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

Leyla Esfandiari,Harold Monbouquette,Anisha Banda,Siqing Wang,Siqi Wang,Gayane Kocharyan,Michael Lorenzini,Jacob Schmidt

doi:10.3390/bios6030037

Leyla Esfandiari, Harold Monbouquette + Show 6 more

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https://doi.org/10.3390/bios6030037

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Journal: Biosensors	Publication Date: Jul 22, 2016
Citations: 40	License type: CC BY 4.0

Affiliation: University of California, Los Angeles

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.

Highlights

95% of deaths in the developing world are due to lack of access to proper diagnostics for infectious diseases [1]
The experiments were designed to investigate the accurate detection of the target E. coli and establish the limit of detection (LOD) of our detector in terms of the concentration of 16S rRNA
The concentration of total RNA extracted from the target E. coli was 364 ng/μL

Summary

Introduction

95% of deaths in the developing world are due to lack of access to proper diagnostics for infectious diseases [1]. Even in the developed world, most infectious disease diagnosis is accomplished by culturing methods that typically take days. There is strong motivation for development of sensitive, robust and portable molecular diagnostic devices that can give rapid yes/no results for the presence of important bacterial pathogens in food, water and body fluids. Many of the existing platforms for NA detection at sufficiently low concentration require amplification by PCR, fluorescent or enzymatic labels, and expensive instrumentation [2,3]. Appealing alternatives to PCR for NA amplification have emerged, including some isothermal schemes that can be accomplished in minutes, they all, like PCR, require expensive reagents (e.g., primers) and reactions that must be controlled [7,8,9,10,11].

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