title: PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor.
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.
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, CA 90095, USA. esfandla@ucmail.uc.edu.
authors.forename: Leyla
authors.initials: L
authors.lastname: Esfandiari
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA. wangsiqing19940@ucla.edu.
authors.forename: Siqing
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA. wangsiqi940103@ucla.edu.
authors.forename: Siqi
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, CA 90095, USA. anisha.banda@gmail.com.
authors.forename: Anisha
authors.initials: A
authors.lastname: Banda
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, CA 90095, USA. mhlorenzini@gmail.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Lorenzini
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, CA 90095, USA. gaiakocharyan@gmail.com.
authors.forename: Gayane
authors.initials: G
authors.lastname: Kocharyan
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA. hmonbouq@ucla.edu.
authors.forename: Harold G
authors.initials: HG
authors.lastname: Monbouquette
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, CA 90095, USA. schmidt@seas.ucla.edu.
authors.forename: Jacob J
authors.initials: JJ
authors.lastname: Schmidt
mesh_terms: D015374:Biosensing Techniques / Q000379:methods / Q000592:standards; D004926:Escherichia coli / Q000235:genetics; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012680:Sensitivity and Specificity
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; E. coli detection; nucleic acid detection; peptide nucleic acid (PNA)