Simple Bead Assay for Detection of Live Bacteria (Escherichia coli)

Bead assays are an important rapid microbial detection technology suitable for extremely low pathogen levels. We report a bead assay for rRNA extracted from Escherichia coli K12 that does not require amplification steps and has readout on an Agilent 2100 Bioanalyzer flow cytometry system. Our assay...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2011-02, Vol.83 (4), p.1443-1447
Main Authors: Butterworth, Philip, Baltar, Henrique T. M. C. M, Kratzmeier, Martin, Goldys, Ewa M
Format: Article
Language:English
Subjects:
Analytical chemistry
Base Sequence
Binding sites
Chemistry
Chromatographic methods and physical methods associated with chromatography
E coli
Escherichia coli K12 - genetics
Escherichia coli K12 - isolation & purification
Exact sciences and technology
Flow Cytometry - methods
Gene amplification
Microbial Viability
Microspheres
Models, Molecular
Nucleic Acid Conformation
Oligonucleotide Probes - chemistry
Oligonucleotide Probes - genetics
Other chromatographic methods
Polystyrenes - chemistry
Ribonucleic acid
RNA
RNA, Ribosomal - analysis
RNA, Ribosomal - chemistry
RNA, Ribosomal - genetics
Spectrometry, Fluorescence
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Summary:Bead assays are an important rapid microbial detection technology suitable for extremely low pathogen levels. We report a bead assay for rRNA extracted from Escherichia coli K12 that does not require amplification steps and has readout on an Agilent 2100 Bioanalyzer flow cytometry system. Our assay was able to detect 125 ng of RNA, which is 16 times less than reported earlier. The specificity was extremely high, with no binding to a negative control organism (Bacillus subtilis). We discuss challenges faced during optimization of the key assay components, such as varying amounts of RNA in the samples, number of beads, aggregation, and reproducibility.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac103109v