PCR-金磁微粒层析法检测MTHFR基因C677T多态性评价
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摘要
目的探讨PCR-金磁微粒层析法检测MTHFR基因C677T多态性进行方法学评价和性能确认,为个性化叶酸补充剂量提供参考依据。方法从检测下限、准确性、抗干扰能力、稳定性方面进行评价和确认。检测下限:分别选取MTHFR基因C677T野生型(677CC)、杂合突变型(677CT)和纯合突变型(677TT)外周血样本,提取基因组DNA,A260/A280在1. 65～1. 95之间,定量并稀释至2. 5 ng/μl、5 ng/μl、10 ng/μl三个浓度,每种基因型各浓度一式三份,然后进行PCR-金磁微粒层析法检测,统计三种基因型各浓度样本的检出率。准确性:将992例PCR-金磁微粒层析法检测的样本,提取基因组DNA,送第三方检测机构进行Sanger测序验证,比较二者的一致性。抗干扰能力:取三种基因型的外周血样本,分别加入终浓度为10 mg/ml的血红蛋白(Hb)、0. 2 mg/ml的胆红素(BIL)和5 mg/ml三酰甘油(TG),然后与不加任何干扰物质的样本同时进行检测,比较干扰组与正常组的检出情况。稳定性:取三种基因型的样本各1例,每种基因型各一式十份做平行检测,比较各基因型的检出情况。结果 (1)三种基因型在2. 5 ng/μl浓度水平均没有被检出,而在5 ng/μl和10 ng/μl浓度水平一式三份全部被检出,灵敏度为5 ng/μl。(2)992例样本的测序结果与PCR-金磁微粒层析法的检出结果完全一致,准确性100%。(3)三种基因型加干扰物的检出结果与不加干扰物的检出结果完全一致,抗干扰能力强。(4)三种基因型样本各1例平行检测10次的结果高度一致,稳定性高。结论 PCR-金磁微粒层析法可有效用于MTHFR基因C677T多态性检测,该法具有灵敏度高、准确性高、抗干扰能力强、稳定性高等特点,且无需特殊大型设备,具有快速、便捷、准确、经济等优势,适合在各级医疗机构开展。
Objective To evaluate and validate MTHFR C677 T gene polymorphism test by PCR-gold microparticle chromatography method,provide a reference basis for individualized supplementation of folic acid.Methods The evaluation and validation were performed through the limit of detection(LOD),accuracy,anti-interference ability,and stability.For LOD,extraction of genomic DNA from three genotypes of MTHFR C677 T gene blood samples was performed to make sure A260/A280 between 1.65-1.95,then quantification and dilution of genomic DNA to 2.5 ng/μl,5 ng/μl,and 10 ng/μl were performed,followed by PCR-gold magnetic particles chromatography test in triplicate for each genotype and calculation of detection rate of each concentration sample of three genotypes.For accuracy,genomic DNA was extracted from 992 samples detected by PCR-gold microparticle chromatography,and Sanger sequencing verification was carried out by third-party testing institutions.For anti-interference ability,certain concentration of interfering substance was added to peripheral blood sample of each genotype respectively,which were 10 mg/ml hemoglobin(HB),0.2 mg/ml bilirubin(BIL),and 5 mg/ml triglycerides(TG),then interference groups and non-interference groups genomic DNA were extracted,followed by PCR-gold magnetic particles chromatography test,the detection results of the two groups were compared.For stability,detection of one sample of each genotype was performed for ten times in parallel,and the detection results of each genotype were compared.Results Three genotypes of MTHFR C677 T gene were not detected at the concentration of 2.5 ng/μl genomic DNA,but they were detected at both 5 ng/μl genomic DNA and 10 ng/μl genomic DNA in triplicate,which demonstrated that LOD of MTHFR C677 T gene polymorphism test by PCR-Gold magnetic particles chromatography was 5 ng/μl genomic DNA.The sequencing results of 992 samples were identical with that of PCR-gold microparticle chromatography test,which verified that the accuracy was as high as 100%.Meanwhile,PCR-gold microparticle chromatography test had strong anti-interference ability,the detection result was highly consistent with that of non-interference groups.Furthermore,the results of ten detection of each genotype in parallel were highly consistent.Conclusion PCR-gold magnetic particles chromatography method can be reliably applied to detect MTHFR gene C677 T polymorphism with high sensitivity,high accuracy,strong anti-interference performance,and high stability characteristics,and this method does not need special large equipment,which have fast,convenient,accurate,economic,easily-use,and other advantages,therefore,it is suitable for promotion in medical institutions at all levels.
Objective To evaluate and validate MTHFR C677 T gene polymorphism test by PCR-gold microparticle chromatography method,provide a reference basis for individualized supplementation of folic acid.Methods The evaluation and validation were performed through the limit of detection(LOD),accuracy,anti-interference ability,and stability.For LOD,extraction of genomic DNA from three genotypes of MTHFR C677 T gene blood samples was performed to make sure A260/A280 between 1.65-1.95,then quantification and dilution of genomic DNA to 2.5 ng/μl,5 ng/μl,and 10 ng/μl were performed,followed by PCR-gold magnetic particles chromatography test in triplicate for each genotype and calculation of detection rate of each concentration sample of three genotypes.For accuracy,genomic DNA was extracted from 992 samples detected by PCR-gold microparticle chromatography,and Sanger sequencing verification was carried out by third-party testing institutions.For anti-interference ability,certain concentration of interfering substance was added to peripheral blood sample of each genotype respectively,which were 10 mg/ml hemoglobin(HB),0.2 mg/ml bilirubin(BIL),and 5 mg/ml triglycerides(TG),then interference groups and non-interference groups genomic DNA were extracted,followed by PCR-gold magnetic particles chromatography test,the detection results of the two groups were compared.For stability,detection of one sample of each genotype was performed for ten times in parallel,and the detection results of each genotype were compared.Results Three genotypes of MTHFR C677 T gene were not detected at the concentration of 2.5 ng/μl genomic DNA,but they were detected at both 5 ng/μl genomic DNA and 10 ng/μl genomic DNA in triplicate,which demonstrated that LOD of MTHFR C677 T gene polymorphism test by PCR-Gold magnetic particles chromatography was 5 ng/μl genomic DNA.The sequencing results of 992 samples were identical with that of PCR-gold microparticle chromatography test,which verified that the accuracy was as high as 100%.Meanwhile,PCR-gold microparticle chromatography test had strong anti-interference ability,the detection result was highly consistent with that of non-interference groups.Furthermore,the results of ten detection of each genotype in parallel were highly consistent.Conclusion PCR-gold magnetic particles chromatography method can be reliably applied to detect MTHFR gene C677 T polymorphism with high sensitivity,high accuracy,strong anti-interference performance,and high stability characteristics,and this method does not need special large equipment,which have fast,convenient,accurate,economic,easily-use,and other advantages,therefore,it is suitable for promotion in medical institutions at all levels.
引文
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[17]Hui W,Zhang S,Zhang C,et al.A novel lateral flow assay based on Gold Mag nanoparticles and its clinical applications for genotyping of MTHFR C677T polymorphisms[J].Nanoscale,2016,8(6):3579-3587.
[18]Kroll MH,Elin RJ.Interference with clinical laboratory analyses[J].Clin Chem,1994,40(11):1996-2005.
[2]Sen S,Reddy PL,Grewal RP,et al.Hyperhomocysteinemia is associated with aortic atheroma progression in stroke/TIA patients[J].Front Neurol,2010,1(1):131.
[3]Ou CY,Stevenson RE,Brown VK,et al.5,10 Methylenetetrahydrofolate reductase genetic polymorphism as a risk factor for neural tube defects[J].Am J Med Genet,1996,63(4):610-614.
[4]Huh HJ,Chi HS,Shim EH,et al.Gene--nutrition interactions in coronary artery disease:correlation between the MTHFR C677T polymorphism and folate and homocysteine status in a Korean population[J].Thromb Res,2006,117(5):501-506.
[5]李克深,郑冬梅,薛雅丽,等.MTHFR基因C677T多态与神经管缺陷及先兆子痫关系的研究[J].中华医学遗传学杂志,2000,17(2):16-18.
[6]姜海鹤,王斌,粟丽.叶酸代谢与人类肿瘤发生的机制及对策[J].中国优生与遗传杂志,2008,27(7):5-6.
[7]焦海燕,薛雅丽.亚甲基四氨叶酸还原酶基因(C677T)多态性研究进展[J].国际遗传学杂志,2002,25(4):207-210.
[8]王红,张向阳.亚甲基四氨叶酸还原酶基因多态性对心血管疾病的影响[J].Chinese Journal of Cardiovascular Rehabilitation Medicine,2010,19(4):450-452.
[9]赵娟,周正艳,蔡超群,等.MTHFR基因单核苷酸多态性与中国南方汉族阿尔茨海默病的相关性分析[J].实用医学杂志,2014,30(11):1722-1724.
[10]钟巧莹,卢礼贤,杨志华,等.血浆同型半胱氨酸水平及MTHFR基因多态性与血管性痴呆的关系[J].解剖学研究,2007,29(6):432-434.
[11]钟震宇.南北方汉族人群同型半胱氨酸及MTHFR基因多态性与冠心病关系[D].中国人民解放军医学院,2012.
[12]Li R,Li Y,Fang X,et al.SNP detection for massively parallel whole-genome resequencing[J].Genome Res,2009,9(6):1124-1132.
[13]Chang HW,Yang CH,Chang PL,et al.SNP-RFLPing:restriction enzyme mining for SNPs in genomes[J].BMC Genomics,2006,7:30.
[14]De la Vega FM,Lazaruk KD,Rhodes MD,et al.Assessment of two flexible and compatible SNP genotyping platforms:TaqMan SNPGenotyping Assays and the SNPlex Genotyping System[J].Mutat Res,2005,573(1-2):111-135.
[15]Gabriel S,Ziaugra L,Tabbaa D.SNP genotyping using the Sequenom Mass ARRAY i PLEX platform[J].Curr Protoc Hum Genet,2009,Chapter 2:Unit 2.12.
[16]Tanaka M,Kamada I,Takahashi J,et al.Genotyping of the ABCG2gene using matrix-associated laser desorption/ionisation,time-offlight mass spectrometry[J].Transfus Med,2018,28(3):255-260.
[17]Hui W,Zhang S,Zhang C,et al.A novel lateral flow assay based on Gold Mag nanoparticles and its clinical applications for genotyping of MTHFR C677T polymorphisms[J].Nanoscale,2016,8(6):3579-3587.
[18]Kroll MH,Elin RJ.Interference with clinical laboratory analyses[J].Clin Chem,1994,40(11):1996-2005.