CN113817820B - Primer group and kit for evaluating sirolimus drug metabolism and evaluation method - Google Patents
Primer group and kit for evaluating sirolimus drug metabolism and evaluation method Download PDFInfo
- Publication number
- CN113817820B CN113817820B CN202111286896.0A CN202111286896A CN113817820B CN 113817820 B CN113817820 B CN 113817820B CN 202111286896 A CN202111286896 A CN 202111286896A CN 113817820 B CN113817820 B CN 113817820B
- Authority
- CN
- China
- Prior art keywords
- sirolimus
- patient
- genotype
- kit
- drug metabolism
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 229960002930 sirolimus Drugs 0.000 title claims abstract description 75
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 title claims abstract description 75
- 230000036267 drug metabolism Effects 0.000 title claims abstract description 14
- 238000011156 evaluation Methods 0.000 title abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 238000011282 treatment Methods 0.000 claims abstract description 3
- 210000004369 blood Anatomy 0.000 claims description 20
- 239000008280 blood Substances 0.000 claims description 20
- 238000002054 transplantation Methods 0.000 claims description 15
- 101150052538 CYP3A5 gene Proteins 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 24
- 229940079593 drug Drugs 0.000 abstract description 17
- 230000004060 metabolic process Effects 0.000 abstract description 12
- 102000004328 Cytochrome P-450 CYP3A Human genes 0.000 abstract description 11
- 108010081668 Cytochrome P-450 CYP3A Proteins 0.000 abstract description 11
- 230000002503 metabolic effect Effects 0.000 abstract description 9
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 5
- 238000011369 optimal treatment Methods 0.000 abstract description 4
- 230000001506 immunosuppresive effect Effects 0.000 abstract description 3
- 238000001647 drug administration Methods 0.000 abstract description 2
- 238000003205 genotyping method Methods 0.000 abstract description 2
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 12
- 238000002156 mixing Methods 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 230000002980 postoperative effect Effects 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 208000020832 chronic kidney disease Diseases 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 102100034343 Integrase Human genes 0.000 description 2
- 101710203526 Integrase Proteins 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- BJDWURMSYDDCRX-UHFFFAOYSA-N ethoxycarbonyl ethyl carbonate;hydrate Chemical compound O.CCOC(=O)OC(=O)OCC BJDWURMSYDDCRX-UHFFFAOYSA-N 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 230000003907 kidney function Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 101100447462 Brugia malayi G3PD gene Proteins 0.000 description 1
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000003409 anti-rejection Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 208000028208 end stage renal disease Diseases 0.000 description 1
- 201000000523 end stage renal failure Diseases 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000005182 global health Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 231100001095 no nephrotoxicity Toxicity 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer group and a kit for evaluating sirolimus drug metabolism and an evaluation method, and belongs to the fields of molecular biology and biomedicine. The kit of the invention comprises 2 pairs of specific primers, can accurately and specifically amplify gene segments of CYP3A5rs15524 and CYP3A5rs4646453, carries out genotyping by a standard PCR-restriction fragment length polymorphism method, and can accurately detect the genotypes of all sites. The evaluation method of the invention selects two SNP loci of CYP3A5rs15524 and CYP3A5rs4646453 of CYP3A5 which are obviously related to the metabolism of sirolimus at different time points (P <0.05) based on the individual difference of the expression conditions of metabolic enzymes related to sirolimus in human bodies, and can predict the drug metabolism condition of patients before the patients take the drugs, thereby individually designing the drug administration scheme of the sirolimus, reducing the time for the patients to adjust the dosage of the sirolimus to reach the optimal treatment concentration, and having positive significance for guiding clinical use of the sirolimus for immunosuppressive treatment and increasing the clinical safety of the sirolimus.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a primer group and a kit for evaluating sirolimus drug metabolism and an evaluation method.
Background
The world health organization releases the report on the global health assessment in 2019 from 2020, 12, 9, showing that chronic kidney disease has risen to one of ten causes of death worldwide. According to the national cross-section epidemiological survey result, the prevalence rate of adult chronic kidney diseases in China is about 10.8%, and the number of patients with chronic kidney diseases in China is about 1.5 hundred million according to the estimation of the total population number in China. Most of the kidney diseases are hidden, the discomfort symptoms of patients in early stage or initial years are mild and are often ignored, and the end stage of the kidney diseases is developed when obvious symptoms appear. The kidney transplantation is the most effective kidney replacement scheme with the longest life cycle for the patients with end-stage renal diseases, the problem after the kidney transplantation is mainly renal rejection reaction, and the postoperative patients only need to take anti-rejection medicines and carry out periodic review, so that the normal work and life are basically not influenced.
Currently, sirolimus is a second-line immunosuppressant after renal transplantation and has the advantages of no nephrotoxicity and neurotoxicity, and the inventor studies early to show that the conversion to a tetrad low-dose immunosuppressive scheme containing sirolimus in the early stage after renal transplantation of a recipient can effectively improve the transplanted renal function without increasing the risk of adverse reaction. However, early postoperative administration of sirolimus may lead to wound healing disorders and surgical complications such as lymphocysts; meanwhile, sirolimus has huge pharmacokinetic differences among different patients, and complicated drug metabolism can cause a series of surgical complications in early stage after renal transplantation, so that the clinical application of sirolimus is always greatly limited. Thus, serum drug concentration monitoring is being widely applied to individualized concentration monitoring in sirolimus use to minimize postoperative early surgical complications of sirolimus while maintaining a sufficient therapeutic amount of sirolimus serum drug concentration.
The existing serum drug concentration monitoring generally adopts chromatography to detect the concentration of drugs which are combined with plasma protein in a blood sample or are free in plasma in whole blood. The detection method is rapid, simple and convenient, and can effectively detect the concentration of the drugs in the current blood sample. However, this method has hysteresis, and only after a patient takes a certain standardized initial dose, the serum drug concentration is detected, and then the drug dosage is adjusted according to the detected concentration. Moreover, since sirolimus-associated metabolic enzymes have large individual differences in their expression in humans, even if the same starting sirolimus dose corrected by a kilogram body weight is administered, some patients will have a sirolimus serum drug concentration that is not within their therapeutic window after renal transplantation, which may lead to the risk of acute rejection or greater risk of suffering from acute rejection when the serum drug concentration is too low; when the serum drug concentration is too high, it will result in an increased risk of associated surgical complications after renal transplantation.
Therefore, a method for predicting the drug metabolism condition of a patient before the patient takes the drug is urgently needed, so that the initial taking dosage of sirolimus is individually designed, the time for the patient to adjust the dosage of sirolimus to reach the optimal treatment concentration is shortened, and the patient is better treated.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems that the existing detection method for the sirolimus medicament concentration in serum can only detect after a patient takes a certain standardized initial dose, and then the medicament use amount is adjusted according to the detection concentration, so that the medicament adjustment has hysteresis, and the risk of acute rejection or postoperative early-stage operation related clinical complications caused by too low or too high serum medicament concentration of the patient is increased, the invention develops an evaluation method for the sirolimus medicament metabolism according to the individual difference of the expression conditions of sirolimus related metabolic enzymes in a human body, can predict the medicament metabolism condition of the patient before the patient takes the medicament, so that the medication scheme of the sirolimus is designed individually, and the time for the patient to adjust the sirolimus use amount to reach the optimal treatment concentration is particularly important.
2. Technical scheme
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention provides a sirolimus drug metabolism evaluation method, which comprises the following steps:
s1: confirming the genotypes of the two loci of the CYP3A5rs15524 and the CYP3A5rs4646453 of the patient;
s2: sirolimus blood concentration was determined according to the following formula:
the blood sirolimus concentration is-24.223-69.272 Xgender-1.368 Xage (year) +0.204 Xtime after transplantation (day) +24.886 Xrs 464646453-4.627 Xrs 15524 Xthe unit weight dosage ratio of the sirolimus taken every day,
wherein, the values of the parameters in the formula are as follows:
sex: female, gender-0, male, gender-1;
rs 464646453 genotype: AA, rs4646453 ═ 11; for CA, rs4646453 ═ 21; CC, rs4646453 ═ 22;
rs15524 genotype: AA, rs15524 ═ 11; AG, rs15524 ═ 13; when GG is GG, rs15524 is 33.
Sirolimus is metabolized in a human body mainly through demethylation and hydroxylation reactions of CYP3A4 and CYP3A5 of cytochrome P450 enzyme systems, so that CYP3A5 plays an important role in sirolimus metabolism, and the genotype of relevant sites is measured, so that the metabolic state of sirolimus in a patient body can be judged. Single Nucleotide Polymorphism (SNP) is called as a third generation DNA genetic marker, has the characteristics of dense distribution, large quantity, high frequency, binary marker and relatively stable existence, and is considered as a genetic marker with the best application prospect. Therefore, by detecting the SNP site of the sirolimus-related metabolic gene, the genotype of the patient is obtained, and the metabolic state of sirolimus in the recipient can be predicted efficiently and stably at an early stage after the renal transplantation. The inventor discovers the mutation of two SNP sites (CYP3A 5rs 464646453 and CYP3A5rs15524) on the CYP3A5 gene through research, and can reveal the metabolism condition of sirolimus in a patient body.
The invention also provides a primer group for detecting the genotypes of the two SNP loci rs15524 and rs 464646453 on the CYP3A5 gene, which comprises a primer pair 1 and a primer pair 2, wherein the sequences of the primer pairs are shown in the table 1:
TABLE 1 PCR primer set sequences
The invention also provides a kit for detecting the genotypes of the two SNP loci rs15524 and rs4646453 on the CYP3A5 gene, and the kit comprises the primer group.
Preferably, the kit further comprises (1) a total RNA extraction reagent; (2) PCR amplification reagents and sequencing reagents.
Preferably, the total RNA extraction reagent comprises: trizol, chloroform, isopropanol, ethanol, deionized formamide.
Preferably, the PCR amplification and sequencing reagents comprise: PCR primer sets at the two sites, dNTP, PCR buffer, Taq enzyme, a primer specific to an internal reference (G3PD), diethyl pyrocarbonate (DEPC) water, double distilled water and the like.
The invention also provides an application method of the kit, which comprises the following steps:
s11: extracting total RNA of the blood sample;
s12: synthesizing cDNA;
s13: PCR amplification of target gene;
s14: and (5) analyzing the genotype result.
Preferably, the above S11: the extraction of the total RNA of the blood sample comprises the following steps:
(1) placing 2mL of the whole blood sample on ice, adding 1mL of trizol for cracking, and standing for 5 min;
(2) adding 0.2mL of chloroform for 5min, mixing, subpackaging into 1.5mL of centrifuge tubes, violently oscillating for 15s by using a vortex instrument, incubating for 2-3 min at 15-30 ℃, centrifuging the centrifuge tubes at 4 ℃ and 12000rpm for 15min, taking the supernatant into a new centrifuge tube, adding isopropanol with the same volume as the supernatant into each tube, and standing for 10min at 15-30 ℃;
(3) centrifuging the centrifuge tube at 4 deg.C and 12000rpm for 10min, discarding the supernatant, adding 75% anhydrous ethanol to clean the precipitate, and centrifuging at 4 deg.C and 7500rpm for 5 min;
(4) centrifuging, removing supernatant, air drying at room temperature, drying, adding deionized formamide into each tube, blowing, mixing, and storing at-70 deg.C.
Preferably, the above S12: the synthesis of cDNA, comprising the following steps:
(1) taking a 0.5mL microcentrifuge tube, adding the total RNA extracted from 1-5 mu g S11, adding a proper amount of diethyl pyrocarbonate water to a constant volume of 11 mu L, adding 10 mu M oligo (dT) of 12-18 mu L, oscillating, uniformly mixing and centrifuging;
(2) heating the centrifugal tube in an environment of 70 ℃ for 10min, inserting the centrifugal tube into an ice block, and cooling at 0 ℃ for more than 1 min;
(3) adding the mixture into a centrifuge tube, shaking and mixing the reagents at room temperature, centrifuging, and immediately placing in an environment at 42 ℃ for 2-5 min, wherein the proportions of the reagents are as follows:
(4) adding 1 mu L of Superscript II into a centrifuge tube, and incubating for 50min in a water bath at 42 ℃;
(5) then transferring the centrifuge tube to a 70 ℃ environment to heat for 15min to terminate incubation;
(6) placing the centrifugal tube on ice, adding 1 mu L of RNase H, then placing the centrifugal tube in an environment at 37 ℃ for incubation for 20min, and finally placing the centrifugal tube at-20 ℃ for storage for later use;
preferably, the above S13: the PCR amplification of the target gene comprises the following steps:
(1) the following reagents were added to a 0.5mL PCR tube in the proportions:
(2) adding double distilled water into a PCR tube, fixing the volume to 50 mu L, shaking and uniformly mixing, and centrifuging at room temperature;
(3) and (3) placing the PCR tube in a PCR instrument, setting a corresponding program for detection and carrying out corresponding analysis on the data.
Preferably, the above S14: genotype result analysis, comprising the following steps:
sequencing the amplified fragment in step S13 to obtain the genotype.
Preferably, the genotype obtained by the above method is analyzed by software. More preferably, by htsnper1.0 software.
The invention also provides a primer group, a kit and an application of the detection method in an evaluation method of sirolimus drug metabolism for detecting the genotypes of two SNP loci (CYP3A 5rs15524 and CYP3A5rs4646453) on the CYP3A5 gene.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the individual difference of the expression conditions of sirolimus-related metabolic enzymes in a human body, the two SNP sites (CYP3A 5rs15524 and CYP3A5rs4646453) of CYP3A5 which are obviously related to the metabolism of sirolimus at different time points (P <0.05) are selected, so that the drug metabolism condition of a patient can be predicted before the patient takes the drug, the drug administration scheme of the sirolimus is individually designed, and the time for the patient to adjust the dosage of the sirolimus to achieve the optimal treatment concentration is reduced.
(2) The detection primer group and the kit provided by the invention have the advantages that 2 pairs of specific primers contained in the detection primer group and the kit can accurately and specifically amplify gene segments of CYP3A5rs15524 and CYP3A5rs4646453, and the genotypes of all sites can be accurately detected by performing genotyping by a standard PCR-restriction fragment length polymorphism method.
(3) The sirolimus drug metabolism evaluation method provided by the invention is based on the obvious correlation of genotypes of two SNP sites, and has positive significance for guiding clinical use of sirolimus for immunosuppressive treatment and increasing clinical safety of sirolimus on the basis of simple genotype determination steps and accurate results.
(4) The sirolimus drug metabolism evaluation method provided by the invention is established on the research result of a large-sample kidney transplant recipient, is accurate and efficient, has higher practicability, and provides a basis for establishing a proper sirolimus administration scheme by integrating the disease condition and pharmacological action characteristics of a patient.
Drawings
FIG. 1: the influence of two different gene mutants at two significant sites on the metabolism of sirolimus at different time points on CYP3A5, wherein A, B, C is rs4646453 site, D, E, F is rs15524 site;
FIG. 2: the ROC curve is used for verifying the prediction capability of the metabolic state of sirolimus in the queue by using the method.
Detailed Description
The invention is further described with reference to specific examples.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, measure or value. The degree of flexibility for a particular variable can be readily determined by one skilled in the art.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limit values of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed as including all such values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or feature being described.
Example 1
This example provides a relationship between the genotypes of the rs4646453 and rs15524 sites of the CYP3a5 gene and the metabolism of sirolimus in patients. CYP3A5 is an important gene related to sirolimus metabolism, wherein the basic information of rs4646453 and rs15524 sites is shown in Table 2.
TABLE 2 basic information of SNP sites
As can be seen from Table 2, the possible genotypes at the rs4646453 and rs15524 sites are shown in Table 3.
TABLE 3 possible genotypes at the individual sites of SNPs
As can be seen from fig. 1 or table 4, mutations at two SNP sites (CYP3A5 rs4646453 and CYP3A5rs15524) on CYP3A5 gene are significantly related to sirolimus metabolism in patients, and the possible genotypes of the SNP sites can reveal the metabolism of sirolimus in patients.
TABLE 4 relationship between SNP sites on the CYP3A5 Gene and sirolimus metabolism
Note that the P value is less than 0.05, which is significant.
Example 2
This example provides an assessment method of the invention for predicting blood sirolimus levels in a patient.
This study, which included 101 recipients who performed kidney transplantation surgery at the present center (kidney transplantation center, first subsidiary hospital of the university of medical Nanjing), was designed to pass the approval of the ethical Committee of the first subsidiary hospital of the university of medical Nanjing.
The method for estimating the blood sirolimus concentration/unit weight dosage ratio of the patient comprises the following steps:
s1: determining patient basic information
Patient, 24 years old, male, did not develop delayed recovery of renal function after surgery.
S2: determining the genotypes of 2 loci of a patient, and detecting by the kit and the method provided by the invention, wherein the method comprises the following steps:
s21: extracting total RNA of the blood sample:
(1) placing 2mL of the whole blood sample on ice, adding 1mL of trizol for cracking, and standing for 5 min;
(2) adding 0.2mL of chloroform for 5min, mixing, subpackaging into 1.5mL of centrifuge tubes, violently oscillating for 15s by using a vortex instrument, incubating for 2-3 min at 15-30 ℃, centrifuging the centrifuge tubes at 4 ℃ and 12000rpm for 15min, taking the supernatant into a new centrifuge tube, adding isopropanol with the same volume as the supernatant into each tube, and standing for 10min at 15-30 ℃;
(3) centrifuging the centrifuge tube at 4 deg.C and 12000rpm for 10min, removing supernatant, adding 75% anhydrous ethanol, cleaning precipitate, and centrifuging at 4 deg.C and 7500rpm for 5 min;
(4) centrifuging, removing supernatant, air drying at room temperature, drying, adding deionized formamide into each tube, blowing, mixing, and storing at-70 deg.C.
S22: synthesis of cDNA:
(1) taking a 0.5mL microcentrifuge tube, adding 1-5 mu g of extracted RNA, adding proper amount of diethyl pyrocarbonate water to fix the volume to 11 mu L, adding 12-18 mu L of 10 mu M oligo (dT), oscillating, uniformly mixing and centrifuging;
(2) heating the centrifugal tube in an environment of 70 ℃ for 10min, inserting the centrifugal tube into an ice block, and cooling at 0 ℃ for more than 1 min;
(3) adding the mixture into a centrifuge tube, shaking and mixing the reagents at room temperature, centrifuging, and immediately placing in an environment at 42 ℃ for 2-5 min, wherein the proportions of the reagents are as follows:
(4) adding 1 mu L of Superscript II into a centrifuge tube, and incubating for 50min in a water bath at 42 ℃;
(5) then transferring the centrifuge tube to a 70 ℃ environment to heat for 15min to terminate incubation;
(6) placing the centrifugal tube on ice, adding 1 μ L of RNase H, then placing the centrifugal tube in an environment of 37 ℃ for incubation for 20min, and finally placing the centrifugal tube at-20 ℃ for storage for later use.
S23: PCR amplification of target genes
(1) The following reagents were added to a 0.5mL PCR tube in the proportions:
(2) adding double distilled water into a PCR tube, fixing the volume to 50 mu L, shaking and uniformly mixing, and centrifuging at room temperature;
(3) and (3) placing the PCR tube in a PCR instrument, setting a corresponding program for detection and carrying out corresponding analysis on the data.
S24: determination of genotype:
the genotypes of the patient at each site were analyzed by htsnper1.0 software as follows: rs15524 genotype: AA; rs 464646453 genotype: and (C).
S3: sirolimus blood concentration was determined according to the following formula:
the blood sirolimus concentration is-24.223-69.272 Xgender-1.368 Xage (year) +0.204 Xtime after transplantation (day) +24.886 Xrs 464646453-4.627 Xrs 15524 Xthe unit weight dosage ratio of the sirolimus taken every day,
wherein, the values of the parameters in the formula are as follows:
sex: female, gender-0, male, gender-1;
rs 464646453 genotype: AA, rs4646453 ═ 11; for CA, rs4646453 ═ 21; CC, rs4646453 ═ 22;
rs15524 genotype: AA, rs15524 ═ 11; AG, rs15524 ═ 13; when GG is GG, rs15524 is 33.
Based on the basic information and the genotype determination results, the parameters were assigned as follows:
for males, sex is 1;
rs15524 genotype is AA, then rs15524 is 11;
rs 464646453 genotype CC, then rs 46453 ═ 22;
the weight of the patient is 76.5kg after the operation for 30 days, and the dosage of the sirolimus is 1 mg/day.
Substitution calculation, the expected trough concentration of sirolimus for this patient 90 days post-surgery was:
-24.223-69.272 × 1-1.368 × 24+0.204 × 90+24.886 × 22-4.627 × 11 × 1/76.5 ═ 388.628 ═ 5.08 ng/mL. The actually measured sirolimus valley concentration of the patient at 90 days after the operation is 5.07ng/mL, which is basically the same as the predicted valley concentration.
Example 3
Another 101 renal transplant recipients were included as a validation group and monitored for postoperative blood sirolimus levels (see table 5 for general patient information) including:
(1) DNA samples of peripheral blood of 101 patients are extracted, and two sites (rs15524 and rs4646453) in a prediction formula are detected by a qRT-PCR method.
(2) And (4) assigning according to the following conditions.
Sex: female, gender-0, male, gender-1;
rs 464646453 genotype: AA, rs4646453 ═ 11; for CA, rs4646453 ═ 21; CC, rs4646453 ═ 22;
rs15524 genotype: AA, rs15524 ═ 11; AG, rs15524 ═ 13; when GG is GG, rs15524 is 33.
(3) Substituting the assignment into a formula to obtain the blood sirolimus concentration:
blood sirolimus concentration of-24.223-69.272 × sex-1.368 × age (year) +0.204 × time after transplantation (large) +24.886 × rs4646453-4.627 × rs15524 × unit dose ratio of sirolimus per large dose,
(4) the sirolimus drug concentration of the 101 patients at the corresponding postoperative time points was collected, and the sirolimus blood drug concentration was calculated.
And (4) analyzing results: the result is shown in fig. 2, the prediction ability (AUC, area under the curve) of the prediction formula is 79.1%, and the 95% confidence interval is 70.4-87.8%. Therefore, the method can effectively predict the metabolic state of sirolimus in a recipient after renal transplantation.
Table 5 general information to validate cohort inclusion in patients
Claims (2)
1. A method for evaluating sirolimus drug metabolism for non-diagnosis and treatment purposes is characterized by comprising the following steps:
s1: confirming the genotypes of the two sites rs15524 and rs4646453 of the CYP3A5 gene of the patient;
s2: determining the blood sirolimus concentration of said patient according to the following formula:
blood sirolimus concentration = -24.223-69.272 × sex-1.368 × age +0.204 × time after transplantation +24.886 × rs4646453-4.627 × rs15524 × unit weight dosage ratio of sirolimus taken per day,
wherein, the values of the parameters in the formula are as follows: the age is calculated in years and the time after transplantation is calculated in days;
sex: female, gender =0, male, gender = 1;
when the genotype of rs4646453 of the patient is AA, rs4646453= 11; when rs4646453 genotype of patient is CA, rs 46453= 21; when rs 464646453 genotype of patient is CC, rs 46453= 22;
rs15524=11 when the rs15524 genotype of the patient is AA; rs15524=13 when the rs15524 genotype of the patient is AG; when the patient's rs15524 genotype is GG, rs15524= 33.
2. Use of a kit for the method for evaluating sirolimus drug metabolism according to claim 1 for non-diagnostic purposes, wherein the kit is used for detecting genotypes of two loci rs15524 and rs 464646453 of the CYP3a5 gene in the step S1 according to claim 1, and comprises a primer pair 1 and a primer pair 2, wherein the sequence of the primer pair 1 is as follows: 5'-TGAAATCTCTGGTGTTCTGGGG-3' for F, 5'-GGATTCAAGAGATGGAACCCT-3' for R; the sequence of the primer pair 2 is as follows: 5'-AGGGAGGGCCTTGTTCTGA-3' for F and 5'-GTGTTGTTCTGCTATGTGGCA-3' for R.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111286896.0A CN113817820B (en) | 2021-11-02 | 2021-11-02 | Primer group and kit for evaluating sirolimus drug metabolism and evaluation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111286896.0A CN113817820B (en) | 2021-11-02 | 2021-11-02 | Primer group and kit for evaluating sirolimus drug metabolism and evaluation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113817820A CN113817820A (en) | 2021-12-21 |
| CN113817820B true CN113817820B (en) | 2022-05-06 |
Family
ID=78919528
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111286896.0A Active CN113817820B (en) | 2021-11-02 | 2021-11-02 | Primer group and kit for evaluating sirolimus drug metabolism and evaluation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113817820B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109563549A (en) * | 2016-06-03 | 2019-04-02 | 新加坡保健服务集团有限公司 | Biomarker of the hereditary variation as the sensibility to disease treatment in antisense long non-coding RNA |
| CN111424087A (en) * | 2020-04-09 | 2020-07-17 | 合肥诺为尔基因科技服务有限公司 | Detection Panel for pan-cancer species detection or targeted drug application based on next-generation sequencing, kit and application |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007027963A2 (en) * | 2005-08-30 | 2007-03-08 | The Board Of Trustees Of The Leland Stanford Junior University | Methods of screening bifunctional molecules for modulated pharmacokinetic properties |
| CN106987906A (en) * | 2017-05-15 | 2017-07-28 | 重庆市肿瘤研究所 | The construction method in oncotherapy cardiac toxic predicted gene abrupt climatic change library |
| CN110358824A (en) * | 2018-08-13 | 2019-10-22 | 上海佰臻生物科技有限公司 | A kind of detection tacrolimus drug personalized medicine gene detecting kit |
| CN112322723B (en) * | 2020-11-16 | 2023-06-13 | 江苏省人民医院（南京医科大学第一附属医院） | Primer group, kit and evaluation method for evaluating tacrolimus drug metabolism |
-
2021
- 2021-11-02 CN CN202111286896.0A patent/CN113817820B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109563549A (en) * | 2016-06-03 | 2019-04-02 | 新加坡保健服务集团有限公司 | Biomarker of the hereditary variation as the sensibility to disease treatment in antisense long non-coding RNA |
| CN111424087A (en) * | 2020-04-09 | 2020-07-17 | 合肥诺为尔基因科技服务有限公司 | Detection Panel for pan-cancer species detection or targeted drug application based on next-generation sequencing, kit and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113817820A (en) | 2021-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lee et al. | Association of polymorphisms of cytochrome P450 2D6 with blood hydroxychloroquine levels in patients with systemic lupus erythematosus | |
| EP3240911B1 (en) | Detection and treatment of disease exhibiting disease cell heterogeneity and systems and methods for communicating test results | |
| WO2016207653A1 (en) | Detection of chromosome interactions | |
| Bonaccorsi-Riani et al. | Molecular characterization of acute cellular rejection occurring during intentional immunosuppression withdrawal in liver transplantation | |
| CN112322723B (en) | Primer group, kit and evaluation method for evaluating tacrolimus drug metabolism | |
| US20160319351A1 (en) | Genetic variants as markers for use in diagnosis, prognosis and treatment of eosinophilia, asthma and myocardial infarction | |
| WO2012066582A1 (en) | Genetic risk factors of sick sinus syndrome | |
| CN111471758B (en) | Use of a product for detecting mutations in the gene locus for the preparation of a product for assessing the metabolic condition of a patient following tacrolimus administration | |
| CN104450707B (en) | A kind of application of Serum miRNA biomarker | |
| CN111662975A (en) | Application of product for detecting gene locus mutation in preparation of product for predicting or evaluating metabolic condition of patient taking tacrolimus | |
| CN105940116A (en) | Method for screening risk of drug-induced toxicity | |
| Tängdén et al. | A genome-wide association study in a large community-based cohort identifies multiple loci associated with susceptibility to bacterial and viral infections | |
| CN108148908B (en) | Application of molecular marker for diagnosing atherosclerotic renal artery stenosis | |
| CN113817820B (en) | Primer group and kit for evaluating sirolimus drug metabolism and evaluation method | |
| Han et al. | Serum miR‐515‐3p, a potential new RNA biomarker, is involved in gastric carcinoma | |
| Wei et al. | Leveraging a Genomic Instability-Derived Signature to Predict the Prognosis and Therapy Sensitivity of Clear Cell Renal Cell Carcinoma | |
| WO2022165069A1 (en) | Methods and systems for characterizing and treating combined hepatocellular cholangiocarcinoma | |
| WO2013150002A1 (en) | Methods and kits for determining if a subject is predisposed to fast progression of liver fibrosis | |
| CN114990208A (en) | Primer group, kit and evaluation method for evaluating mycophenolic acid drug metabolism | |
| Lin et al. | The differentially expressed genes of human sporadic cerebral cavernous malformations | |
| CN111566229A (en) | Breast cancer molecular typing and distant metastasis risk gene group, diagnosis product and application | |
| CN109504763A (en) | For predicting the molecular labeling of interferon alfa hepatitis B patient curative effect | |
| CN104372004B (en) | One detection method and its application with the susceptible relevant miR-27a Polymorphisms site of myocardial infarction | |
| CN113817818B (en) | Tool for diagnosing allergic airway inflammation | |
| Lin et al. | Differential Diagnosis of Osteoarthritis and Rheumatoid Arthritis by Bioinformatics Analysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |