, after the reaction is finished, the 96-well plate is transferred to a fluorescent quantitative PCR reader to read the result, and the ABCB13435C > T gene locus mutation of a person to be tested is TT typing;
(3) determining site mutation typing of ABCB11236C > T gene: the method is the same as the example 2, after the reaction is finished, the 96-well plate is transferred to a fluorescent quantitative PCR reader to read the result, and the locus of the ABCB11236C gene of the person to be detected is mutated into TT parting;
(5) according to the formula P ═ es/(1+es) The P value is calculated from S6.66 +0.32 × M1+0.18 × M2-0.07 × M3, where M1 is 1, M2 is 1, M3 is 84, S is 1.28, and P is 0.78, so that the subject is cyclosporine a slow metabolic type.
Comparative example 1 measurement of cyclosporin A metabolism type by Using the prior art
(1) Collecting 10ml of blood from the 3 testees (A, female, 37 years old; B, female, 82 years old; C, male, 84 years old) by using Streck noninvasive blood collection tubes, transferring the blood into 15ml centrifuge tubes, centrifuging at the low temperature of 4 ℃ for 10min at 2000g, taking about 4-6ml of upper plasma in a new 15ml centrifuge tube, and continuously centrifuging at the low temperature of 4 ℃ for 10min at 4000 g; taking 4-6ml of upper plasma, putting the upper plasma into a new 15ml centrifuge tube, taking 4ml of plasma, and extracting total free DNA by using a QIAamp Circulating Nucleic Acid Kit of QIAGEN company;
(2) DNA sequencing is carried out on the 3 groups of DNA templates by adopting a conventional gene sequencing method;
(3) according to the analysis result, A belongs to CC type, B belongs to CT type, and C belongs to TT type.
Effect example 1 comparison of Cyclosporin A metabolism Gene detection kit with conventional method
Based on the results of the tests of examples 2-4, we can obtain the following results compared to the conventional gene sequencing test method, as shown in the following table:
table 4 comparison of the test results of the present application with those of the conventional method
As can be seen from the summary of the above table, the detection kit provided by the present application can refine the metabolic type of cyclosporine a of a subject into a slow metabolic type, an intermediate metabolic type or a fast metabolic type, and the determination criteria of the present application are: when P <0.813, it is of the cyclosporin a weak metabotropic type; cyclosporine a intermediate metabotropic form when 0.813< P < 0.852; when P >0.852, it is a fast cyclosporin A metabotropic form. The existing method can only detect the mutation typing of ABCB13435C > T gene locus for the cyclosporine A metabolism type, and then judges the intensity or weakness of the subject to the cyclosporine A metabolism level according to the typing according to the existing research result. Although the results of the three subjects tested in the test of the invention are consistent with the prior art, the advantages of the invention are obviously embodied in the subsequent large sample test.
Effect example 2 Cyclosporin A metabolism Gene detection kit Large sample test
The purpose of the test is as follows: in order to verify the difference between the cyclosporine A metabolism gene detection kit prepared by the invention and the existing method for determining the cyclosporine A metabolism type by detecting the site mutation and typing of ABCB13435C > T gene, the detection accuracy is improved.
The test method comprises the following steps: 100 subjects were recruited to participate in the trial, simultaneously in the experimental, control and standard reference groups, experimental groups: oral epithelial cells of the subject were harvested and total free DNA extracted as described in example 2, and ABCB13435C was detected separately>T gene locus mutation typing and ABCB11236C>T gene site mutation typing according to the formula P ═ es/(1+es) Calculating P value according to P6.66 +0.32 XM 1+0.18 XM 2-0.07 XM 3<0.813 belongs to the cyclosporin A slow metabolic form, 0.813<P<0.852 belongs to the cyclosporin A intermediate metabotropic form, P>0.852 belongs to the criteria for fast cyclosporin A metabolome the metabolic patterns of the subjects were classified. Control group: since ABCB13435C has been detected>And (3) carrying out T gene locus mutation typing, wherein CC type is fast metabolism, CT type is normal metabolism, and TT type is slow metabolism, and classifying the subjects again. Standard reference group: the clinical metabolism type of the subjects is detected by measuring the blood concentration according to the cyclosporine A taken by the subjects according to the instruction of the medicine, and the clinical metabolism type is taken as the reference standard.
And (3) test results: the type of cyclosporin A metabolism of the test subjects after examination by 3 methods is shown in the following table.
TABLE 5 Large sample test subjects Cyclosporin A Metabolic types
| Test group | Cyclosporin A fast metabolizing forms | Cyclosporin A intermediate metabotropic form | Cyclosporin A slow metabolism pattern |
| Number of people | 14 | 80 | 6 |
| Control group | CC type (fast metabolism) | CT type (Normal) | TT type (Slow metabolism) |
| Number of people | 19 | 71 | 10 |
| Set of standard references | Cyclosporin A fast metabolizing forms | Is normal | Cyclosporin A slow metabolism pattern |
| Number of people | 9 | 87 | 4 |
The result shows that the detection rate of the standard reference group is 87%, the detection rate of the test group is 80% and the detection rate of the control group is 71% based on the detection ratio of the normal metabolic type in the standard reference group. Therefore, the accuracy rate of only detecting ABCB13435C > T genotyping and judging the metabolic type is only about 80%, and the accuracy rate can reach about 92% by detecting ABCB13435C > T and ABCB11236C > T in a combined manner and judging the metabolic type through an algorithm.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
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aagggtctga acctg 15
Claims (4)
1. A cyclosporine A metabolic gene detection kit comprises a primer and a probe, wherein the primer and the probe consist of a primer pair shown in SEQ ID No.1-2 and a probe pair shown in SEQ ID No.3-4 for detecting the mutation of ABCB13435C > T gene locus, a primer pair shown in SEQ ID No.5-6 and a probe pair shown in SEQ ID No.7-8 for detecting the mutation of ABCB11236C > T gene locus,
the application method of the kit comprises the following steps:
(1) taking total free DNA of a sample to be detected as a template, adding a primer pair of SEQ ID No.1-2 and a probe pair of SEQ ID No.3-4 to prepare PCR reaction liquid, transferring the prepared reaction liquid to a 96-well plate, and carrying out PCR reaction; after the reaction is finished, transferring the 96-well plate into a fluorescent quantitative PCR reader, reading the result and determining mutation typing of the ABCB13435C > T gene locus;
(2) taking total free DNA of a sample to be detected as a template, adding a primer pair of SEQ ID No.5-6 and a probe pair of SEQ ID No.7-8 to prepare PCR reaction liquid, transferring the prepared reaction liquid to a 96-well plate, and carrying out PCR reaction; after the reaction is finished, transferring the 96-well plate into a fluorescent quantitative PCR reader, reading the result and determining mutation typing of the ABCB11236C > T gene locus;
(3) according to the formula P ═ es/(1+es) The value of P is calculated and,
wherein, S is 6.66+0.32 XM 1+0.18 XM 2-0.07 XM 3,
m1 is related to ABCB13435C > T gene locus mutation typing, wherein M1 is 1 when the typing is CT or TT, and M1 is 2 when the typing is CC;
m2 is related to ABCB11236C > T gene locus mutation typing, wherein M2 is 1 when the typing is CT or TT, and M2 is 2 when the typing is CC;
m3 is age;
when P <0.813, is of the cyclosporine a slow metabolic type;
when 0.813< P <0.852, it belongs to the cyclosporin a intermediate metabotropic pattern;
when P >0.852, it belongs to the cyclosporin A fast metabolic form.
2. The kit according to claim 1, wherein the probes for detecting gene mutation sites are labeled with different fluorophores at 5 'ends of SEQ ID Nos. 3-4 and labeled with MGB at 3' ends, and are labeled with different fluorophores at 5 'ends of SEQ ID Nos. 7-8 and labeled with MGB at 3' ends.
3. The kit of claim 2, wherein the probe pair is labeled with FAM fluorophore and VIC fluorophore at 5 'ends of SEQ ID Nos. 3 to 4, respectively, and the probe pair is labeled with FAM fluorophore and VIC fluorophore at 5' ends of SEQ ID Nos. 7 to 8, respectively.
4. The kit of claim 1, further comprising real-time qPCR MasterMix and ultrapure water.
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| AU2006315760A1 (en) * | 2005-11-10 | 2007-05-24 | Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Materials and methods for ABCB1 polymorphic variant screening, diagnosis, and treatment |
| JP2013090622A (en) * | 2011-10-03 | 2013-05-16 | Arkray Inc | Probe for polymorphism detection, polymorphism detection method, drug efficacy determination method, and kit for polymorphism detection |
| CN104630342A (en) * | 2014-11-26 | 2015-05-20 | 中国医科大学 | Kit for detecting ABCB1 C3435T/A at room temperature |
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| CN109554477A (en) * | 2019-01-04 | 2019-04-02 | 中山大学达安基因股份有限公司 | A kind of kit of Multiple detection chemotherapeutics toxicity related gene parting |
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