Xu Hongli, Deng Rentang, Mei-lian Chen, Chen Zaixin, Zhi-Hong Huang, Bo Situ, G. Kong, L. Lisha, Lei Zheng, Wen-jin Fu
{"title":"液相芯片技术快速检测CYP2C9、CYP2C19、CYP4F2、VKORC1和ABCB1基因多态性","authors":"Xu Hongli, Deng Rentang, Mei-lian Chen, Chen Zaixin, Zhi-Hong Huang, Bo Situ, G. Kong, L. Lisha, Lei Zheng, Wen-jin Fu","doi":"10.3760/CMA.J.ISSN.1009-9158.2019.12.013","DOIUrl":null,"url":null,"abstract":"Objective \nTo establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P450 2C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology. \n \n \nMethods \nMethod establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results. \n \n \nResults \nThe results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4% and 10.9%, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results. \n \n \nConclusion \nA method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology. \n \n \nKey words: \nLiquid phase chip technology; Allele-specific primer extension; Warfarin; Clopidogrel; Single nucleotide polymorphism","PeriodicalId":10096,"journal":{"name":"中华检验医学杂志","volume":"41 1","pages":"1042-1050"},"PeriodicalIF":0.0000,"publicationDate":"2019-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Rapid detection of CYP2C9, CYP2C19,CYP4F2,VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology\",\"authors\":\"Xu Hongli, Deng Rentang, Mei-lian Chen, Chen Zaixin, Zhi-Hong Huang, Bo Situ, G. Kong, L. Lisha, Lei Zheng, Wen-jin Fu\",\"doi\":\"10.3760/CMA.J.ISSN.1009-9158.2019.12.013\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Objective \\nTo establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P450 2C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology. \\n \\n \\nMethods \\nMethod establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results. \\n \\n \\nResults \\nThe results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4% and 10.9%, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results. \\n \\n \\nConclusion \\nA method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology. \\n \\n \\nKey words: \\nLiquid phase chip technology; Allele-specific primer extension; Warfarin; Clopidogrel; Single nucleotide polymorphism\",\"PeriodicalId\":10096,\"journal\":{\"name\":\"中华检验医学杂志\",\"volume\":\"41 1\",\"pages\":\"1042-1050\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-12-11\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"中华检验医学杂志\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3760/CMA.J.ISSN.1009-9158.2019.12.013\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"Health Professions\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"中华检验医学杂志","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3760/CMA.J.ISSN.1009-9158.2019.12.013","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Health Professions","Score":null,"Total":0}
Rapid detection of CYP2C9, CYP2C19,CYP4F2,VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology
Objective
To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P450 2C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology.
Methods
Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results.
Results
The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4% and 10.9%, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results.
Conclusion
A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.
Key words:
Liquid phase chip technology; Allele-specific primer extension; Warfarin; Clopidogrel; Single nucleotide polymorphism
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