Department of Bioengineering, University of California, Los Angeles, CA 90095, USA.
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
Biosensors (Basel). 2016 Jul 22;6(3):37. doi: 10.3390/bios6030037.
A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100-1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids.
一种无 PCR、无光学器件的设备可用于检测大肠杆菌 (E. coli) 16S rRNA，其检测限低至 10 fM，对应于约 100-1000 个菌落形成单位/毫升 (CFU/mL)，具体取决于细胞内 rRNA 水平。人们迫切需要开发一种快速、灵敏且经济有效的核酸检测平台，用于检测食品、水和体液中的致病微生物。由于 16S rRNA 序列具有物种特异性，并且在活细胞中以高拷贝数存在，因此这些核酸为微生物病原体检测方案提供了有吸引力的目标。在这里，我们使用一种基于机电信号转导的概念简单方法，以总 RNA 为背景，检测 10 fM 浓度的大肠杆菌目标 16S rRNA，其中通过孔的离子电流发生阶跃式减少表明，电泳迁移的珠-肽核酸探针缀合物与目标核酸杂交后发生阻塞。我们研究了细菌物种特异性 16S rRNA 在 1 pM 至 1 fM 浓度下的浓度检测极限，发现我们的设备检测限为 10 fM，这与我们之前用类似长度的单链 DNA 得到的结果一致。此外，用对照 RNA 未获得假阳性反应，用目标 16S rRNA 也未获得假阴性反应，即使目标 16S rRNA 的浓度低至检测限 (LOD) 10 fM 也是如此。因此，这种检测方案有望集成到便携式、低成本系统中，用于快速检测食品、水和体液中的致病微生物。
