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 mic
作者
何学虎
郭雅琪
梁小燕
张玉英
赵倩颖
董洁
贾伟
HE Xue-Hu;GUO Ya-Qi;LIANG Xiao-Yan(Clinical Laboratory Center,General Hospital of Ningxia Medical University,Yinchuan,Ningxia 750004,China)
出处
《中国妇幼保健》
CAS
2019年第1期130-134,共5页
Maternal and Child Health Care of China
基金
宁夏医科大学校级科研项目(XY2017124)
