A ratiometric fluorescent biosensor for rapid detection of Burkholderia pseudomallei by dual CRISPR/Cas12a trans-cleavage assisted signal enhancement
A ratiometric fluorescent biosensor for rapid detection of Burkholderia pseudomallei by dual CRISPR/Cas12a trans-cleavage assisted signal enhancement
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DOI：
10.1016/j.snb.2022.133204
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发表时间：
2022-12
期刊：
影响因子：
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通讯作者：
Shoujia Lin;Yingzi Lin;Jie Wu;Guangming Li;Xiaotian Wu;Nini Luo;Wenting Li;Chuanlong Zhu;Rui Liu;Qiqi Xu;Qianfeng Xia;H. Ju
中科院分区：
文献类型：
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作者：
Shoujia Lin;Yingzi Lin;Jie Wu;Guangming Li;Xiaotian Wu;Nini Luo;Wenting Li;Chuanlong Zhu;Rui Liu;Qiqi Xu;Qianfeng Xia;H. Ju
由假鼻疽伯克霍尔德氏菌(BP)感染引起的类鼻疽病在热带和亚热带地区死亡率很高。迫切需要一种快速、灵敏的检测方法。为此，设计了一种具有双靶点识别位点的比率荧光传感器，用于BpDNA的灵敏检测。该传感器以TAMRA标记的DNA为探针，在酸性pH条件下吸附在聚合物点(Pdots)上，通过荧光共振能量转移(FRET)在Pdots的激发波长产生TAMRA荧光。在靶DNA存在的情况下，CRISPR/Cas12a的反式切割活性被双重识别位点激活，从而消化探针DNA以削弱FRET。以Pdots与Tamra的荧光比值为检测信号，实现了比例荧光生物传感。这种双重CRISPR/Cas12a反式切割辅助信号增强的方法可以检测到1.0~10nM的目标DNA，并可成功地用于检测BpDNA提取物，检测时间为40min。该方法灵敏度高、特异度好、准确度高、分析时间短、操作方便，在临床诊断类鼻疽病中具有潜在的应用价值。
Melioidosis caused by Burkholderia pseudomallei (Bp) infection has high mortality in tropical and subtropical regions. A rapid and sensitive method for its detection is urgently needed. Herein, a ratiometric fluorescent sensor with dual recognition sites of target was designed for sensitive detection ofBpDNA. The sensor was constructed with a TAMRA-labeled DNA as probe, which could adsorb on polymer dots (Pdots) at acidic pH to produce TAMRA fluorescence at the excitation wavelength of Pdots via fluorescence resonance energy transfer (FRET). In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a was activated with the dual recognition sites, which digested the probe DNA to weaken the FRET. With the fluorescence ratio of Pdots to TAMRA as the detection signal, a ratiometric fluorescent biosensing was thus achieved. This method with dual CRISPR/Cas12a trans-cleavage assisted signal enhancement showed a detectable range of target DNA from 1.0 pM to 10 nM, and could be successfully used to detectBpDNA extract with a detection time of 40 min. The excellent performance such as high sensitivity, good specificity, acceptable accuracy and short analytical time along with convenient operation demonstrated its potential in clinic diagnosis of melioidosis.