CN110643698A - Tacrolimus metabolic gene detection kit and application method thereof - Google Patents
Tacrolimus metabolic gene detection kit and application method thereof Download PDFInfo
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Abstract
The invention discloses a tacrolimus metabolism gene detection kit, which comprises a primer pair shown as SEQ ID No.1-2 for detecting CYP3A56986A > G gene locus mutation and a probe pair shown as SEQ ID No.3-4, a primer pair shown as SEQ ID No.5-6 for detecting ABCB11236C > T gene locus mutation and a probe pair shown as SEQ ID No.7-8 for detecting POR1508C > T gene locus mutation and a primer pair shown as SEQ ID No.9-10 and a probe pair shown as SEQ ID No. 11-12. The kit can accurately judge the tacrolimus metabolism type of a person to be tested, and can judge the tacrolimus metabolism degree 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 tacrolimus 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.
Tacrolimus is a clinical common medicine for preventing rejection reaction after organ transplantation, and is one of immunosuppressant medicines. About 150 million patients need organ transplantation every year in China, tacrolimus is used as a strong and effective immunosuppressant, the individual difference is large, the therapeutic index is narrow, the clinical adverse reaction is obvious, and the most important is nephrotoxicity and neurotoxicity. The same administered dose will vary in blood concentration and in vivo metabolic rate for different patients. The blood concentration of the tacrolimus in the patient with strong metabolism is low, so that the treatment effect is influenced; and the drug toxicity reaction can occur when the required concentration of the tacrolimus in the patient with the weak metabolism type is too high. Therefore, it is necessary to make a reasonable individualized and safe treatment scheme according to the specific condition of the patient to reduce the occurrence of adverse reactions.
Studies have shown that CYP3a5 is the major enzyme in tacrolimus metabolism, and its extent of metabolism directly affects the blood level of tacrolimus in patients, thereby affecting the efficacy of treatment. The research finds that the gene polymorphism of CYP3A5 influences the expression of CYP3A5 enzyme activity. An SNP rs776746(CYP3a5 x 3, 6986A > G) in the CYP3a5 gene (CYP3a5 x 3, 6986A > G) may cause the mRNA of the CYP3a5 gene to be cut too short to form an "incomplete" CYP3a5 protein, thereby causing the activity of the CYP3a5 enzyme protein to be reduced or even disappear. Patients carrying the CYP3a5 mutant allele performed significantly differently during clinical drug treatment than wild-type carriers. When the CYP3A5 gene is mutated, the blood concentration of tacrolimus in a patient is greatly increased, and a series of drug toxic and side effects such as nephrotoxicity, neurotoxicity, hypertension, gastrointestinal disorder and the like are caused. According to statistics, the CYP3A5 x 3 mutant frequency is found to be as high as 75% in Chinese population at present, so that the detection of CYP3A5 genotyping in clinic has good guiding effect on realizing individualized administration and safe administration.
On the basis, the researchers of the invention believe that CYP450 oxidoreductase (POR) is the only electron donor of all liver microsomal cytochrome P450 oxidases and plays an important role in CYP 450-mediated drug metabolism, and the mutation of a specific gene site, such as POR1508C > T, can influence the metabolic level of tacrolimus in a patient by influencing the activity of the CYP 450. The ABCB1 gene is located on human chromosome 7 and contains 28 exons and encodes a transmembrane glycoprotein P-gp of 170 kDa. P-gp protein is considered to be an important factor affecting the absorption and excretion of drugs in vivo. Therefore, the mutation on the ABCB11236C > T gene site can cause the expression level of P-gp to change significantly, and further influences the absorption and metabolism of tacrolimus in human body.
The research focus of the prior art on tacrolimus in-vivo metabolism types only lies in detecting the CYP3A5SNP rs776746 typing, and other points which can influence the tacrolimus in-vivo metabolism are ignored. For example, patent document CN201310316741.6 discloses a primer set and a kit for detecting CYP3a5 genotyping, wherein the kit can detect CYP3a5 genotyping and guide clinical administration schedule of tacrolimus to patients. For another example, patent document CN201510784899.5 discloses a primer set for detecting CYP3a5 gene, which is also used for guiding clinical medication by detecting CYP3a5 genotyping.
In addition, in the prior art, sequencing or PCR-RFLP methods are mostly adopted for detecting the genotyping, and the two methods need to detect samples such as blood, tissues and the like, have high detection cost and are difficult to popularize in clinic. 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 breaking away from the operation requirements and the instrument limitation of the conventional gel imaging and greatly reducing the detection cost.
In summary, the invention provides a tacrolimus metabolism-related gene detection kit and an application method thereof, the conventional CYP3A5 genotyping is combined with POR gene and ABCB1 gene, the metabolic type of an organism to be detected for tacrolimus is jointly determined, and the detection specificity is higher. The invention carries out detection by a fluorescent quantitative PCR platform, has short detection time and low detection cost.
Disclosure of Invention
An object of the present invention is to provide a tacrolimus metabolism gene detection kit; the invention also aims to provide an application method of the tacrolimus metabolism gene detection kit.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention provides a tacrolimus 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 CYP3A56986A > G 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, a primer pair shown in SEQ ID No.9-10 and a probe pair shown in SEQ ID No.11-12 for detecting site mutation of POR1508C > T gene.
The sequence of SEQ ID No.1 is: 5'-AGTATTTTAAACATATAAAAC-3'
The sequence of SEQ ID NO.2 is: 5'-TTCTAGTTCATTAGGGTGTGA-3'
The sequence of SEQ ID NO.3 is: 5'-TTCAATATCTCTTCC-3'
The sequence of SEQ ID NO.4 is: 5'-TTCAGTATCTCTTCC-3'
The sequence of SEQ ID NO.5 is: 5'-ACAGTGATAAATGATTAATCAA-3'
The sequence of SEQ ID NO.6 is: 5'-ATGTGACTGCTGATCACCGCAG-3'
The sequence of SEQ ID NO.7 is: 5'-AGGGCCTGAACCTGA-3'
The sequence of SEQ ID NO.8 is: 5'-AGGGTCTGAACCTGA-3'
The sequence of SEQ ID NO.9 is: 5'-AACGGGACTTGGGGCCGGGGCT-3'
The sequence of SEQ ID NO.10 is: 5'-GCAGCCAGGCCCGCTCCTGGAT-3'
The sequence of SEQ ID NO.11 is: 5'-CCTGCCGGGGAGAAC-3'
The sequence of SEQ ID NO.12 is: 5'-CCTGTCGGGGAGAAC-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 tacrolimus metabolism gene detection kit, which comprises the steps of (1) determining a sample to be detected CYP3A56986A>G、ABCB1 1236C>T and POR1508C>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 5.38-0.24 XM 1-0.37 XM 2-0.19 XM 3-0.03 XM 4,
m1 is related to CYP3A56986A > G gene locus mutation typing, wherein M1 is equal to 1 when the typing is AA, and M1 is equal to 2 when the typing is AG or GG;
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 associated with POR1508C > T locus mutation typing, M3 is 1 when the typing is CC, and M3 is 2 when the typing is CT or TT;
m4 is age.
When P is less than 0.93, the slow tacrolimus metabolism type is obtained;
(ii) is of tacrolimus intermediate metabolic type when 0.93< P < 0.967;
when P is greater than 0.967, it belongs to tacrolimus rapid metabolism type.
In the present invention, the typing of the 3 gene site mutations is shown in the following table:
TABLE 13 typing Table of Gene site mutations
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 the mutation typing of the CYP3A56986A > G 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) taking total free DNA of a sample to be detected as a template, adding a primer pair of SEQ ID No.9-10 and a probe pair of SEQ ID No.11-12 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 the mutation typing of POR1508C > T gene locus;
(4) according to the formula P ═ es/(1+es) The value of P is calculated and,
wherein, S is 5.38-0.24 XM 1-0.37 XM 2-0.19 XM 3-0.03 XM 4, and the value of M1-4 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 a use of genetic locus mutation in the preparation of a product for detecting tacrolimus metabolism type, wherein the genetic locus mutation comprises CYP3A56986A > G gene locus mutation, ABCB11236C > T gene locus mutation and POR1508C > T gene locus mutation.
In a fourth aspect, the invention provides a primer pair and a probe pair for preparing a product for detecting tacrolimus metabolism type, which comprise a primer pair shown as SEQ ID No.1-2 and a probe pair shown as SEQ ID No.3-4 for detecting CYP3A56986A > G gene locus mutation, a primer pair shown as SEQ ID No.5-6 and a probe pair shown as SEQ ID No.7-8 for detecting ABCB11236C > T gene locus mutation, and a primer pair shown as SEQ ID No.9-10 and a probe pair shown as SEQ ID No.11-12 for detecting POR1508C > T gene locus mutation.
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 tacrolimus metabolism gene detection kit can subdivide the tacrolimus metabolism types of a person to be detected into 3 types, namely, a slow tacrolimus metabolism type, an intermediate tacrolimus metabolism type and a fast tacrolimus metabolism type. The traditional method for detecting the CYP3A5 genotyping can only roughly judge the tacrolimus metabolism speed, and cannot classify the tacrolimus in detail. The conclusion calculated by the algorithm based on the three gene detection results is more accurate than that obtained by singly detecting one gene, for example, the accuracy rate is only about 60 percent when the CYP3A5 gene typing is used for judging the metabolic type, and the accuracy rate can reach more than 90 percent when the CYP3A56986A > G, ABCB11236C > T and POR1508C > T gene typing is detected and the metabolic type is judged by the algorithm. Therefore, the detection method using the kit provided by the application has the advantages of 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 Tacrolimus metabolism Gene assay kit
A tacrolimus metabolism gene detection kit comprises:
the primers and the probes for detecting the site mutation of the CYP3A56986A > G gene:
an upstream primer: 5'-AGTATTTTAAACATATAAAAC-3'
A downstream primer: 5'-TTCTAGTTCATTAGGGTGTGA-3'
1, probe 1: 5'-TTCAATATCTCTTCC-3', 5 '-end labeled FAM, 3' -end linked MGB
And (3) probe 2: 5'-TTCAGTATCTCTTCC-3', 5 '-end mark VIC, 3' -end connection MGB
Primers and probes for detecting site mutation of ABCB11236C > T gene:
an upstream primer: 5'-ACAGTGATAAATGATTAATCAA-3'
A downstream primer: 5'-ATGTGACTGCTGATCACCGCAG-3'
1, probe 1: 5'-AGGGCCTGAACCTGA-3', 5 '-end labeled FAM, 3' -end linked MGB
And (3) probe 2: 5'-AGGGTCTGAACCTGA-3', 5 '-end mark VIC, 3' -end connection MGB
Primers and probes for detecting POR1508C > T gene site mutation:
an upstream primer: 5'-AACGGGACTTGGGGCCGGGGCT-3'
A downstream primer: 5'-GCAGCCAGGCCCGCTCCTGGAT-3'
1, probe 1: 5'-CCTGCCGGGGAGAAC-3', 5 '-end labeled FAM, 3' -end linked MGB
And (3) probe 2: 5'-CCTGTCGGGGAGAAC-3', 5 'end mark VIC, 3' end connecting MGB,
in addition, the kit also comprises real-time qPCR MasterMix and ultrapure water.
Specifically, the reaction system for detecting site mutation of CYP3A56986A > G gene is shown in the following table:
TABLE 2 CYP3A56986A > G 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 |
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 |
The reaction system for detecting POR1508C > T gene site mutation is shown in the following table:
TABLE 4 POR1508C > T Gene site mutation detection System
Example 2 tacrolimus metabolism type detection Using tacrolimus metabolism Gene detection kit
(1) Collecting oral epithelial cells of human body (A, male, 34 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 CYP3A56986A > G gene site mutation typing: 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 CYP3A56986A G gene locus of the person to be detected into AA subtype;
(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 CC typing;
(4) determination of POR1508C > T Gene site mutation typing: adding 0.5 mu L of upstream primer (SEQ ID No.9), 0.5 mu L of downstream primer (SEQ ID No.10), 0.5 mu L of probe 1(SEQ ID No.11), 0.5 mu L of probe 2(SEQ ID No.12), 10 mu L of 0.5 mu L, real-time qPCR MasterMix 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 results, and mutating POR1508C > T gene locus of a person to be tested into CC typing;
(5) according to the formula P ═ es/(1+es) And calculating a P value by using the method, wherein the P value is calculated by using S-5.38-0.24 XM 1-0.37 XM 2-0.19 XM 3-0.03 XM 4, wherein M1-1, M2-2, M3-1, M4-34, S-3.19 and P-0.96, so that the to-be-detected person belongs to tacrolimus intermediate metabolic patterns.
Example 3 tacrolimus metabolism type detection Using tacrolimus metabolism Gene detection kit
(1) Human (B, female, 86 years old) oral epithelial cells were collected and total free DNA was extracted: the procedure is as in example 2;
(2) determining CYP3A56986A > G gene site mutation typing: 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 CYP3A56986A G gene locus of the person to be detected is mutated into AG 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;
(4) determination of POR1508C > T Gene site mutation typing: 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 POR1508C > T gene locus of the person to be tested is mutated into CC typing;
(5) according to the formula P ═ es/(1+es) And calculating a P value by using the S-5.38-0.24 xM 1-0.37 xM 2-0.19 xM 3-0.03 xM 4, wherein M1-2, M2-2, M3-1, M4-86, and calculating to obtain S-1.39 and P-0.80, so that the to-be-tested person belongs to tacrolimus slow metabolism.
Example 4 tacrolimus metabolism type detection Using tacrolimus metabolism Gene detection kit
(1) Human (C, male, 10 years old) oral epithelial cells were collected and total free DNA was extracted: the procedure is as in example 2;
(2) determining CYP3A56986A > G gene site mutation typing: 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 CYP3A56986A G gene locus of the person to be detected is mutated into AG 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;
(4) determination of POR1508C > T Gene site mutation typing: 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 POR1508C > T gene locus of the person to be detected is mutated into TT typing;
(5) according to the formula P ═ es/(1+es) And calculating a P value by using the formula S-5.38-0.24 XM 1-0.37 XM 2-0.19 XM 3-0.03 XM 4, wherein M1-2, M2-2, M3-2, M4-84, and calculating to obtain S-3.48 and P-0.97, so that the to-be-tested person belongs to tacrolimus fast metabolism type.
Comparative example 1 detection of tacrolimus metabolism type Using the Prior Art
(1) Collecting 10ml of blood from the 3 testees (A, male, 34 years old; B, female, 86 years old; C, male, 10 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) as a result of the analysis, A was of type AA, B was of type AG, and C was of type AG.
Effect example 1 Tacrolimus metabolism gene assay kit and 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 5 comparison of the results of the tests of the present application with those of the conventional methods
It can be seen from the summary of the above table that the detection kit provided by the present application can be refined into a slow metabolic type, an intermediate metabolic type or a fast metabolic type for the metabolic type of tacrolimus of the subject, and the judgment criteria of the present application are: when P is less than 0.93, the slow tacrolimus metabolism type is obtained; (ii) is of tacrolimus intermediate metabolic type when 0.93< P < 0.967; when P is greater than 0.967, it belongs to tacrolimus rapid metabolism type. However, the existing method can only detect CYP3A56986A > G gene site mutation typing for tacrolimus metabolic types, and then judge the strength or weakness of the tacrolimus metabolic level of a subject according to the typing according to the existing research results, generally, the blood concentration of the GG type mutant is higher than that of AA type and AG type when the same dosage of tacrolimus is taken due to the deletion of metabolic enzyme. Although no GG type appears in the test subjects, the subjects B and C are AG type, the subject A belongs to AA type, and the tacrolimus metabolism levels of the three subjects are equivalent according to the conventional method, the method provided by the invention can judge that the tacrolimus metabolism levels of the three subjects are different, particularly the subjects A and C have small difference in P value, but the two subjects are respectively classified into an intermediate metabolism type and a fast metabolism type according to the judgment standard provided by the invention. Therefore, the method provided by the application can be used for detecting results to classify the metabolic types of tacrolimus more accurately.
Effect example 2 Tacrolimus metabolism Gene detection kit Large sample test
The purpose of the test is as follows: in order to verify the difference between the tacrolimus metabolism gene detection kit prepared by the invention and the existing method for determining the tacrolimus metabolism type by detecting CYP3A56986A > G gene site mutation typing in the aspect of detection accuracy.
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 a subject were collected and total free DNA was extracted as described in example 2, and CYP3A56986A was detected separately>G Gene site mutation typing, ABCB11236C>T locus mutational typing and POR1508C>T gene site mutation typing according to the formula P ═ es/(1+es) Calculating P value according to P5.38-0.24 XM 1-0.37 XM 2-0.19 XM 3-0.03 XM 4<0.93 belongs to the slow tacrolimus metabolism type, 0.93<P<0.967 belongs to the intermediate metabolic form of tacrolimus, P>0.967 belongs to the criteria for tacrolimus rapid metabolism type to classify the metabolic type of the subject. Control group: since alreadyCYP3A56986A was detected>The subjects were reclassified by mutational typing at the G gene locus, by slow metabolism in the GG type and normal metabolism in the AG and AA type. Standard reference group: the clinical metabolism type of the subjects is detected by measuring the blood concentration according to the instruction of the medicine, and the tacrolimus is taken as the reference standard.
And (3) test results: the tacrolimus metabolism patterns of the subjects in the group after the 3 methods were examined are shown in the following table.
TABLE 6 Tacrolimus metabolism patterns of large sample test subjects
| Test group | Tacrolimus rapid metabolism type | Tacrolimus intermetabolic form | Tacrolimus slow metabolism type |
| Number of people | 7 | 79 | 14 |
| Control group | AA type (Normal) | AG type (Normal) | GG type (Slow metabolism) |
| Number of people | 37 | 54 | 9 |
| Set of standard references | Tacrolimus rapid metabolism type | Is normal | Tacrolimus slow metabolism type |
| Number of people | 9 | 76 | 15 |
Compared with the clinical standard reference result, the overall detection result of the test group is closer to that of the standard reference group, and the error of the control group is larger. Taking the detection rate of the slow metabolism type as an example, the slow metabolism proportion of a standard reference group is 15%, the detection rate of the kit prepared by the invention is 14%, the detection rate of a control group is 9%, the detection accuracy of the metabolism type of the control group is only about 60%, and the detection accuracy of a test group can reach more than 90%, which indicates that the detection method for judging the metabolism type by combining CYP3A56986A > G gene locus mutation typing, ABCB11236C > T gene locus mutation typing and POR1508C > T gene locus mutation typing has higher accuracy.
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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Claims (10)
1. A tacrolimus metabolism gene detection kit comprises a primer pair shown in SEQ ID No.1-2 for detecting site mutation of CYP3A56986A > G gene, a probe pair shown in SEQ ID No.3-4, a primer pair shown in SEQ ID No.5-6 for detecting site mutation of ABCB11236C > T gene, a probe pair shown in SEQ ID No.7-8, a primer pair shown in SEQ ID No.9-10 for detecting site mutation of POR1508C > T gene, and a probe pair shown in SEQ ID No. 11-12.
2. The kit of claim 1, wherein each set of probes for detecting gene mutation sites is labeled with different fluorophores at their 5 'ends and with MGB at their 3' ends.
3. The kit of claim 2, wherein the 5' ends of the probe pairs are labeled with FAM fluorophore and VIC fluorophore, respectively.
4. The kit of claim 1, further comprising real-time qPCR MasterMix and ultrapure water.
5. AA method for using the tacrolimus metabolism gene detection kit of claim 1, which comprises the following steps: (1) determination of test sample CYP3A56986A>G、ABCB1 1236C>T and POR1508C>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 5.38-0.24 XM 1-0.37 XM 2-0.19 XM 3-0.03 XM 4,
m1 is related to CYP3A56986A > G gene locus mutation typing, wherein M1 is equal to 1 when the typing is AA, and M1 is equal to 2 when the typing is AG or GG;
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 associated with POR1508C > T locus mutation typing, M3 is 1 when the typing is CC, and M3 is 2 when the typing is CT or TT;
m4 is age.
6. The method for use according to claim 5, wherein the sample to be tested is selected from saliva or oral epithelium.
7. The use method according to claim 5, wherein the use 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 the mutation typing of the CYP3A56986A > G 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) taking total free DNA of a sample to be detected as a template, adding a primer pair of SEQ ID No.9-10 and a probe pair of SEQ ID No.11-12 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 the mutation typing of POR1508C > T gene locus;
(4) according to the formula P ═ es/(1+es) The value of P is calculated and,
wherein, S is 5.38-0.24 XM 1-0.37 XM 2-0.19 XM 3-0.03 XM 4, and the value of M1-4 is shown in claim 5.
8. Use of a genetic locus mutation in the manufacture of a product for assessing tacrolimus metabolic patterns, said genetic locus mutation comprising a CYP3a56986A > G gene locus mutation, ABCB11236C > T gene locus mutation and POR1508C > T gene locus mutation.
9. A primer pair and a probe pair for preparing products for evaluating tacrolimus metabolism types comprise a primer pair shown in SEQ ID No.1-2 and a probe pair shown in SEQ ID No.3-4 for detecting CYP3A56986A > G gene site mutation, a primer pair shown in SEQ ID No.5-6 and a probe pair shown in SEQ ID No.7-8 for detecting ABCB11236C > T gene site mutation, and a primer pair shown in SEQ ID No.9-10 and a probe pair shown in SEQ ID No.11-12 for detecting POR1508C > T gene site mutation.
10. The probe pair of claim 9, wherein each set of probes for detecting gene mutation sites is labeled with different fluorophores at their 5 'ends and labeled with MGB at their 3' ends.
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