基于核酸识体的滚环DNA扩增反应用于PDGF-BB的检测  

Aptamer-based PDGF-BB detection using rolling circle DNA amplification

作　　者：周龙[1] 欧丽娟[1] 陈珂[1] 

机构地区：[1]湖南师范大学化学化工学院,湖南长沙410081

出　　处：《化学传感器》2006年第4期12-17,共6页Chemical Sensors

摘　　要：该文提出了一种新颖的、高灵敏的基于抗体-抗原-核酸识体夹心法和DNA滚环扩增(RCA)的方法用于血小板源生长因子BB(PDGF-BB)的检测。在核酸识体的3'端连接RCA反应的引物,在29DNA聚合酶、脱氧核糖核苷酸及环形模板存在下,经过滚环扩增反应后,可得到一条与环形DNA模板互补的重复序列的DNA单链,利用生物素标记的互补寡核苷酸探针与扩增产物杂交,再用亲和素标记的碱性磷酸酶与之反应,将反应后的产物在底物溶液中进行银沉积反应,最后用电化学方法检测电极表面沉积的银量,通过检测银量来达到检测PDGF-BB的目的。考察了抗体固定化浓度、RCA反应时间和其它蛋白质对测定信号的影响。结果表明,RCA扩增反应能显著的提高检测的灵敏度,降低检测下限。PDGF-BB在0.5pg/mL～8000pg/mL范围内呈良好的线性关系,检测下限为0.2pg/mL。该方法在蛋白质的检测和医学诊断等方面有很大的应用前景。A novel and high sensitive method for the detection of platelet - derived growth factor BB （PDGF BB） based on antibody - antigen - aptamer sandwich assay by using rolling circle amplification has been reported in this paper. The product of the RCA was hybridized with the 3＇ - terminal of the aptamer. With 629 DNA polymerase, deoxyribonucleic acid and circle template, rolling circle amplification can perform. The reaction form a long single chain DNA which repetitive sequence is complementary with circle template DNA sequence. The product of RCA hybridized with biotinylated oligonucleotide probe. Following with avidin - ALP and subsequent Ag deposition in substrate solution, electrochemical detection was performed. The effect of antibody concentration and RCA reaction time on the signal has been investigated. The results showed that RCA can improve remarkably the sensitivity and lower the detection limit. The linear ranges for the PDGF - BB is 0. 5 pg/mL -8 000 pg/mL and the lower limit is 0. 2 pg/mL. It will provide wide application of detecting of protein and diagnosing disease.

关 键 词：核酸识体 血小板源生长因子BB 滚环DNA扩增 银沉积 

分 类 号：Q503[生物学—生物化学]