高分辨熔解曲线技术检测HLA-G14bp插入/缺失突变方法的建立及初步应用  

Establishment and application of high-resolution melting curve analysis in detection of HLA-G 14 bp insertion /deletion

作　　者：陈晓晴[1] 余玲玲[1] 王慧燕[1] 田可港[1] 郑晓群[1] 

机构地区：[1]温州医科大学附属第二医院检验科,浙江温州325027

出　　处：《中国卫生检验杂志》2014年第5期719-722,725,共5页Chinese Journal of Health Laboratory Technology

基　　金：浙江省重点科技创新团队(2012R10048-11);浙江省医药卫生科技计划项目(2010KYA137);温州市科技计划项目(Y20100258)

摘　　要：目的建立高分辨率熔解(HRM)技术检测人类白细胞抗原G(HLA-G)14 bp插入/缺失突变的方法,并初步探讨其临床应用价值。方法对HRM进行方法学评价。对100例EV71感染重症手足口病(HFMD)患儿、80例EBV感染传染性单核细胞增多症(IM)患儿和100例对照组进行HLA-G 14 bp插入/缺失突变的检测;用聚合酶链反应结合聚丙烯酰胺凝胶电泳(PCR-PAGE)法和产物测序法验证HRM分型。结果不同基因型HRM检测的批内、批间熔解温度(Tm)值的变异系数(CV)均<0.1%;HRM与PAGE及直接测序所得基因型相符。100份EV71感染HFMD患儿DNA样本经检测,10份为14 bp+/+纯合子基因型,42份为14 bp+/-杂合子基因型,48份为14 bp-/-纯合子基因型,与对照组比较差别有统计学意义(χ2=6.181,P<0.05),80份EBV感染IM患儿DNA样本与对照组比较差别亦有统计学意义(χ2=8.531,P<0.05)。结论本研究建立的HRM技术可用于HLA-G 14 bp插入/缺失基因突变的检测。Objective To establish a method for detection of gene mutations in HLA - G 14 bp insertion/deletion by high - resolution melting （HRM） and explore the preliminary clinical application. Methods A method for detection of HLA - G 14 bp insertion/deletion based on HRM was evaluated by methodology. One hundred severe HFMD children infected with EV71 （ EV71 group）, 80 infectious mononucleosis（IM） children infected by EBV （EBV group） and 100 controls （control group） were involved to detect HLA - G 14 bp insertion/deletion by PCR - HRM. The HRM results were further confirmed by PCR - PAGE and sequencing. Results The coefficients of variation of within - run and between - run Tm values were all less than 0.1% in HLA - G of different genotypes by HRM, showing coincident genotypes with PAGE and direct DNA sequencing. There were ten cases of 14 bp ＋/＋ homozygous genotype, forty -two cases of 14 bp ＋/- heterozygous genotypes and forty -eight cases of 14 bp -/- homozygous genotype in the EVT1 group, and there was significant statistical difference in the frequencies of the HLA- G 14bp genotypes between the EV71 group and control group （χ2 = 6. 181, P 〈 0.05）. And the significant statistical difference was also observed between the EBV group and control group （χ2 = 8.531, P 〈 0.05 ）. Conclusion HRM could be applied to analyze the HLA - G 14 bp insertion/deletion polymorphism.

关 键 词：高分辨熔解曲线技术 人类白细胞抗原G 基因突变 EV71病毒 Epstein—Barr病毒 

分 类 号：Q754[生物学—分子生物学]