Visual detection of bacterial pathogens via PNA-based padlock probe assembly and isothermal amplification of DNAzymes.
Visual detection of bacterial pathogens via PNA-based padlock probe assembly and isothermal amplification of DNAzymes.
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DOI：
10.1021/ac5018748
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发表时间：
2014-12-16
影响因子：
7.4
通讯作者：
Smolina, Irina
中科院分区：
文献类型：
--
作者：
Gomez, Anastasia;Miller, Nancy S.;Smolina, Irina
我们开发了一种自报告等温系统，用于单碱基分辨率的视觉细菌病原体检测。新的DNA诊断是基于肽核酸（PNA）技术、滚动圈扩增（RCA）和DNAzymes的结合。PNAs被用作极具选择性的化学工具，在非变性条件下以预定序列结合基因组DNA。在PNA-DNA结构组装完成后，在自由链上循环一个挂锁探针。该探针包含一个g -四重体结构，两侧是切口酶识别位点。组装的环作为一种新型杂交RCA策略的模板，允许指数扩增和产生短单链DNA片段。这些DNA片段折叠成g -四重体结构，当与血红蛋白结合时，就成为功能性的DNA酶。每个DNAzyme单元的催化活性导致比色检测并提供第二个扩增步骤。PNA， RCA和DNAzymes的组合允许用肉眼观察到的比色输出对序列特异性和高度敏感的细菌检测。本文将该方法应用于大肠杆菌、鼠伤寒沙门菌和艰难梭菌基因组的鉴别。
We have developed a self-reporting isothermal system for visual bacterial pathogen detection with single base resolution. The new DNA diagnostic is based on combination of peptide nucleic acid (PNA) technology, rolling circle amplification (RCA) and DNAzymes. PNAs are used as exceedingly selective chemical tools that bind genomic DNA at a predetermined sequence under nondenaturing conditions. After assembly of the PNA-DNA construct a padlock probe is circularized on the free strand. The probe incorporates a G-quadruplex structure flanked by nicking enzyme recognition sites. The assembled circle serves as a template for a novel hybrid RCA strategy that allows for exponential amplification and production of short single-stranded DNA pieces. These DNA fragments fold into G-quadruplex structures and when complexed with hemin become functional DNAzymes. The catalytic activity of each DNAzyme unit leads to colorimetric detection and provides the second amplification step. The combination of PNA, RCA, and DNAzymes allows for sequence-specific and highly sensitive detection of bacteria with a colorimetric output observed with the naked eye. Herein, we apply this method for the discrimination of Escherichia coli, Salmonella typhimurium, and Clostridium difficile genomes.
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