摘要
目的建立以TaqMan-MGB荧光探针为特点的荧光定量聚合酶链反应(PCR),用于检测人载脂蛋白A5(apo A5)基因。方法针对人apo A5基因设计特异性引物和TaqMan-MGB荧光探针,以重组克隆质粒pPCR-Script-apo A5为DNA模板,在荧光定量PCR仪上建立TaqMan-MGB荧光定量PCR检测方法和标准曲线,进行灵敏度、重复性、特异性实验。结果建立的定量标准曲线阈值循环数(Ct)与模板拷贝数呈良好线性关系(r=0.998);最低检测浓度为103拷贝/μL;扩增效率(E)为110.2%,且重复性好。结论成功建立检测人apo A5基因的TaqMan-MGB荧光定量PCR。该法具有较好的灵敏度、特异性及重复性。
Objective To establish fluorescence quantitation polymerase chain reaction（PCR） based on TaqMan-MGB probe for detecting apolipoprotein A5（apoA5）gene.Methods The assay,which was based on specific primers and TaqMan-MGB probe from apoA5 gene,was performed to reconstruct DNA fragment of pPCR-Script-apoA5.The fluorescence quantitation PCR with TaqMan-MGB probe and the standard curve were established.The sensitivity,specificity and repeatability of the TaqMan-MGB probe fluorescence quantitation PCR were determined.Results A fine linear relationship between the threshold cycle（Ct） values of the developed standard curve and the copy number of template was observed（r=0.998）.The sensitivity of the assay was 103 copies/μL,the amplification efficiency of assay was 110.2%,and the repeatability was good.Conclusions The fluorescence quantitation PCR based on TaqMan-MGB probe for detecting the apoA5 gene is successfully developed with high sensitivity,specificity and repeatability.
出处
《检验医学》
CAS
2012年第5期400-403,共4页
Laboratory Medicine
基金
南京市科技发展重点项目(zkx09030)
关键词
聚合酶链反应
载脂蛋白A5
2型糖尿病
Polymerase chain reaction
Apolipoprotein A5
Type 2 diabetes mellitus
