基于核酸外切酶Ⅲ信号放大的比率型荧光传感器检测致病菌基因

摘要

Food-borne pathogenic bacteria seriously threaten public health. Based on the mechanism of fluorescence resonance energy transfer (FRET), a ratiometric fluorescence biosensor was constructed by integration of Exo III-based signal amplification strategy. The Cy3 labeled R1-DNA firstly hybridized with Cy5 labeled R2-DNA to form duplex of R1/R2. Cy3 showed a low fluorescence response while Cy5 showed a high fluorescence response. The addition of target pathogenic bacterial gene (Lac Z gene) could de-hybridize the R1/R2,resulting in the fluorescence decreasing of Cy5 and the fluorescence recovering of Cy3. Under the assistance of Exo III, the signal change was further amplified. The detection of limit reached as low as 5.29 pmol/L. The linear detection range was from 10 pmol/L to 2000 pmol/L. The developed ratiomtric detection strategy significantly reduced the possibility of false-positive and false-negative detection results.%食源性致病菌严重威胁着公众健康.本研究基于荧光共振能量转移原理,以Cy3和Cy5作为荧光供体和受体基团,利用核酸外切酶Ⅲ(Exo Ⅲ)增强检测信号,构建了比率型荧光传感器,用于高灵敏度检测致病菌基因.分别标记有Cy3的R1-DNA和标记Cy5的R2-DNA形成双链R1/R2,在Cy3的激发光波长激发下,由于发生荧光共振能量转移,Cy3的荧光被猝灭而产生Cy5的荧光信号.致病菌靶基因(大肠杆菌Lac Z 基因)的存在可将R1/R2双链解旋,使得Cy3远离Cy5,导致Cy3的荧光恢复而Cy5的荧光信号降低.在ExoⅢ作用下,Cy3与 Cy5的信号变化进一步增大.在优化的实验条件下,荧光信号变化与靶基因在10~2000 pmol/L浓度范围内呈现良好的线性关系,检出限为5.29 pmol/L.所采用的比率型信号检测策略极大地降低了假阴性、假阳性检测结果的产生,增强了检测特异性.