CN101760528A - Medicine metabolic relevant loci detection method - Google Patents
Medicine metabolic relevant loci detection method Download PDFInfo
- Publication number
- CN101760528A CN101760528A CN200810208219A CN200810208219A CN101760528A CN 101760528 A CN101760528 A CN 101760528A CN 200810208219 A CN200810208219 A CN 200810208219A CN 200810208219 A CN200810208219 A CN 200810208219A CN 101760528 A CN101760528 A CN 101760528A
- Authority
- CN
- China
- Prior art keywords
- mark
- seq
- taqman probe
- gene
- 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.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a medicine metabolic relevant loci detection method, which comprises the following steps: extracting genome DNA from human samples; respectively designing a Taqman probe pair and a primer pair according to at least two medicine metabolic relevant genes; respectively marking the 5' end and the 3'end of the Taqman probe pair with fluorescence reporting genes and fluorescence quenching genes; carrying out fluorescence quantitative PCR augmentation on the genome DNA; and judging whether the medicine metabolic relevant genes have the mutation according to the fluorescence quantitative PCR augmentation results. Preferably, the number of the medicine metabolic relevant genes is four, the Taqman probe pair and the primer pair are used for detecting a loci rs1057910 of a gene CYP2C9, a loci rs4244285 of a gene CYP2C19, a loci rs4986893of a gene CYP2C19, a loci rs1065852 of a gene CYP2D6 and a loci rs28371759 of a gene CYP3A4. The invention has the advantages of ingenious design, simple operation and accurate and reliable detection results, and provides the reference frame for determining whether professional doctors are needed to be consulted so as to make sure the medicine can be taken or not or the proper dosage and the like when a certain medicine is taken.
Description
Technical field
The present invention relates to function related locus detection technique field, more specifically, relate to medicament metabolism ability related locus detecting method technical field, be meant a kind of drug metabolism related locus detecting method especially.
Background technology
Cytochrome P 450 enzymes (cytochrome P450, CYP), claim mixed-function oxidase (mixed functionoxidase) and monooxygenase (monooxygenase) again, mainly be present in the hepatomicrosome, play crucial effect in the exogenous compounds bio-transformation of (comprising medicine and poisonous substance), the metabolic rate of its activity decision medicine has direct relation with the clearance rate of medicine, be the phasel enzyme of drug metabolism, thereby be called drug metabolism enzyme again.Its energy metabolism and/or activate many kinds of medicines, and procarcinogen, preceding poisonous substance and mutagen.The gene of P450 enzyme system mainly contains CYP1, CYP2, and three families of CYP3, relevant have following several important P450 enzyme: CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4.
Human many drug metabolism CYP450 enzymes have heritable variation, studies show that, many hypotypes of CYP450 enzyme as: the activity of enzymes such as CYP1A1, CYP1A2, CYP2A6, CYP2C9, CYP2D6 and CYP2E1 distributes the crowd and is genetic polymorphism.The polymorphism of cytochrome P 450 enzymes is to cause individual important factor to medicament metabolism ability difference, we are divided into the liver metabolism ability according to person under inspection's genotype: not good figure metabolizer (PM), osculant metabolizer (IM), extensively four types of type metabolizer (EM) and super type metabolizer (UM), the liver metabolism ability is weak more, various kinds of drug is big more to hepatotoxicity, and the power of liver drug metabolic capacity is determined by each subtype gene type of cytochrome P 450 enzymes.The medicine that the different loci of cytochrome P 450 enzymes is corresponding different, associated metabolic medicine as the CYP2C9 gene comprises anti-inflammation analgesia medicine (diclofenac, for sieve former times health, Ibuprofen BP/EP, Naproxen Base, piroxicam, mefenamic acid, flurbiprofen, indomethacin), antiepileptic drug (Phenytoin Sodium Salt, Carbamzepine), anticoagulant (warfarin, temparin), hypoglycemic agents (tolbutamide, Glipizide, Glyburide, glimepiride), other (losartan, torasemide, tamoxifen, dronabinol, amitriptyline) etc., and the enzyme clearance rate of CYP2C9 variation has only 15%～20% of wild-type, easily produce the substrate Side effects of pharmaceutical drugs, especially warfarin, Phenytoin Sodium Salt and some antidiabetic drug medicines are (as Glyburide, glimepiride), cause bleeding as the S-Warfarin, tolbutamide causes hypoglycemic shock.Therefore, as run under the situation of not good figure metabolizer (PM) and osculant metabolizer (IM), medication please consulting profession doctor suggestion.
Therefore, in order earlier to understand medicament metabolism ability, and then when taking some drugs, accomplish to shoot the arrow at the target, avoid some drugs to cause side effect, need to go out by the accurate examination of gene test in advance the situation of said medicine metabolism related locus, thereby understand individual gene physique and liver metabolism ability metabolism intensity to medicine, assist the possible drug reaction of prediction, judgement via medical practitioner, the most suitable your list of medications is provided, and the untoward reaction of avoiding may unconformable medicine being caused takes place.
Summary of the invention
Main purpose of the present invention is exactly the problems and shortcomings at above existence, a kind of drug metabolism related locus detecting method is provided, the design of this detection method is ingenious, simple to operate, detected result accurately and reliably, whether need when taking some drugs the consulting profession doctor to provide reference frame with determine whether to take and taking dose etc.
To achieve these goals, the technical solution used in the present invention is as follows:
This drug metabolism related locus detecting method is characterized in, comprises step:
A. extract genomic dna from people's sample;
B. design a pair of Taqman probe respectively to right according at least 2 drug metabolism genes involveds with a pair of primer, 5 ' end that described Taqman probe is right and 3 ' end be mark fluorescent reporter group and fluorescent quenching group respectively, respectively described genomic dna is carried out fluorescent quantitative PCR;
C. judge according to the fluorescent quantitative PCR result whether described drug metabolism genes involved undergos mutation.
Preferably, the number of described drug metabolism genes involved is 4.
More preferably, described drug metabolism genes involved is CYP2C9 gene, CYP2C19 gene, CYP2D6 gene and CYP3A4 gene.
Further, described Taqman probe to described primer to being used to detect the rs1057910 site of CYP2C9 gene, the rs4244285 site of CYP2C19 gene, the rs4986893 site of CYP2C19 gene, the rs1065852 site of CYP2D6 gene, the rs28371759 site of CYP3A4 gene.
Preferably, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:1 and the SEQ ID NO:2,5 ' end difference flag F AM mark and the HEX mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4.
Preferably, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:5 and the SEQ ID NO:6,5 ' end difference flag F AM mark and the HEX mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:7 and the SEQ ID NO:8.
Preferably, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:9 and the SEQ ID NO:10,5 ' end difference flag F AM mark and the HEX mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:11 and the SEQ ID NO:12.
Preferably, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:13 and the SEQ ID NO:14,5 ' end difference flag F AM mark and the HEX mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:15 and the SEQ ID NO:16.
Preferably, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:17 and the SEQ ID NO:18,5 ' end difference flag F AM mark and the TET mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:19 and the SEQ ID NO:20.
Innovative point of the present invention is that the relevant marquis of several drug metabolisms examines the integration in site, examine the site by the marquis who chooses a plurality of drug metabolisms, according to the CYP2C9 gene, the CYP2C19 gene, CYP2D6 gene and CYP3A4 gene design Auele Specific Primer and Taqman probe carry out fluorescent quantitative PCR to the genomic dna from people's sample, can know that according to the amount of the fluorescence that produces and the kind of fluorescence the marquis examines the genotype in site, design ingenious, simple to operate, detected result accurately and reliably, thereby understand individual gene physique and liver metabolism ability metabolism intensity as early as possible to medicine, assist the possible drug reaction of prediction, thereby when needs are taken some drugs, running under the situation of not good figure metabolizer (PM) and osculant metabolizer (IM), the suggestion of medical consultation medical practitioner, judgement via medical practitioner, optimal list of medications is provided, and the untoward reaction of avoiding the unconformable medicine of possibility to be caused takes place.Generally speaking, be the osculant of certain medicine or good figure metabolizer not if the gene test result shows the person under inspection, this person under inspection answers decrement to take when using this medicine; If the person under inspection is extensive type metabolizer, this person under inspection please takes by doctor's advice when using this medicine; If the person under inspection is super type metabolizer, when using this medicine, this person under inspection please use by dosage.
Embodiment
Content for a better understanding of the present invention is described further below in conjunction with specific embodiment.
1. extracting genome DNA:
1.1 reagent and instrument:
1.1.1 reagent and consumptive material: genome DNA extraction test kit (TIANamp BLOOD DNA kit, DP318-03; TIANamp Micro micro-example genome DNA extracting reagent kit, DP316; TIANamp buccal swab genome DNA extracting reagent kit DP322-03) contains: cell pyrolysis liquid CL; Damping fluid GA; Damping fluid GS; Damping fluid GB; Damping fluid GD; Rinsing liquid PW; Elution buffer TB; Carrier RNA; RNase-free ddH2O; Proteinase K (20mg/ml); Adsorption column; Collection tube (2ML); 1.5ML aseptic collection tube; Dehydrated alcohol.
1.1.2 instrument: DENVILLE260 whizzer, XW-80A vortex mixer, H.H.S pointer-type electric-heated thermostatic water bath.
1.2 extraction step
From whole blood, extract genomic dna
1.2.1 the anticoagulated whole blood of getting 200 μ l is to the Eppendorf pipe of 1.5ml, if the amount of sample is less than 200 μ l, then adds damping fluid GS and is supplemented to 200 μ l.If sample size is more than 200 μ l, need to handle with cell pyrolysis liquid CL, concrete steps are as follows: add the cell pyrolysis liquid CL of 1-2.5 times of volume in sample, put upside down mixing, 10, centrifugal 1 minute of 000rpm, draw supernatant, stay nucleus precipitation (, can repeat above step once) if cracking is not thorough, add 200 μ l damping fluid GS in the centrifugal nucleus precipitation of collecting, vibration is to thorough mixing.
1.2.2 add the Proteinase K solution of 20 μ l, mixing adds the damping fluid GB of 200 μ l again, fully puts upside down mixing at once.56 ℃ of water-bath samples 10 minutes were please softly put upside down the mixing sample up and down every 3 minutes in the water-bath process, the solution strain is limpid.(thoroughly do not become limpid as solution, can prolong the cracking time to solution becomes limpid till).
1.2.3 add 200 μ l dehydrated alcohols, fully put upside down mixing, at this moment flocks may appear.
1.2.4 previous step gained solution and flocks are all added in the adsorption column (adsorption column is put into collection tube), 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, adsorption column is put into collection tube.
1.2.5 in adsorption column, add 500 μ l damping fluid GD (please checking to have added dehydrated alcohol whether earlier before using), 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, adsorption column is put into collection tube.
1.2.6 in adsorption column, add 700 μ l rinsing liquid PW (please checking to have added dehydrated alcohol whether earlier before using), 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, adsorption column is put into collection tube.1.2.7 in adsorption column, add 500 μ l rinsing liquid PW, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube.
1.2.8 adsorption column is put back in the collection tube, 12,000rpm (～13,400 * g) centrifugal 2 minutes, outwell waste liquid.Adsorption column places room temperature to place several minutes, thoroughly to dry rinsing liquid remaining in the sorbing material.
1.2.9 adsorption column is changed in the clean centrifuge tube, and to adsorption film mid-way unsettled Dropwise 5 0-200 μ l elution buffer TB, room temperature was placed 2-5 minute, and 12,000rpm (～13,400 * g) centrifugal 2 minutes, solution is collected in the centrifuge tube.
Attention: the elution buffer volume should not be less than 50 μ l, the too small organic efficiency that influences of volume.For increasing the yield of genomic dna, the centrifugal solution that obtains can be added in the adsorption column again room temperature placement 2 minutes, 12,000rpm (～13,400 * g) centrifugal 2 minutes.The pH of elutriant has a significant impact for elution efficiency.
Should guarantee that its pH value (can be transferred to this scope with the pH value of water with NaOH) in the 7.0-8.5 scope if water is cooked elutriant, the pH value is lower than 7.0 and understands the reduction elution efficiencies; And the DNA product should be kept at-20 ℃, in case dna degradation.
2.Taqman probe method detects the marquis and examines the site:
2.1 experiment reagent and instrument
2.1.1 experiment reagent
Fluorescent quantitation reagent: ABI TaqMan 2 * PCR Master mix (P/N:4326614, Lot:G15502).
Regular-PCR reagent: the Ex Taq archaeal dna polymerase of precious biotechnology (Dalian) company limited (P/N:DRR100B, Lot:CKA1801A).
2.1.2 laboratory apparatus: ABI 9700 type PCR instrument and ABI 7900HT type quantitative real time PCR Instrument.
2.1.3 detection site probe and primer table
2.2 experimental procedure
2.2.1 the concentration of all sample DNAs is adjusted to 20ng/ul～50ng/ul.
2.2.2 the site is detected
The a.PCR reaction system:
The b.PCR cycling condition:
2.2.3 judge the genotype of corresponding candidate locus according to the kind of the amount of the fluorescence that discharges in the reaction process and fluorescence.
3. interpretation of result:
Analyze according to the final terminal point fruit somatotype figure that reads to harden, the result is as follows:
Reaction 1: be used to detect the rs1057910 site of CYP2C9 gene, the associated metabolic medicine comprises: anti-inflammation analgesia medicine (diclofenac, for sieve former times health, Ibuprofen BP/EP, Naproxen Base, piroxicam, mefenamic acid, flurbiprofen, indomethacin), antiepileptic drug (Phenytoin Sodium Salt, Carbamzepine), anticoagulant (warfarin, temparin), hypoglycemic agents (tolbutamide, Glipizide, Glyburide, glimepiride), other (losartan, torasemide, tamoxifen, dronabinol, amitriptyline etc.).There are three kinds of genotype: AA in this time inspection site in the crowd, AC and CC, wherein carrying the AA genotype is that normal population is that extensive type metabolizer is represented with EM, the liver metabolism ability that the crowd who carries AC carries the genotypic crowd of AA is wanted weak and is represented with IM, the crowd who carries CC carries that preceding two kinds of genotype crowds' liver metabolism ability is the most weak to be represented with PM, the size of liver metabolism ability power is AA type＞AC type＞CC type, somatotype map analysis result shows that the rs1057910 site is AA, is extensive type metabolizer EM;
Reaction 2: be used to detect the rs4244285 site of CYP2C19 gene, the associated metabolic medicine comprises: antimalarial drug (chloroguanide), thymoleptic (amitriptyline, clomipramine, imipramine, citalopram), anticonvulsive drug (mephenytoin, Phenytoin Sodium Salt, ethotoin, diazepam, phenylethyl barbituric acid, Mephogarbital), anti-ulcerative drug (omeprazole, lansoprazole), other (Hexobarbital, Proprasylyte, carisoprodol, thioridazine, warfarin etc.).There are three kinds of genotype: GG in this time inspection site in the crowd, AG and AA, wherein carrying the GG genotype is that normal population is that extensive type metabolizer is represented with EM, carrying liver metabolism ability that the genotypic crowd of AG carries the genotypic crowd of GG wants weak and represents with IM, the crowd who carries AA carries that preceding two kinds of genotype crowds' liver metabolism ability is the most weak to be represented with PM, the size of liver metabolism ability power is GG type＞AG type＞AA type, somatotype map analysis result shows that the rs4244285 site is GA, is osculant metabolizer IM;
Reaction 3: be used to detect the rs4986893 site of CYP2C19 gene, the associated metabolic medicine comprises: antimalarial drug (chloroguanide), thymoleptic (amitriptyline, clomipramine, imipramine, citalopram), anticonvulsive drug (mephenytoin, Phenytoin Sodium Salt, ethotoin, diazepam, phenylethyl barbituric acid, Mephogarbital), anti-ulcerative drug (omeprazole, lansoprazole), other (Hexobarbital, Proprasylyte, carisoprodol, thioridazine, warfarin etc.).There are three kinds of genotype: GG in this time inspection site in the crowd, AG and AA, wherein carrying the GG genotype is that normal population is that extensive type metabolizer is represented with EM, carrying liver metabolism ability that the genotypic crowd of AG carries the genotypic crowd of GG wants weak and represents with IM, the crowd who carries AA carries that preceding two kinds of genotype crowds' liver metabolism ability is the most weak to be represented with PM, the size of liver metabolism ability power is GG type＞AG type＞AA type, somatotype map analysis result shows that the rs4986893 site is GG, is extensive type metabolizer EM;
Reaction 4: be used to detect the rs1065852 site of CYP2D6 gene, the associated metabolic medicine comprises: anodyne (oxycodone, dihydrocodeine, morphine monomethyl ether, U-26225A), antipsychotic drug (risperidone, thioridazine, trilafon, leoponex, trifluperidol, Fluphenazine, chlorpromazine, haloperidol), thymoleptic (clomipramine, nortriptyline, amitriptyline, imipramine, Trimipramine, Pertofran, zolpidem, amiflamine, fluoxetine, paroxetine, fluvoxamine, tomoxetine (treatment hyperkinetic syndrome), trazodone, Venlafaxine), normal medicine (the Propafenone of anti-rhythm of the heart husband, mexiletine, flecainide, En Nika, sparteine, Ajmaline, Lilly 99170), beta-blockers (metoprolol, pindolol, timolol, carvedilol, bufuralol, Proprasylyte, bufetolol, Bopindolol, alprenolol), antihypertensive drug (Debrisoquine, Indoramine, envacar, urapidil, nicergoline), other (Dextromethorphane Hbrs, Ethylmorphine, indolapril, tacrine, piperazine Ke Xilin, phenformin, Loratadine, ondansetron, promethazine etc.).There are three kinds of genotype: CC in this time inspection site in the crowd, CT and TT, carry CC and CT genotypic for normal population be that extensive type metabolizer is represented with EM, carrying liver metabolism ability that the genotypic crowd of TT carries CC and the genotypic crowd of CT wants weak and represents with IM, the size of liver metabolism ability power is CC type and CT type＞TT type, somatotype map analysis result shows that the rs1065852 site is CT, is extensive type metabolizer EM;
Reaction 5: the rs28371759 site that is used to detect the CYP3A4 gene, associated metabolic medicine: microbiotic (erythromycin, clarithromycin, troleomycin, Azythromycin, Rifampin), antifungal drug (itraconazole, KETOKONAZOL), antiviral drug (Indinavir, ritonavir, Saquinavir, zidovudine), anodyne (alfentanil fentanyl, morphine monomethyl ether, Cocaine, dihydrocodeine, oxycodone, methadone, paracetamol, U-26225A), antipsychotic drug (haloperidol, pimozide, leoponex), thymoleptic (amitriptyline, imipramine, clomipramine, nefazodone, Sertraline, trazodone, citalopram, cyclobenzaprine), anticonvulsive drug (Carbamzepine, Trimethadione, felbamate), sedative hypnotic (triazolam, diazepam, zolpidem), normal medicine (the amiodarone of anti-rhythm of the heart husband, disopyramide, lignocaine, Quinidine, Propafenone), calcium antagonist (felodipine, nifedipine, nitrendipine, amlodipine, nicardipine, Isrodipine, verapamil, Odizem, Mibefradil), lipid regulating agent (Simvastatin, lovastatin, fluvastatin, Cerivastatin, bezafibrate, fenofibrate, gemfibrozil), antihistaminic (astemizole, terfenadine, Loratadine, ebastine, azelastine), hormones (testosterone, androsterone, cortisone, gestodene, estradiol, dexamethasone, mifepristone, methylprednisolone, Progesterone, prednisolone), antitumour drug (endoxan, ifosfamide, vinealeucoblastine(VLB), tamoxifen, vincristine(VCR), purple refined alcohol), immunosuppressor (ciclosporin, tacrolimus), other (chloroguanide, colchicine, grapefruit juice (beverage has interaction with multiple medicine), Virga (vigour, viagra), Glyburide, the R--warfarin, zileuton, ondansetron, cisapride, finasteride, flutamide, Thiopental Sodium, Sha Tuosiqiong, losartan, quinine, ropivacaine, omeprazole, dapsone, Dextromethorphane Hbr, Ethylmorphine etc.).There are three kinds of genotype: TT in this time inspection site in the crowd, CT and CC, carry TT and CT genotypic for normal population be that extensive type metabolizer is represented with EM, carrying liver metabolism ability that the genotypic crowd of CC carries CT and the genotypic crowd of TT is eager to excel and represents with UM, the size of liver metabolism ability power is CC type＞TT type and CT type, somatotype map analysis result shows that the rs28371759 site is TT, is extensive type metabolizer EM;
According to above-mentioned detected result, can judge the rs1057910 site of above-mentioned inspected CYP2C9 gene, the rs4986893 site of CYP2C19 gene, the rs1065852 site of CYP2D6 gene and the rs28371759 site of CYP3A4 gene are normal, be extensive type metabolizer EM, only the rs4244285 site of CYP2C19 gene is unusual, belong to osculant metabolizer IM, the person under inspection needs suitably to note antimalarial drug (chloroguanide), thymoleptic (amitriptyline, clomipramine, imipramine, citalopram), anticonvulsive drug (mephenytoin, Phenytoin Sodium Salt, ethotoin, diazepam, phenylethyl barbituric acid, Mephogarbital), anti-ulcerative drug (omeprazole, lansoprazole), other (Hexobarbital, Proprasylyte, carisoprodol, thioridazine, warfarin) etc. medicine takes, best consulting profession doctor during medication, judgement via medical practitioner, optimal list of medications is provided, and the untoward reaction of avoiding the unconformable medicine of possibility to be caused takes place.
In sum, drug metabolism related locus detecting method of the present invention design is ingenious, simple to operate, detected result accurately and reliably, whether need when taking some drugs the consulting profession doctor to provide reference frame with determine whether to take and taking dose etc.
Need to prove that all documents of mentioning are in the present invention quoted as a reference in this application, are just quoted as a reference separately as each piece document.Should understand in addition, above-described is specific embodiments of the invention and the know-why used, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications and not deviate from spirit of the present invention and scope the present invention, and these equivalent form of values fall within the scope of the invention equally.
Sequence table
＜110〉Jikang Biotechnology Co Ltd, Shanghai
＜120〉a kind of drug metabolism related locus detecting method
<160>20
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(18)
＜223〉the Taqman probe that designs according to people CYP2C9 gene order
<400>1
agagatacat?tgaccttc 18
<210>2
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(17)
＜223〉the Taqman probe that designs according to people CYP2C9 gene order
<400>2
agataccttg?accttct 17
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(22)
＜223〉upstream primer that designs according to people CYP2C9 gene order
<400>3
ccaggaagag?attgaacgtg?tg 22
<210>4
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(25)
＜223〉downstream primer that designs according to people CYP2C9 gene order
<400>4
cggtgatggt?agaggtttaa?aaatg 25
<210>5
<211>16
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(16)
＜223〉the Taqman probe that designs according to people CYP2C19 gene order
<400>5
ttatttcccg?ggaacc 16
<210>6
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(17)
＜223〉the Taqman probe that designs according to people CYP2C19 gene order
<400>6
tttcccagga?acccata 17
<210>7
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(28)
＜223〉upstream primer that designs according to people CYP2C19 gene order
<400>7
ttctcttaga?tatgcaataa?ttttccca 28
<210>8
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(28)
＜223〉downstream primer that designs according to people CYP2C19 gene order
<400>8
ccaaaatatc?actttccata?aaagcaag 28
<210>9
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(22)
＜223〉the Taqman probe that designs according to people CYP2C19 gene order
<400>9
accccctgga?tccaggtaag?gc 22
<210>10
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(23)
＜223〉the Taqman probe that designs according to people CYP2C19 gene order
<400>10
accccctgaa?tccaggtaag?gcc 23
<210>11
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(28)
＜223〉upstream primer that designs according to people CYP2C19 gene order
<400>11
aaaattgaat?gaaaacatca?ggattgta 28
<210>12
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(25)
＜223〉downstream primer that designs according to people CYP2C19 gene order
<400>12
aatgtacttc?agggcttggt?caata 25
<210>13
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(20)
＜223〉the Taqman probe that designs according to people CYP2D6 gene order
<400>13
tgcacgctac?ccaccaggcc 20
<210>14
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(22)
＜223〉the Taqman probe that designs according to people CYP2D6 gene order
<400>14
ctgcacgcta?ctcaccaggc?cc 22
<210>15
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(21)
＜223〉upstream primer that designs according to people CYP2D6 gene order
<400>15
ccatttggta?gtgaggcagg?t 21
<210>16
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(20)
＜223〉downstream primer that designs according to people CYP2D6 gene order
<400>16
tggaagtcca?catgcagcag 20
<210>17
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(21)
＜223〉the Taqman probe that designs according to people CYP3A4 gene order
<400>17
cacgagctcc?ggatcggaca?g 21
<210>18
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(24)
＜223〉the Taqman probe that designs according to people CYP3A4 gene order
<400>18
acgagctcca?gatcggacag?agct 24
<210>19
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(25)
＜223〉upstream primer that designs according to people CYP3A4 gene order
<400>19
atgccctaca?ttgatctgat?ttacc 25
<210>20
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(20)
＜223〉according to the downstream primer of people CYP3A4 sequences Design
<400>20
cgtggtttca?tagccagcaa 20
Claims (9)
1. a drug metabolism related locus detecting method is characterized in that, comprises step:
A. extract genomic dna from people's sample;
B. design a pair of Taqman probe respectively to right according at least 2 drug metabolism genes involveds with a pair of primer, 5 ' end that described Taqman probe is right and 3 ' end be mark fluorescent reporter group and fluorescent quenching group respectively, respectively described genomic dna is carried out fluorescent quantitative PCR;
C. judge according to the fluorescent quantitative PCR result whether described drug metabolism genes involved undergos mutation.
2. drug metabolism related locus detecting method according to claim 1 is characterized in that, the number of described drug metabolism genes involved is 4.
3. drug metabolism related locus detecting method according to claim 2 is characterized in that, described drug metabolism genes involved is CYP2C9 gene, CYP2C19 gene, CYP2D6 gene and CYP3A4 gene.
4. drug metabolism related locus detecting method according to claim 3, it is characterized in that, described Taqman probe to described primer to being used to detect the rs1057910 site of CYP2C9 gene, the rs4244285 site of CYP2C19 gene, the rs4986893 site of CYP2C19 gene, the rs1065852 site of CYP2D6 gene, the rs28371759 site of CYP3A4 gene.
5. drug metabolism related locus detecting method according to claim 1, it is characterized in that, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:1 and the SEQ ID NO:2,5 ' end difference flag F AM mark and the HEX mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:3 and the SEQID NO:4.
6. drug metabolism related locus detecting method according to claim 1, it is characterized in that, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:5 and the SEQ ID NO:6,5 ' end difference flag F AM mark and the HEX mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:7 and the SEQID NO:8.
7. drug metabolism related locus detecting method according to claim 1, it is characterized in that, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:9 and the SEQ ID NO:10,5 ' end difference flag F AM mark and the HEX mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:11 and the SEQ ID NO:12.
8. drug metabolism related locus detecting method according to claim 1, it is characterized in that, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:13 and the SEQ ID NO:14,5 ' end difference flag F AM mark and the HEX mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:15 and the SEQ ID NO:16.
9. drug metabolism related locus detecting method according to claim 1, it is characterized in that, described Taqman probe is to being the nucleotide sequence shown in SEQ ID NO:17 and the SEQ ID NO:18,5 ' end difference flag F AM mark and the TET mark that described Taqman probe is right, the equal mark TAMRA mark of 3 ' end, described primer is to being the nucleotide sequence shown in SEQ ID NO:19 and the SEQ ID NO:20.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810208219A CN101760528B (en) | 2008-12-26 | 2008-12-26 | Medicine metabolic relevant loci detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810208219A CN101760528B (en) | 2008-12-26 | 2008-12-26 | Medicine metabolic relevant loci detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101760528A true CN101760528A (en) | 2010-06-30 |
| CN101760528B CN101760528B (en) | 2012-09-05 |
Family
ID=42491906
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810208219A Expired - Fee Related CN101760528B (en) | 2008-12-26 | 2008-12-26 | Medicine metabolic relevant loci detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101760528B (en) |
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173443A (en) * | 2013-03-22 | 2013-06-26 | 卫生部北京医院 | CYP2C9 gene fragment containing 1300A>T mutation, protein fragment encoded through same and application thereof |
| CN104156632A (en) * | 2014-07-21 | 2014-11-19 | 金华市中心医院 | Method for intelligently designing drug metabolic enzyme detecting gene chip probes |
| CN104232757A (en) * | 2014-07-29 | 2014-12-24 | 杭州艾迪康医学检验中心有限公司 | Method and oligonucleotide for detecting mutation site of CYP2C19*2 |
| CN104561301A (en) * | 2014-12-31 | 2015-04-29 | 中国医学科学院阜外心血管病医院 | Detection method for CYP2C9*3 gene polymorphism, as well as nucleic acid probe and kit for method |
| CN104561300A (en) * | 2014-12-31 | 2015-04-29 | 中国医学科学院阜外心血管病医院 | Detection method for CYP2C9*2 gene polymorphism, as well as nucleic acid probe and kit for method |
| CN105648082A (en) * | 2016-03-01 | 2016-06-08 | 常州市中医医院 | Primer-probe combination and kit for detecting associated genotyping of warfarin medication |
| CN105779601A (en) * | 2016-04-07 | 2016-07-20 | 江苏苏博生物医学科技有限公司 | Drug metabolic enzyme related gene SNP fluorescence labeling composite amplification kit |
| JP2016192939A (en) * | 2015-04-01 | 2016-11-17 | 東洋鋼鈑株式会社 | Probe for detecting cyp2c19*2 and probe for detecting cyp2c19*3 |
| CN106367479A (en) * | 2016-08-25 | 2017-02-01 | 杭州百迈生物股份有限公司 | Detection composition for guiding hypertension medication, applications of detection composition, kit and detection method |
| CN106754991A (en) * | 2016-12-28 | 2017-05-31 | 温州医科大学 | Including 972C>CYP3A4 genetic fragments, coded protein fragments and its application that A is mutated |
| CN107523612A (en) * | 2017-05-08 | 2017-12-29 | 韩林志 | Primer sets, kit and method for the detection of children's safety medication related gene |
| CN107760778A (en) * | 2017-10-25 | 2018-03-06 | 广州和康医疗技术有限公司 | A kind of detection method and its kit that genotype detection is carried out to CYP2C9 sites |
| CN107937506A (en) * | 2017-11-16 | 2018-04-20 | 南京迪安医学检验所有限公司 | A kind of kit of molecular beacon probe method detection mankind's CYP2C19 gene pleiomorphisms, method and its application |
| CN108929904A (en) * | 2018-07-26 | 2018-12-04 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs1057910 |
| CN108998515A (en) * | 2018-08-24 | 2018-12-14 | 北京青航基因科技有限公司 | SNP combination and the application in the product of preparation detection drug response related gene |
| CN108998512A (en) * | 2018-08-14 | 2018-12-14 | 上海芯超生物科技有限公司 | The hands-free kit and detection method for CYP2C19 genetic polymorphism detection taken |
| CN109055553A (en) * | 2018-08-23 | 2018-12-21 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs4244285 |
| CN109182485A (en) * | 2018-10-22 | 2019-01-11 | 北京华夏时代生物工程有限公司 | Detection method is sequenced in the fluorescence in situ hybridization of CYP gene mononucleotide polymorphism |
| CN109706234A (en) * | 2017-10-26 | 2019-05-03 | 山东中科翰康医学检验有限公司 | Antipyretics object personalization gene detecting kit |
| CN112501265A (en) * | 2020-09-24 | 2021-03-16 | 杭州百迈生物股份有限公司 | Reagent, detection method and kit for detecting CYP2D6 gene |
| CN113249467A (en) * | 2021-06-08 | 2021-08-13 | 北京大学第一医院 | Metabolic typing method of CYP2D6 gene related to psychotropic drugs in Chinese population |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908188B (en) * | 2005-08-01 | 2011-01-19 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for detecting genotype of gene CYP1A2 associated with medicament metabolism ability and liver cancer susceptibility |
| CN101054601A (en) * | 2006-04-13 | 2007-10-17 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Oligonucleotide for detecting cytochrome P450 enzyme series mutation site and gene chip |
| CN101525654A (en) * | 2008-03-07 | 2009-09-09 | 樊世斌 | Fluorescence quantitative detecting method for CYP2C19 genotyping |
-
2008
- 2008-12-26 CN CN200810208219A patent/CN101760528B/en not_active Expired - Fee Related
Cited By (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173443A (en) * | 2013-03-22 | 2013-06-26 | 卫生部北京医院 | CYP2C9 gene fragment containing 1300A>T mutation, protein fragment encoded through same and application thereof |
| CN103173443B (en) * | 2013-03-22 | 2016-08-24 | 卫生部北京医院 | Including 1300A > T sudden change CYP2C9 genetic fragment, coded protein fragments and application thereof |
| CN104156632A (en) * | 2014-07-21 | 2014-11-19 | 金华市中心医院 | Method for intelligently designing drug metabolic enzyme detecting gene chip probes |
| CN104232757B (en) * | 2014-07-29 | 2016-08-17 | 杭州艾迪康医学检验中心有限公司 | The method in detection CYP2C19*2 mutational site and oligonucleotide |
| CN104232757A (en) * | 2014-07-29 | 2014-12-24 | 杭州艾迪康医学检验中心有限公司 | Method and oligonucleotide for detecting mutation site of CYP2C19*2 |
| CN104561301A (en) * | 2014-12-31 | 2015-04-29 | 中国医学科学院阜外心血管病医院 | Detection method for CYP2C9*3 gene polymorphism, as well as nucleic acid probe and kit for method |
| CN104561300A (en) * | 2014-12-31 | 2015-04-29 | 中国医学科学院阜外心血管病医院 | Detection method for CYP2C9*2 gene polymorphism, as well as nucleic acid probe and kit for method |
| JP2016192939A (en) * | 2015-04-01 | 2016-11-17 | 東洋鋼鈑株式会社 | Probe for detecting cyp2c19*2 and probe for detecting cyp2c19*3 |
| CN105648082A (en) * | 2016-03-01 | 2016-06-08 | 常州市中医医院 | Primer-probe combination and kit for detecting associated genotyping of warfarin medication |
| CN105648082B (en) * | 2016-03-01 | 2019-01-25 | 常州市中医医院 | It is a kind of for detecting the primer combination of probe and kit of warfarin medication related gene parting |
| CN105779601A (en) * | 2016-04-07 | 2016-07-20 | 江苏苏博生物医学科技有限公司 | Drug metabolic enzyme related gene SNP fluorescence labeling composite amplification kit |
| CN108690876A (en) * | 2016-08-25 | 2018-10-23 | 杭州百迈生物股份有限公司 | Detect primer, probe and application, kit and the detection method of ACE gene pleiomorphisms |
| CN106367479A (en) * | 2016-08-25 | 2017-02-01 | 杭州百迈生物股份有限公司 | Detection composition for guiding hypertension medication, applications of detection composition, kit and detection method |
| CN106754991A (en) * | 2016-12-28 | 2017-05-31 | 温州医科大学 | Including 972C>CYP3A4 genetic fragments, coded protein fragments and its application that A is mutated |
| CN106754991B (en) * | 2016-12-28 | 2020-11-10 | 温州医科大学 | CYP3A4 gene segment containing 972C > A mutation, protein segment coded by same and application thereof |
| CN107523612A (en) * | 2017-05-08 | 2017-12-29 | 韩林志 | Primer sets, kit and method for the detection of children's safety medication related gene |
| CN107760778A (en) * | 2017-10-25 | 2018-03-06 | 广州和康医疗技术有限公司 | A kind of detection method and its kit that genotype detection is carried out to CYP2C9 sites |
| CN109706234A (en) * | 2017-10-26 | 2019-05-03 | 山东中科翰康医学检验有限公司 | Antipyretics object personalization gene detecting kit |
| CN107937506A (en) * | 2017-11-16 | 2018-04-20 | 南京迪安医学检验所有限公司 | A kind of kit of molecular beacon probe method detection mankind's CYP2C19 gene pleiomorphisms, method and its application |
| CN108929904A (en) * | 2018-07-26 | 2018-12-04 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs1057910 |
| CN108998512A (en) * | 2018-08-14 | 2018-12-14 | 上海芯超生物科技有限公司 | The hands-free kit and detection method for CYP2C19 genetic polymorphism detection taken |
| CN109055553A (en) * | 2018-08-23 | 2018-12-21 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs4244285 |
| CN108998515A (en) * | 2018-08-24 | 2018-12-14 | 北京青航基因科技有限公司 | SNP combination and the application in the product of preparation detection drug response related gene |
| CN109182485A (en) * | 2018-10-22 | 2019-01-11 | 北京华夏时代生物工程有限公司 | Detection method is sequenced in the fluorescence in situ hybridization of CYP gene mononucleotide polymorphism |
| CN112501265A (en) * | 2020-09-24 | 2021-03-16 | 杭州百迈生物股份有限公司 | Reagent, detection method and kit for detecting CYP2D6 gene |
| CN113249467A (en) * | 2021-06-08 | 2021-08-13 | 北京大学第一医院 | Metabolic typing method of CYP2D6 gene related to psychotropic drugs in Chinese population |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101760528B (en) | 2012-09-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101760528B (en) | Medicine metabolic relevant loci detection method | |
| Horstmann et al. | Pharmacogenomics of antidepressant drugs | |
| Geurts et al. | Altered gut microbiota and endocannabinoid system tone in obese and diabetic leptin-resistant mice: impact on apelin regulation in adipose tissue | |
| CN106222281A (en) | Test kit, application and method of based on the gene pleiomorphism accurate medication of guiding children patient | |
| CN105256019B (en) | MTHFR and MTRR genetic polymorphism detection primer sets and kit | |
| CN105779601A (en) | Drug metabolic enzyme related gene SNP fluorescence labeling composite amplification kit | |
| JP2021531040A (en) | Kits and methods for detecting polymorphic sites of CYP3A4 and CYP3A5 | |
| CN107523612A (en) | Primer sets, kit and method for the detection of children's safety medication related gene | |
| CN111808943A (en) | Gene detection method for individual medication of mental | |
| Pardo et al. | Exposure to air pollution interacts with obesogenic nutrition to induce tissue-specific response patterns | |
| CN107893114A (en) | For primer pair, kit and the method for instructing fentanyl class medicine personalized medicine related gene to detect | |
| CN106987618A (en) | The multiple gene mononucleotide polymorphism Sites Combinations related to statins personalized medicine and its application | |
| Chuang et al. | IL-10 promoter gene polymorphisms and sustained response to combination therapy in Taiwanese chronic hepatitis C patients | |
| CN101962669A (en) | Gene combination, primer and probe for detecting alcoholic liver disease susceptibility and application | |
| CN110079597A (en) | The susceptible related gene inspecting reagent kit of nonalcoholic fatty liver | |
| Dallio et al. | PNPLA3, TM6SF2, and MBOAT7 influence on nutraceutical therapy response for non-alcoholic fatty liver disease: a randomized controlled trial | |
| Pae et al. | Heat-shock protein-70 genes and response to antidepressants in major depression | |
| Fatima et al. | Role of δ-tocotrienol and resveratrol supplementation in the regulation of micro RNAs in patients with metabolic syndrome: A randomized controlled trial | |
| CN110241197A (en) | Primer combination of probe and kit and application for instructing Atorvastatin drug personalized medicine related gene to detect | |
| CN103131776A (en) | A719G single nucleotide polymorphism detection kit of thiopurine methyltransferase (TPMT) * 3C | |
| CN104774950B (en) | A kind of kit based on HSP90AA1 genetype for predicting glucocorticoid treatment SLE curative effects | |
| Xie et al. | Small intestinal flora graft alters fecal flora, stool, cytokines and mood status in healthy mice | |
| CN110295224A (en) | Primer combination of probe and kit and application for instructing Rosiglitazone drug personalized medicine related gene to detect | |
| CN101760530B (en) | Method for detecting stroke related locus | |
| Cui et al. | Relationship between the polymorphisms in KCNQ1 and type 2 diabetes in Chinese Kazakh population |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20151112 Address after: 201203 Shanghai city Pudong New Area Bing 67 Lane Road No. 15 on the third. Patentee after: SHANGHAI CHROMYSKY MEDICAL RESEARCH CO., LTD. Address before: Bing Lu Pudong New Area Zhangjiang hi tech park Shanghai 201203 Lane 67 No. 15 Patentee before: Shanghai Genecore Biological Technology Co., Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120905 Termination date: 20201226 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |