PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor.
PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor.
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DOI：
10.3390/bios6030037
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发表时间：
2016-07-22
期刊：
影响因子：
--
通讯作者：
Schmidt JJ
中科院分区：
文献类型：
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作者：
Esfandiari L;Wang S;Wang S;Banda A;Lorenzini M;Kocharyan G;Monbouquette HG;Schmidt JJ
一种无PCR、无光学装置用于检测大肠杆菌（E。coli）16 S rRNA，其对应于约100-1000菌落形成单位/mL（CFU/mL），这取决于细胞rRNA水平。寻求开发一种快速、灵敏和具有成本效益的核酸检测平台，用于检测食品、水和体液中的病原微生物。由于16 S rRNA序列是物种特异性的，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靶标。在此，靶向E.使用基于机电信号转导的概念上简单的方法相对于总RNA背景检测10 fM浓度的大肠杆菌，由此通过孔的离子电流的阶跃变化降低表明被与靶核酸杂交的电泳动员的珠-肽核酸探针缀合物堵塞。我们研究了细菌物种特异性16 S rRNA在1 pM至1 fM的浓度检测限，发现我们的设备的检测限为10 fM，这与我们之前对相似长度的单链DNA的发现一致。此外，对照RNA未获得假阳性响应，靶16 S rRNA在低至10 fM的检测限（LOD）时未获得假阴性。因此，该检测方案显示出集成到便携式、低成本系统中用于快速检测食品、水和体液中的病原微生物的前景。
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.