基于金属增强荧光的高通量碱基突变的筛查

High throughput mutation screening based on metal-enhanced fluorescence

目的开发简单特异的金属增强荧光方法以区分含突变位点和完全互补配对的核酸序列。方法靶标能通过toehold调节的链置换反应将固定在96孔板表面的发卡结构核酸打开，再加入5’端修饰了羧基荧光素的信号探针，该探针能与另一段打开的捕捉探针杂交，通过检测荧光信号实现对靶标的分析。结果相对于一般方法，金属增强荧光（MEF）能明显区分互补配对靶标和含突变靶标，并具有显著的信号放大作用（对于100nmol／L的靶标，信号放大-13倍），能检测出0．1nmol／L级别的靶标（普通方法5nmol／L）。结论为核酸突变检测提供了一种简便高通量的初筛手段。

Objective To develop a highly sensitive and selective method for mutation detection utilizing metal enhanced fluorescence （MEF）. Methods Strand-displacement was initiated by introducing the target ssDNA and resuiting in the unfolding of the capture probe. A signal probe coupled with carboxy fluorescein （FAM） was further hybridizing with the unfolding capture probe and generating fluorescent signal. Results Comparing to normal method, the MEF design could discriminate complementary target from targets with mismatch, and holds noticeable specificity and highly sensitivity （ -13 fold signal amplification with 100 nmol/L target） with the detection limit of 0. 1 nmol/L in silver-coated 96-well plate （control group is 5 nmol/L）. Conclusions The MEF assay provides an alternative screening method for mutation detection.