CYP2C19基因多态性PCR扩增体系的正交优化与验证
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摘要
本实验通过建立CYP2C19*2、*3和*17基因多态性PCR反应体系和条件,筛选出4μL体系中各因素最佳水平。采用SNaPshot技术对CYP2C19基因3个SNP位点*2、*3和*17同时进行复合扩增检测,利用L9(34)正交实验设计,对影响PCR反应体系和条件的3个因素(PCR Mix, Taq DNA聚合酶,循环次数)在3个水平上进行优化,结果采用综合评分法和极差分析法进行分析。用3组已知样本对正交优化所得条件进行重复性和稳定性验证。结果表明CYP2C19*2、*3和*17基因PCR扩增体系的影响因素依次为:PCR Mix>循环数>Taq DNA聚合酶。最佳反应体系为PCR Mix 2.0μL、Taq DNA聚合酶0.2μL、循环次数32次。3组样本验证效果满意。优化的CYP2C19*2、*3和*17基因PCR反应体系稳定性高,重复性和经济性较好,为该基因多态性的大规模调查奠定了基础。
This study established the PCR reaction system and conditions for investigating of CYP2 C19*2, *3 and*17 gene polymorphisms, and screened the optimal level of each factors in 4 μL system. Three SNPs locus of *2, *3 and*17 in CYP2 C19 gene were applied the complex amplification detection by SNa Pshot method at the same time. Three factors(PCR Mix, Taq DNA polymerase and cycles) influencing the PCR reaction system were optimized with L9(34)orthogonal experimental design on three levels. Then the results were analyzed by methods of comprehensive evaluation and range analysis. Finally, the repeatability and stability of optimized conditions were verified by three groups of known samples. The results showed that the influence factors of the PCR amplification system of CYP2 C19*2, *3 and *17 gene were PCR Mix, then the number of cycles and last the Taq DNA polymerase. Optimal reaction system was 2.0 μL PCR Mix, 0.2 μL Taq DNA polymerase and 32 times of cycles. Three groups of known samples were verified satisfactory. The optimized PCR system of CYP2 C19*2, *3 and *17 gene was testified with high stability and better repeatability and economy, which would lay the foundation for large-scale investigation of the gene polymorphism.
This study established the PCR reaction system and conditions for investigating of CYP2 C19*2, *3 and*17 gene polymorphisms, and screened the optimal level of each factors in 4 μL system. Three SNPs locus of *2, *3 and*17 in CYP2 C19 gene were applied the complex amplification detection by SNa Pshot method at the same time. Three factors(PCR Mix, Taq DNA polymerase and cycles) influencing the PCR reaction system were optimized with L9(34)orthogonal experimental design on three levels. Then the results were analyzed by methods of comprehensive evaluation and range analysis. Finally, the repeatability and stability of optimized conditions were verified by three groups of known samples. The results showed that the influence factors of the PCR amplification system of CYP2 C19*2, *3 and *17 gene were PCR Mix, then the number of cycles and last the Taq DNA polymerase. Optimal reaction system was 2.0 μL PCR Mix, 0.2 μL Taq DNA polymerase and 32 times of cycles. Three groups of known samples were verified satisfactory. The optimized PCR system of CYP2 C19*2, *3 and *17 gene was testified with high stability and better repeatability and economy, which would lay the foundation for large-scale investigation of the gene polymorphism.
引文
Chuwongwattana S.,Jantararoungtong T.,Chitasombat M.N.,Puangpetch A.,Prommas S.,Dilokpattanamongkol P.,Watcharananan S.P.,and Sukasem C.,2016,A prospective observational study of CYP2C19,polymorphisms and voriconazole plasma level in adult Thai patients with invasive aspergillosis Drug Metab Pharmacokinet,31(2):117-122
Ciftci H.S.,Karadeniz M.S.,Tefik T.,Caliskan Y.,Yaz1c1 H.Demir E.,Turkmen A.,Nane I.,Oguz F.S.,and Aydin F.,2017Influence of proton pump inhibitors on mycophenolic acid pharmacokinetics in patients with renal transplantation and the relationship with cytochrome 2C19 gene polymorphism Transplantation Proceedings,49(3):490-496
Fondevila M.,B?rsting C.,Phillips C.,de la Puente M.,Consortium E.N.,Carracedo A.,Morling N.,and Lareu M.V.,2017,Forensic SNP genotyping with SNaPshot:Technical considerations for the development and optimization of multiplexed SNP assays,Forensic Science Review,29(1):57
Ghodke Y.,Joshi K.,Arya Y.,Radkar A.,Chiplunkar A.,Shintre P.,and Patwardhan B.,2007,Genetic polymorphism of CYP-2C19 in Maharashtrian population,European Journal of Epidemiology,22(12):907-915
Jing L.,Wang Y.X.,and Wang H.P.,2017,Distribution of CYP2-C19polymorphisms in Mongolian and Han nationals and the choice of specific antiplatelet drugs,International Journal of Clinical Pharmacy,39(4):1-7
Lang J.E.,Holbrook J.T.,Mougey E.B.,Wei C.Y.,Wise R.A.,Teague W.G.,and Lima J.J.,2015,Lansoprazole is associated with worsening asthma control in children with the CYP2C19 poor metabolizer phenotype,Annals of the American Thoracic Society,12(6):878
Larsen J.B.,and Rasmussen J.B.,2017,Pharmacogenetic testing revisited:5'nuclease real-time polymerase chain reaction test panels for genotyping CYP2D6 and CYP2C19,Pharmgenomics Pers.Med.,10:115-128
M?rginean A.,B?nescu C.,Moldovan V.,Scridon A.,M?rginean M.,B?lasa R.,Maier S.,T?rusi M.,and Dobreanu M.,2017,The impact of CYP2C19 loss-of-function polymorphisms,clinical,and demographic variables on platelet response to clopidogrel evaluated using impedance aggregometry,Clinical&Applied Thrombosis/hemostasis Official Journal of the International Academy of Clinical&Applied Thrombosis/hemostasis,23(3):255
Scott S.A.,Sangkuhl K.,Stein C.M.,Hulot J.S.,Mega J.L.,Roden D.M.,Klein T.E.,Sabatine M.S.,Johnson J.A.,and Shuldiner A.R.,2013,Clinical pharmacogenetics implementation consortium guidelines for CYP2C19 genotype and clopidogrel therapy:2013 update,Clinical Pharmacology&Therapeutics,94(3):317-323
Settin A.,Abdalla A.F.,Al-Hussaini A.S.,El-Baz R.,and Galal A.,2014,Cure rate of helicobacter pylori infection in Egyptian children related to CYP2C19 gene polymorphism,Indian Journal of Gastroenterology,33(4):1-6
Sim S.C.,Risinger C.,Dahl M.L.,Aklillu E.,Christensen M.,Bertilsson L.,and Ingelman-Sundberg M.,2006,A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants,Clinical Pharmacology&Therapeutics,79(1):103-113
Uckun Z.,Baskak B.,Ozel-Kizil E.T.,Ozdemir H.,Devrimci Ozguven H.,and Suzen H.S.,2016,The impact of CYP2C19polymorphisms on cital opram metabolism in patients with major depressive disorder,Journal of Clinical Pharmacy&Therapeutics,40(6):672-679
Wang S.C.,Guo Y.J.,Ren Y.,Wu H.J.,and Liu J.X.,2016,Optimization of ethanol mixed-extraction technology of red ginseng and salvia miltiorrhiza in Xinlikang Granules by multiple indexes comprehensive evaluation method combined with orthogonal test,Zhongguo Yaofang(Orthogonal Force China Pharmacy)19:2678-2681(王少辰,郭宇洁,任烨,吴红金,刘建勋,2016多指标综合评分法结合正交试验优化心力康颗粒中红参与丹参的混合醇提工艺,中国药房,19:2678-2681)
Yamamoto Y.,Takahashi Y.,Imai K.,Miyakawa K.,Nishimura S.,Kasai R.,Ikeda H.,Takayama R.,Mogami Y.,Yamaguchi T.,Terada K.,Matsuda K.,Inoue Y.,and Kagawa Y.,2013Influence of CYP2C19 polymorphism and concomitant antiepileptic drugs on serum clobazam and N-desmethyl clobazam concentrations in patients with epilepsy,Therapeutic Drug Monitoring,35(3):305-312
Yu S.X.,Zeng J.,Jin Y.Z.,Jin Y.Z.,Wan H.J.,Zhai D.,Xing Y.M.,and Cheng B.W.,2017,Genotyping of ABO blood group in partial population of Yunnan Province by SNaPshot technology,Fayixue Zazhi(Journal of Forensic Medicine),33(3):277-280(于书欣,曾发明,金岩章,万洪静,翟滇,邢豫明,程宝文,2017,SNaPshot技术应用于云南省部分人群ABO血型基因分型,法医学杂志,33(3):277-280)
Zivin K.,Pfeiffer P.N.,Bohnert A.S.,Ganoczy D.,Blow F.C.,Nallamothu B.K.,and Kales H.C.,2013,Evaluation of the FDA warning against prescribing citalopram at doses exceeding 40 mg,American Journal of Psychiatry,170(6):642-650
Ciftci H.S.,Karadeniz M.S.,Tefik T.,Caliskan Y.,Yaz1c1 H.Demir E.,Turkmen A.,Nane I.,Oguz F.S.,and Aydin F.,2017Influence of proton pump inhibitors on mycophenolic acid pharmacokinetics in patients with renal transplantation and the relationship with cytochrome 2C19 gene polymorphism Transplantation Proceedings,49(3):490-496
Fondevila M.,B?rsting C.,Phillips C.,de la Puente M.,Consortium E.N.,Carracedo A.,Morling N.,and Lareu M.V.,2017,Forensic SNP genotyping with SNaPshot:Technical considerations for the development and optimization of multiplexed SNP assays,Forensic Science Review,29(1):57
Ghodke Y.,Joshi K.,Arya Y.,Radkar A.,Chiplunkar A.,Shintre P.,and Patwardhan B.,2007,Genetic polymorphism of CYP-2C19 in Maharashtrian population,European Journal of Epidemiology,22(12):907-915
Jing L.,Wang Y.X.,and Wang H.P.,2017,Distribution of CYP2-C19polymorphisms in Mongolian and Han nationals and the choice of specific antiplatelet drugs,International Journal of Clinical Pharmacy,39(4):1-7
Lang J.E.,Holbrook J.T.,Mougey E.B.,Wei C.Y.,Wise R.A.,Teague W.G.,and Lima J.J.,2015,Lansoprazole is associated with worsening asthma control in children with the CYP2C19 poor metabolizer phenotype,Annals of the American Thoracic Society,12(6):878
Larsen J.B.,and Rasmussen J.B.,2017,Pharmacogenetic testing revisited:5'nuclease real-time polymerase chain reaction test panels for genotyping CYP2D6 and CYP2C19,Pharmgenomics Pers.Med.,10:115-128
M?rginean A.,B?nescu C.,Moldovan V.,Scridon A.,M?rginean M.,B?lasa R.,Maier S.,T?rusi M.,and Dobreanu M.,2017,The impact of CYP2C19 loss-of-function polymorphisms,clinical,and demographic variables on platelet response to clopidogrel evaluated using impedance aggregometry,Clinical&Applied Thrombosis/hemostasis Official Journal of the International Academy of Clinical&Applied Thrombosis/hemostasis,23(3):255
Scott S.A.,Sangkuhl K.,Stein C.M.,Hulot J.S.,Mega J.L.,Roden D.M.,Klein T.E.,Sabatine M.S.,Johnson J.A.,and Shuldiner A.R.,2013,Clinical pharmacogenetics implementation consortium guidelines for CYP2C19 genotype and clopidogrel therapy:2013 update,Clinical Pharmacology&Therapeutics,94(3):317-323
Settin A.,Abdalla A.F.,Al-Hussaini A.S.,El-Baz R.,and Galal A.,2014,Cure rate of helicobacter pylori infection in Egyptian children related to CYP2C19 gene polymorphism,Indian Journal of Gastroenterology,33(4):1-6
Sim S.C.,Risinger C.,Dahl M.L.,Aklillu E.,Christensen M.,Bertilsson L.,and Ingelman-Sundberg M.,2006,A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants,Clinical Pharmacology&Therapeutics,79(1):103-113
Uckun Z.,Baskak B.,Ozel-Kizil E.T.,Ozdemir H.,Devrimci Ozguven H.,and Suzen H.S.,2016,The impact of CYP2C19polymorphisms on cital opram metabolism in patients with major depressive disorder,Journal of Clinical Pharmacy&Therapeutics,40(6):672-679
Wang S.C.,Guo Y.J.,Ren Y.,Wu H.J.,and Liu J.X.,2016,Optimization of ethanol mixed-extraction technology of red ginseng and salvia miltiorrhiza in Xinlikang Granules by multiple indexes comprehensive evaluation method combined with orthogonal test,Zhongguo Yaofang(Orthogonal Force China Pharmacy)19:2678-2681(王少辰,郭宇洁,任烨,吴红金,刘建勋,2016多指标综合评分法结合正交试验优化心力康颗粒中红参与丹参的混合醇提工艺,中国药房,19:2678-2681)
Yamamoto Y.,Takahashi Y.,Imai K.,Miyakawa K.,Nishimura S.,Kasai R.,Ikeda H.,Takayama R.,Mogami Y.,Yamaguchi T.,Terada K.,Matsuda K.,Inoue Y.,and Kagawa Y.,2013Influence of CYP2C19 polymorphism and concomitant antiepileptic drugs on serum clobazam and N-desmethyl clobazam concentrations in patients with epilepsy,Therapeutic Drug Monitoring,35(3):305-312
Yu S.X.,Zeng J.,Jin Y.Z.,Jin Y.Z.,Wan H.J.,Zhai D.,Xing Y.M.,and Cheng B.W.,2017,Genotyping of ABO blood group in partial population of Yunnan Province by SNaPshot technology,Fayixue Zazhi(Journal of Forensic Medicine),33(3):277-280(于书欣,曾发明,金岩章,万洪静,翟滇,邢豫明,程宝文,2017,SNaPshot技术应用于云南省部分人群ABO血型基因分型,法医学杂志,33(3):277-280)
Zivin K.,Pfeiffer P.N.,Bohnert A.S.,Ganoczy D.,Blow F.C.,Nallamothu B.K.,and Kales H.C.,2013,Evaluation of the FDA warning against prescribing citalopram at doses exceeding 40 mg,American Journal of Psychiatry,170(6):642-650