强直性脊柱炎HLA-B27基因荧光定量诊断方法的建立  

Research of fluorogenic quantitative polymerase chain reaction method to quantify HLA-B27 gene for diagnosis of ankylosing spondylitis

作　　者：张建英 徐冉行[2] 毛海红[2] 姚亚萍[2] 袁建树[2] 刘东海 

机构地区：[1]宁波市眼科医院,浙江宁波315040 [2]宁波市第六医院,浙江宁波315040 [3]宁波市第五医院,浙江宁波315010

出　　处：《中国卫生检验杂志》2010年第12期3316-3318,共3页Chinese Journal of Health Laboratory Technology

基　　金：宁波市自然基金项目(2010A610064)

摘　　要：目的:建立一种强直性脊柱炎基因(HLA-B27)的荧光定量PCR(FQ-PCR)方法。方法:扩增强直性脊柱炎患者白细胞中的HLA-B27基因与质粒PGEM-TEasy Vector重组,转化大肠杆菌E.coli DH5α,获得克隆的HLA-B27基因标准模板。用DA7600 PCR仪检测FQ-PCR扩增产物制成标准曲线来定量检测标本中HLA-B27的浓度。结果:FQ-PCR扩增产物呈"S"形动力学曲线;ct(循环阈值)与PCR体系中起始模板拷贝数的对数值之间有较好的线性关系,显示了FQ-PCR定量的准确性。结论:FQ-PCR方法用于定量检测外周静脉血HLA-B27基因表达水平,具有较好的灵敏度、特异性,且操作简便、快速,易于普及,有良好的应用前景。Objective：To establish a fluorogenic quantitative polymerase chain reaction（FQ-PCR） method for the routine quantification of HLA-B27 gene.Methods： The HLA-B27 standard gene was obtained by in vitro amplification of cloned HLA-B27 fragment in plasmid PGEM-T Easy Vector.FQ-PCR product was detected using a DA 7600 sequence detector system and obtained HLA-B27 standard curve to quantity HLA-B27 of unknown sample.Results： FQ-PCR generated ＂S＂ kinetics curve of PCR amplification constructed by relating the fluorescence signal intensity（△Rn） to the cycle number;The standard curve of HLA-B27 was constructed by the linear relationship between the cycle threshold（ct） and the log of starting copy number.The high correlation（0.999） reveal the reliability of FQ-PCR.Conclusion： The FQ-PCR is a rapid,sensitive and reliable method for quantification of HLA-B27 gene.

关 键 词：HLA-B27 基因 荧光定量-PCR 

分 类 号：R593.23[医药卫生—内科学]