一种基于磁富集靶序列PCR的扩增方法及其灵敏度检测  

A Method of PCR Amplification Based on Target Sequence Magnetic Enrichment and Sensitivity Detection

作　　者：牟贤波[1] 刘洪娜[2] 李松[1,2] 刘丽赏[3] 

机构地区：[1]湖南工业大学绿色包装与生物纳米技术应用湖南省重点实验室,湖南株洲412007 [2]东南大学生物电子学国家重点实验室,江苏南京210096 [3]庆熙大学化学工程系

出　　处：《包装学报》2012年第2期27-31,共5页Packaging Journal

摘　　要：以亲和素修饰的磁性纳米颗粒γ-Fe2O3为载体,提出了基于磁富集靶序列PCR扩增方法。首先将结合有生物素标记特异性引物的靶序列富集到亲和素修饰的γ-Fe2O3纳米颗粒表面,然后通过变性获取单链的靶序列,再进行PCR扩增。同时,优化了靶序列和特异性引物杂交的最适温度和磁性纳米颗粒γ-Fe2O3的最佳用量,并对该方法的灵敏度进行了检测。通过实验得出:该方法中,最适的杂交温度为53℃,磁性纳米颗粒的最佳用量为90μg,靶序列的最低检出浓度为5×10-10ng/mL。In this research,a method of magnetic enrichment of PCR amplification was built.First,target sequence was combined with biotin-modified specific primer and enriched to the surface of γ-Fe2O3.Then single target sequence was gained through degeneration,and performed PCR amplification.Simultaneously,the experiment conditions were optimized,such as the optimal hybridization temperature of target sequence and specific primers and the dosage of magnetic nanoparticles γ-Fe2O3.Besides,the sensitivity of the method was detected.Through the experiment,the optimum temperature of hybridization was 53℃,the optimum dosage of magnetic nanoparticles γ-Fe2O3 was 90μg,and the lowest concentration of target sequence could be detected was 5×10^-7 ng/mL.

关 键 词：Γ-FE2O3 磁富集 杂交 PCR 

分 类 号：O657.2[理学—分析化学]