CRISPR/Cas12a联合催化发夹自组装的mi RNA21检测

Detection for MiRNA21 by combining CRISPR/Cas12a with catalytic hairpin assembly

构建了一种基于CRISPR/Cas12a和催化发夹自组装(CHA)的miRNA21检测方法。目标物miRNA21可引发CHA,产生大量的双链DNA复合物。作为Cas12a的底物,双链DNA复合物包含原型间隔序列毗邻基序(5′-TTTN-3′),可激发Cas12a的附属切割活性,剪切一端带有荧光基团、另一端带有猝灭基团的单链DNA荧光报告探针,产生可检测的荧光信号。考察了CHA反应温度、发夹探针浓度、荧光报告探针浓度和Cas12a切割时间的影响。在优化条件下,荧光信号与miRNA21浓度的对数呈良好的线性关系,线性相关系数R2=0.9924,检出限为1.1 pmol/L。利用本方法对miRNA21加标的胎牛血清样品进行分析,20 pmol/L和100 pmol/L miRNA21的回收率分别为(92.0±4.2)%和(94.1±4.7)%。CRISPR/Cas12a联合催化发夹自组装的miRNA检测方法,使miRNA检测摆脱了热循环仪器,操作简单,通用性强,具有实际应用潜力。

A detection strategy for miRNA21 based on CRISPR/Cas12a and catalytic hairpin self-assembly(CHA)was constructed.The target miRNA21 could trigger CHA and produce a large number of double-stranded DNA complexes.As the substrate of Cas12a,the double-stranded DNA complex contained a prototype spacer sequence adjacent to the motif(5'-TTTN-3'),which could stimulate the accessory cleavage activity of Cas12a.It cleaved a single-stranded DNA fluorescence reporter with a fluorescent group at one end and a quenching group at the other end,thereby leading to the generation of appreciable fluorescence signals.The effects of reaction temperature for CHA,hairpin concentration,fluorescent reporter concentration and Cas12a cleavage time were investigated.Under the optimized conditions,the fluorescence signal and the logarithm of miRNA21 concentration showed a good linear relationship,along with the correlation coefficient(R)of 0.9924 and the detection limit of 1.1 pmol/L.The spiked recoveries of miRNA21 in fetal bovine serum were(92.0±4.2)%and(94.1±4.7)%for 20 pmol/L and 100 pmol/L of miRNA21,respectively.This strategy constructed by combining CRISPR/Cas12a and CHA,made miRNA detection simple,universal and nonuse of thermal cycling instruments,showing great potential for practical application.