CN114350788B - A set of probes and a kit for constructing a library for detecting polymorphism of CYP3A5 gene related to pharmacogenomics by utilizing hybridization capture method - Google Patents
A set of probes and a kit for constructing a library for detecting polymorphism of CYP3A5 gene related to pharmacogenomics by utilizing hybridization capture method Download PDFInfo
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Abstract
The invention provides a group of probes and a kit containing the same, which are used for manufacturing a library-building kit for detecting polymorphism of CYP3A5 gene related to pharmacogenomics by utilizing a hybrid capture method. The genotype and the drug metabolism capability of CYP3A5 can be rapidly obtained by one-time detection, and meanwhile, the kit is simple and convenient to operate, consumes less time, is extremely easy to realize automatic detection, has good clinical application prospect in guiding an organ transplant patient to take an initial dose of tacrolimus drug treatment, and is convenient for clinical popularization.
Description
Technical Field
The invention relates to a group of probes and a database-establishing kit for detecting polymorphism of CYP3A5 gene related to pharmacogenomics by utilizing a hybridization capture method.
Background
With the gradual popularization of accurate medical treatment in recent years, pharmacogenomics (PGx) has been rapidly developed as a powerful means for achieving personalized medicine administration. PGx the main research on gene polymorphism affecting individual differences of drug reactions (including curative effects and adverse reactions) relates to genes encoding drug metabolizing enzymes, transporters, action targets and the like.
The cytochrome P450 (CYP 450) family is a family of isoenzymes associated with 90% of clinical drug metabolism. CYP450 metabolic enzymes are mainly responsible for the drug clearance phase I response and are the rate limiting step in drug metabolism, so genes encoding the CYP450 family are the most relevant genetic factors to substrate pharmacokinetics. The human has 18 CYP450 enzyme gene families and 43 subfamilies, and enzyme systems related to the metabolism of most medicaments mainly comprise three families of CYP1, CYP2 and CYP 3. Among them, CYP3A subfamily members have prominent actions, maximum content and large substrate spectrum, and are the most main rate-limiting enzymes in drug metabolism reaction.
The human CYP3A5 gene is located on chromosome 7, has a total length of 31.8kb, comprises 13 exons, and encodes a protein consisting of 502 amino acids. It is one of the most active enzymes in the P4503A family, and it has become a medical research focus because the Single Nucleotide Polymorphism (SNP) of the gene can lead to different metabolic activities of the drug, and different SNPs may also be associated with genetic susceptibility to certain diseases.
The results of the study on genetics suggest that 20% -95% of individual differences in drug response and primary treatment are caused by genetic factors. The CYP3A5 gene and phenotype of the primary patient can be detected to predict the curative effect, prognosis, adverse reaction and the like of the drug treatment, so that the individual drug administration is guided, and the optimal treatment purpose is achieved. For organ transplantation patients, the CyP3A5 genotype is used as the basis of the immunosuppressant personalized treatment, so that the initial dosage of the tacrolimus drug is determined, passive monitoring is changed into active prediction, the incidence rate of acute rejection after transplantation and the toxicity and adverse reaction of the drug can be effectively reduced, and the survival rate of transplanted organs is further improved.
Currently, methods commonly used for detecting CYP3A5 gene polymorphism include DNA sequencing, restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP), high Performance Liquid Chromatography (HPLC), capillary electrophoresis (Capillary Electrophoresis), and the like. The methods have the defects of complex operation, long time consumption, high cost, low detection sensitivity, easiness in causing false positive or false negative and the like.
With the continuous accumulation of pharmacogenomic knowledge and the continuous development of molecular detection techniques, multi-site/multi-drug personalized drug detection is being accepted by the industry. The genetic information of the drug metabolizing enzyme and the action target gene of the common drugs or the drugs (such as cardiovascular and immunosuppression) can be obtained by one-time detection, and the genetic information has decisive influence on the formulation of the personalized medication scheme of drug dosage, drug type selection, drug interaction and the like.
The currently PGx in-vitro diagnostic reagent for obtaining three types of evidence lot cannot cover the gene locus required to be detected clinically, and along with the popularization of the second-generation sequencing technology, high-throughput sequencing can simultaneously sequence millions or even billions of DNA fragments, so that the detection of hundreds of related genes, whole exons and whole genome of pharmacogenomics can be carried out at one time at lower cost, and the required sample size is not increased. The high-throughput sequencing technology has wide application value in the detection of drug metabolizing enzyme and drug action target gene due to the advantages of throughput, cost and efficiency.
Disclosure of Invention
The invention describes a kit for detecting polymorphism of a pharmacogenomics related gene CYP3A5 by utilizing a hybrid capture method, and the kit uses extremely low initial amount of genomic DNA as a template to realize enzyme cutting disruption, tail end repair and A tail addition by one-step reaction; then adding an adapter and magnetic beads for purification; the library construction can be completed through short index amplification, and then the library is quickly hybridized through a personalized custom probe CYP3A5 Panel, so that the hybridization efficiency which can be achieved through the conventional overnight reaction can be achieved in 15 minutes; and then capturing, enriching, amplifying and purifying the hybridization product by using streptomycin magnetic beads to obtain the final targeted library. And then carrying out high-throughput sequencing on the library, and after the data to be tested is taken off, rapidly completing the accurate typing of the CYP3A5 gene by using relevant bioinformatics analysis software and a pharmacogenomics relevant database through bioinformatics analysis and comparison.
The invention provides a group of probes, which comprises at least one of the base sequences SEQ ID NO. 1-372.
Preferably, the set of probes is used to make a pooling kit for detecting polymorphism of the pharmacogenomics-related gene CYP3A5 using a hybrid capture method.
The invention also provides a database creation kit for detecting the polymorphism of the gene CYP3A5 related to pharmacogenomics by utilizing a hybrid capture method, wherein the database creation kit comprises a group of probes, and the group of probes comprises at least one of base sequences SEQ ID NO. 1-372.
The invention also provides a library-building kit for guiding an organ transplant patient to take an initial dose of tacrolimus drug treatment, the library-building kit comprises a group of probes, and the group of probes comprises at least one of base sequences SEQ ID NO. 1-372.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the database creation kit manufactured by the invention can detect the polymorphism of the gene CYP3A5 related to pharmacogenomics by utilizing a hybridization capture method; only a trace amount of genomic DNA is required for constructing a DNA library, and the template initiation amount can be as low as 10ng; the enzyme cutting breaking, tail end filling and A tail adding are completed in a system, and then the autonomous design probe CYP3A5 Panel is utilized to carry out hybridization capturing and enrichment on the library, so that the time consumption is short and the efficiency is high; secondly, the kit index is up to 384 pairs, so that simultaneous construction and on-machine sequencing can be realized; in addition, the rapid hybridization system of the kit can realize the efficiency of general overnight hybridization with the shortest hybridization time of 15min, and finally the obtained library fragments have the size of about 300-400bp and can be compatible with various sequencing platforms of Illumina for sequencing; by a simple and easy-to-use solution of NGS sequencing while synthesizing, the CYP3A5 gene which can influence the absorption, distribution, metabolism and excretion of drugs and compounds is rapidly and accurately typed; the genotype and the drug metabolism capability of CYP3A5 can be rapidly obtained by one-time detection, meanwhile, the kit is simple and convenient to operate, consumes less time, is extremely easy to realize automatic detection, has good clinical application prospect in guiding an organ transplant patient to take an initial dose of tacrolimus drug treatment, and is convenient for clinical popularization.
Drawings
FIG. 1 shows the results of fragment analysis of the library constructed in example 1.
Detailed Description
The invention is described in further detail below with reference to the drawings and the detailed description.
Embodiment 1: construction of libraries
Library construction by enzymatic cleavage
DNA disruption, end repair and addition of A tail (amplified region)
First, the PCR instrument was turned on and the PCR instrument hot cap temperature was set to 70 ℃.
1.0.1DNA the initial quantity of the warehouse building is 10-100 ng, and the mixture is fully and evenly mixed and immediately separated and then is on ice for standby.
1.0.2 to a reagent preparation area, taking 1 new 1.5ml centrifuge tube to prepare enzyme digestion reaction mixed liquor. After 10x Fragmentation Buffer and 5x Fragmentation Enzyme (Twist Bioscience) were thawed on ice, the mixture was prepared according to the following table, and after mixing and centrifugation, the mixture was split into eight-tube and then transferred to the amplification zone through a transfer window. (simultaneously, the DNA purification magnetic bead solution used in the next purification step can be taken out of a refrigerator, fully and uniformly mixed by shaking, and then transferred to an amplification area together, and balanced for at least 30min at room temperature)
| Reagent(s) | Single reaction volume |
| 10x Fragmentation Buffer | 5ul |
| 5x Fragmentation Enzyme | 10ul |
| Total | 40ul |
1.0.3 DNA samples to be detected are respectively added into eight-joint tubes for preparing reaction mixed solution, mixed evenly by light vibration, quickly put into a PCR instrument after transient separation, and the temperature of a thermal cover of the PCR instrument is determined to be 70 ℃, and then the following procedures are adopted:
connection Universal adapter
1.0.4 Each reaction system after completion of the above steps was added with 5. 5uL Universal adapter (Twist Bioscience), respectively. Mixing with light vibration, centrifuging, and placing on ice for use.
1.0.5 to a reagent preparation area, and preparing a connection reaction mixed solution. After DNA Ligation Buffer and DNALation Mix (Twist Bioscience) were thawed on ice, a mixture was prepared according to the following table, and the mixture was centrifuged on ice and transferred to the amplification zone for use.
| Reagent(s) | Single reaction volume |
| Water | 15ul |
| DNA Ligation Buffer | 20ul |
| DNA Ligation Mix | 10ul |
| Total | 45ul |
1.0.6 to each of the samples prepared in 1.0.3 steps, 45ul of the ligation mixture prepared in 1.1.2 steps was added. Mix by gentle shaking, centrifuge and place into PCR instrument rapidly, connect for 15 minutes at 20deg.C (hot lid closed). After the completion, the sample was taken out, and immediately, the magnetic bead purification was started.
First step of magnetic bead purification (amplification region)
1.1.1 library purification magnetic beads (Vazyme) need to be taken out from 4 ℃ before use, fully vortex, shake and mix uniformly, and equilibrate at room temperature for at least 30min for later use.
1.1.2 the purified bead suspension (Vazyme) equilibrated at room temperature was mixed by shaking for 1min and dispensed into 1.5ml centrifuge tubes at 80ul (0.8 times the sample volume) per sample.
1.1.3 transferring all the connection products into the magnetic beads corresponding to the centrifuge tube, and gently shaking and uniformly mixing to avoid generating bubbles.
1.1.4 after leaving the centrifuge tube to stand at room temperature for 5min, it was placed on a magnetic rack for 5min until the supernatant was clear. The supernatant was carefully removed.
1.1.5 holding centrifuge tubes on a magnetic rack, 200ul of freshly prepared 80% ethanol was added to each tube, and left to stand at room temperature for 1min, and the supernatant was pipetted off.
1.1.6 repeating step 1.2.5, placing the centrifuge tube back to the magnetic rack after instantaneous centrifugation, and sucking all residual ethanol solution at the bottom of the tube as much as possible by using a 10ul pipette.
1.1.7 centrifuge tube is left on a magnetic rack, and is uncapped for 5min for drying at room temperature, so that the ethanol is volatilized completely, and excessive drying is forbidden.
1.1.8 to each tube, 17ul of enzyme-free water was added, and the mixture was mixed up and down 10 times using a pipette. Incubate at room temperature for 5min.
1.1.9 Each tube was placed on a magnetic rack for 5min until the supernatant was clear.
UDI linker primer PCR amplification
1.2.1 step 1.2.9 the standing period can be reached to a reagent preparation area, according to the sample number, eight-way pipes are prepared on an ice box, 25ul KAPA HiFiHotStartReadyMix (KAPA) is taken and packaged into each hole of the eight-way pipes.
1.2.2 adding 10ul of pre-set UDI primers of different indices (Twist Bioscience, REF of UDI primers 101308, 101309, 101310, 101311, each REF containing 96 pairs of UDI primers, split into 96 well plates, 4 REFs corresponding to 4 plates, 384 pairs total) to each well of the octal tube. And then transferred to the amplified I region through a transfer window.
1.2.3 according to the set position sequence, sucking up 15ul of purified supernatant obtained in the step 1.2.9, adding into each hole of the split-packed PCR reaction mixture, covering a tube cover, gently shaking, and briefly centrifuging to collect the liquid.
1.2.4 transfer the formulated PCR reaction tube to the amplification zone through a transfer window for PCR reaction. The reaction procedure was set up in the following table at 105℃on the hot lid.
Second step of magnetic bead purification (amplification region)
1.3.1 library purification magnetic beads (Vazyme) need to be taken out from 4 ℃ before use, fully vortex, shake and mix uniformly, and equilibrate at room temperature for at least 30min for later use.
1.3.2 the purified bead suspension at room temperature was mixed by shaking for 1min and dispensed into 1.5ml centrifuge tubes at 50ul (equal sample volume)/sample.
1.3.3 taking out PCR reaction products, centrifuging for 30s by a centrifuge, transferring all the products into magnetic beads corresponding to the centrifuge tube, and gently shaking to avoid generating bubbles.
1.3.4 standing at room temperature for 5min, and placing on a magnetic separation frame for 5min until the supernatant is clear. The supernatant was carefully removed.
1.3.5 placed on a magnetic separation rack, 200ul of freshly prepared 80% ethanol was added, and left to stand at room temperature for 1min, and the supernatant was aspirated off.
1.3.6 repeat step 1.4.5 and remove as much as possible all residual ethanol solution.
1.3.7 centrifuge tube is left on the magnetic rack, and is uncapped for 5min for drying at room temperature, so that all ethanol is ensured to volatilize completely.
1.3.8 22ul of enzyme-free water was added and gently shaken. Incubate at room temperature for 5min.
1.3.9 placed on a magnetic rack for 5min, 20ul of supernatant (library) was taken to enter the next step of library enrichment. After 10-fold dilution of 1ul, library concentration and fragment size measurements were performed using a Qubit and fragment analyzer, respectively, and the average fragment length of the detected library should be around 350 bp.
Hybrid capture
Library hybridization
2.0.1 pool DNA mixing protocol: the library pool often contains a plurality of different sample libraries, and the library pool entrance is calculated according to the library DNA concentration, and the library pools are obtained by mixing. The total amount of DNA in the library pool is 1.5-4 mug, and a mass sample is recommended to establish the library pool. The amounts of DNA library used are referred to in the following table:
| number of mixed samples | The amount of each library | Total amount of each reaction library |
| 1 | 1000ng | 1000ng |
| 2 | 1000ng | 2000ng |
| 3 | 1000ng | 3000ng |
| 4 | 800ng | 3200ng |
| 8 | 400ng | 3200ng |
2.0.2 calculating the amounts of the different libraries, mixing the libraries in a 1.5ml centrifuge tube, gently shaking, and instantaneously separating for later use.
2.0.3 the following pre-hybridization reagents are added to the mixed sample, and the mixture is uniformly mixed and centrifuged instantaneously, so that bubbles are not generated as much as possible.
2.0.4 opening the tube cover of the mixed prehybridization reagent tube, placing the tube in a vacuum concentrator, balancing, and drying at 45 ℃ until the solution in the tube is completely dried. The drying process is about 30 minutes to 1 hour.
2.0.5 drying to the end, fast Hybridization Mix (Twist Bioscience) at 65 ℃ can be incubated for 10min or until all the precipitate is dissolved, then the PCR instrument procedure is set up according to the following table, hot cap 85 ℃:
| temperature (temperature) | Time | |
| 95 | 5min | |
| 60℃ | 15min～4h |
After 2.0.6 drying, the centrifuge tube was removed from the desiccator, the sealing film was peeled off, fast Hybridization Mix (Twist Bioscience) was swirled rapidly and 20uL was taken and added to the tube to resuspend the sample (please not return the hybridization solution to room temperature), the fingertip was flicked and mixed well, air bubbles were avoided, and the tube was allowed to stand at room temperature for 5min. The pipette is used for blowing and sucking for 10 times up and down, and after DNA is completely dissolved, all the solutions in the tube are transferred into a new PCR tube as far as possible, so that bubbles are avoided.
2.0.7 to the tube was again added 30uL of paraffin oil to cover the inner reagent surface and then centrifuged briefly to remove air bubbles.
2.0.8 the PCR tube is put into a preheated PCR instrument for hybridization and incubated for 15 min-2 h. (Streptavidin Binding Beads (Twist Bioscience) for the next capture can be removed from the refrigerator during this period to the reagent preparation area and equilibrated to room temperature for at least 30min after thoroughly shaking and mixing).
Library capture and washing
2.1.1 to reagent preparation zone, the enrichment reagents (single enrichment reaction reagent dose) were dispensed into 1.5ml centrifuge tubes as follows, and then the components and equilibrated magnetic bead components (Streptavidin Binding Beads) were transferred to the amplification zone through a transfer window.
| Component (A) | The required volume |
| Fast Binding Buffer(Twist Bioscience) | 900ul |
| Fast Wash Buffer 1(Twist Bioscience) | 450ul |
| Wash Buffer 2(Twist Bioscience) | 700ul |
2.1.2 entering an amplification zone, preheating 450ulFast Wash Buffer 1 at 66 ℃ on a constant temperature metal bath; 700ulWash Buffer 2 is preheated at 48 ℃.
2.1.3 shaking pre-equilibrated Binding Beads (Twist Bioscience) until completely mixed, taking 100uL of magnetic Beads into a 1.5mL centrifuge tube;
2.1.4 adding 200uLFast Binding Buffer into the centrifuge tube, and blowing and uniformly mixing by using a gun head;
2.1.5 placing the centrifuge tube on a magnetic rack for 1min or clarifying the solution, discarding the supernatant, and taking down the centrifuge tube;
2.1.6 repeating the above 2.1.4-2.1.5 washing steps 2 times, 3 times in total;
2.1.7, after the third cleaning, adding 200uLFast Binding Buffer, and shaking and re-suspending to fully and uniformly mix;
2.1.8 after hybridization in step 2.0.8, the cover of the PCR instrument is opened and the hybridization solution is rapidly transferred to the well-balanced bead solution. Note that: the rapid transfer of reagents from the PCR instrument to the beads is a critical step and must be performed directly on the PCR instrument without removing the hybridization tube to prevent the hybridization solution from decreasing in temperature.
2.1.9 the tube is capped, then the mouth of the tube is sealed by a sealing film, and then the tube is placed on a Biocate oscillator at 1200rpm, and fully mixed for 30min at room temperature.
2.1.10 taking off the centrifuge tube from the mixer, placing the centrifuge tube on a magnetic rack for 1min after quick centrifugation, removing the supernatant, taking off the tube, adding 200uL of preheated Fast Wash Buffer 1 (without taking the centrifuge tube out of the metal bath), and gently shaking and mixing uniformly;
2.1.11 the centrifuge tube is rapidly placed at 66 ℃ for 5min of incubation;
2.1.12 placing the centrifuge tube on a magnetic rack for 1min after instantaneous separation, removing the supernatant, taking down the tube, adding 200ul of preheated Fast Wash Buffer 1 again, and gently shaking and mixing uniformly;
2.1.13 the centrifuge tube is rapidly placed at 66 ℃ for 5min of incubation;
2.1.14 transferring all liquid in the centrifuge tube to a new 1.5ml centrifuge tube after the centrifuge tube is instantaneously detached; placing on a magnetic rack for 1min, and removing supernatant;
2.1.15 remove the tube, add 200uL of preheated Wash Buffer 2 (without removing it from the metal bath), gently shake and mix;
2.1.16 the centrifuge tube is rapidly placed at 48 ℃ for 5min of incubation;
2.1.17 placing the centrifuge tube on a magnetic rack for 1min after instantaneous separation, removing the supernatant, and taking down the tube;
2.1.18 repeating (steps 2.1.15-2.1.17) the washing 2 times for a total of three times;
2.1.19 the centrifuge tube is instantaneously separated and then placed on a magnetic rack, and a 10uL gun head is used for sucking clean washing liquid;
2.1.20 the centrifuge tube is removed from the magnetic rack, 45uL of non-ribozyme water is added, gently shaken and mixed, the solution is incubated on ice, and if PCR amplification is not performed temporarily, the magnetic bead mixture can be stored at 20 ℃.
PCR amplification
2.2.1 into the reagent preparation area, 1 PCR tube was taken, a mixture was prepared, and (25 ul KAPA HiFiHotStartReadyMix (KAPA) +2.5ul Amplification Primers (Twist Bioscience) =22.5 ul) was added to the tube, respectively, and then the PCR tube was transferred to the amplification I area through a transfer window. ( Note that: at this time, DNA Purification Beads (Vazyme) is taken out of the refrigerator, mixed by shaking, transferred to the amplification region together with the PCR tube, and equilibrated to room temperature for at least 30min )
2.2.2 taking 22.5ul of the magnetic bead suspension obtained in step 2.1.20, adding into a PCR tube, gently shaking, mixing and instantaneous centrifugation. The remaining 22.5ul may be stored to-20℃for further use.
2.2.3 PCR tubes were placed on a PCR instrument, hot-capped 105℃and amplified according to the following reaction procedure:
magnetic bead purification (amplification zone)
2.3.1 vortex fully mixing the pre-equilibrated DNA purification magnetic beads (Vazyme), taking a 1 clean 1.5ml centrifuge tube, adding 90ul (1.8 x) DNA purification magnetic beads, after PCR is finished, instantly centrifuging a PCR tube, then fully transferring all the solutions in the PCR tube into the 1.5ml centrifuge tube, and fully mixing the solutions by vortex;
2.3.2 incubating the centrifuge tube at room temperature for 5min;
2.3.3 placing the centrifuge tube on a magnetic rack for 1min, and removing the supernatant after the solution is clarified;
2.3.4 holding centrifuge tube on magnetic rack, adding newly prepared 80% ethanol 200ul, incubating for 1min, and discarding supernatant; repeat one 80% ethanol wash (2 total);
2.3.5 placing the centrifuge tube on a magnetic rack after instantaneous centrifugation, carefully removing residual ethanol by using a 10ul gun head, and standing for 5-10min at room temperature or until the magnetic beads are dried, wherein dry cracking is avoided;
2.3.6 removing the tube from the magnetic rack, adding 32ul of non-ribozyme water, gently shaking, mixing, and incubating at room temperature for 5min;
2.3.7 placing the centrifuge tube on a magnetic rack for 3min or clarifying the solution;
2.3.8 transfer 30ul of supernatant to a clean 1.5ml centrifuge tube, cover the tube with the designation of library name and date of construction, and transfer to library quality control area. And (3) injection: the enriched library after purification can be stored below-20 ℃ for 30 days if it cannot be immediately sequenced on-machine.
2.3.9 1ul of the mixture is taken and used
dsDNA HS Assay Kit (Vazyme) the library concentration was determined and the library concentration should be greater than 0.5ng/ul. The fragment length distribution of the library can be detected by using a 2100 fragment analyzer (Agilent), and the average fragment length should be 300-400bp.
The CYP3A5 Panel hybridization capture probe sequence is as follows:
example 2 clinical sample testing
4 volunteer whole blood samples were taken 1 for each, and the 4 samples were examined for CYP3A5 gene polymorphism. The genomic DNA of the whole blood of 4 volunteers was extracted first, and then a library was constructed according to the method described in example 1, and a library was successfully constructed (see fig. 1), and the library was sequenced on Illumina high throughput sequencer, sequencing parameters PE: 2X 150. And then performing quality control on the off-machine data, performing bioinformatics analysis on the data by utilizing a related database such as PharmGKB, CPIC and the like to obtain the CYP3A5 gene polymorphism distribution of each sample, and verifying each site by one-generation sequencing, wherein the coincidence rate is 100%.
Volunteer 1 test results:
volunteer 2 test results:
volunteer 3 test results:
| gene | Detection result | Genotype of the type | Metabolic phenotype |
| CYP3A5 | c.219-237A>G:C/C | *3/*3 | Slow metabolism (pool) Metabolizer) |
Volunteer 4 test results:
from the detection result, the kit can detect the polymorphism of the CYP3A5 gene and accurately type the CYP3A5 gene. The CYP3A5 Panel hybridization capture probe can accurately capture CYP3A5 genes. Detection of conventional samples shows that the probe, the method and the kit can detect CYP3A5 gene polymorphism.
Claims (4)
1. The set of probes is characterized by comprising base sequences SEQ ID NO. 1-372.
2. The set of probes according to claim 1, wherein the set of probes is used to create a pooling kit for detecting polymorphism of the pharmacogenomic-associated gene CYP3A5 using a hybrid capture method.
3. A library construction kit for detecting polymorphism of a pharmacogenomics related gene CYP3A5 by utilizing a hybrid capture method, wherein the library construction kit comprises a group of probes, and is characterized in that the group of probes consists of base sequences SEQ ID NO. 1-372.
4. A kit for the construction of a library for directing an organ transplant patient to take an initial dose of tacrolimus drug therapy, said kit comprising a set of probes, characterized in that said set of probes consists of the base sequences SEQ ID No. 1-372.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009027982A (en) * | 2007-07-27 | 2009-02-12 | Toray Ind Inc | Estimation of drug interaction and blood immunosuppressant concentration variation in organ transplant patient to whom immunosuppressant is administered in combination with proton pump inhibitor |
| KR101633783B1 (en) * | 2016-02-29 | 2016-07-08 | 서울대학교산학협력단 | Method of prediction the blood concentration of tacrolimus using SNP in CYP3A5 and CYP3A7 genes |
| CN106399528A (en) * | 2016-10-14 | 2017-02-15 | 上海市第人民医院 | Tacrolimus metabotropic diagnostic marker and application thereof |
| US9938576B1 (en) * | 2012-09-21 | 2018-04-10 | Ohio State Innovation Foundation | Materials and methods for determining metabolizer status in humans |
| CN108179180A (en) * | 2017-10-25 | 2018-06-19 | 广州和康医疗技术有限公司 | A kind of method and kit that genotype detection is carried out to CYP3A5*3 sites |
| CN112322723A (en) * | 2020-11-16 | 2021-02-05 | 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 |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009027982A (en) * | 2007-07-27 | 2009-02-12 | Toray Ind Inc | Estimation of drug interaction and blood immunosuppressant concentration variation in organ transplant patient to whom immunosuppressant is administered in combination with proton pump inhibitor |
| US9938576B1 (en) * | 2012-09-21 | 2018-04-10 | Ohio State Innovation Foundation | Materials and methods for determining metabolizer status in humans |
| KR101633783B1 (en) * | 2016-02-29 | 2016-07-08 | 서울대학교산학협력단 | Method of prediction the blood concentration of tacrolimus using SNP in CYP3A5 and CYP3A7 genes |
| CN106399528A (en) * | 2016-10-14 | 2017-02-15 | 上海市第人民医院 | Tacrolimus metabotropic diagnostic marker and application thereof |
| CN108179180A (en) * | 2017-10-25 | 2018-06-19 | 广州和康医疗技术有限公司 | A kind of method and kit that genotype detection is carried out to CYP3A5*3 sites |
| CN112322723A (en) * | 2020-11-16 | 2021-02-05 | 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 |
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