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Biosensors 2016, 6(3), 37; doi:10.3390/bios6030037

PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor

1
Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
2
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA
3
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA
Present address: Department of Electrical and Computing Systems, Department of Biomedical, Chemical and Environmental Engineering University of Cincinnati, Cincinnati, OH 45221, USA
*
Authors to whom correspondence should be addressed.
Academic Editor: Laurent A. Francis
Received: 12 May 2016 / Revised: 8 July 2016 / Accepted: 15 July 2016 / Published: 22 July 2016
(This article belongs to the Special Issue Systems for the Early Detection of Pathogenic Bacteria)
View Full-Text   |   Download PDF [709 KB, uploaded 22 July 2016]   |  

Abstract

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. View Full-Text
Keywords: nucleic acid detection; peptide nucleic acid (PNA); 16S rRNA; E. coli detection nucleic acid detection; peptide nucleic acid (PNA); 16S rRNA; E. coli detection
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Esfandiari, L.; Wang, S.; Wang, S.; Banda, A.; Lorenzini, M.; Kocharyan, G.; Monbouquette, H.G.; Schmidt, J.J. PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor. Biosensors 2016, 6, 37.

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