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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Veröffentlicht in:	Analytical chemistry (Washington) 2011-02, Vol.83 (4), p.1443-1447
Hauptverfasser:	Butterworth, Philip, Baltar, Henrique T. M. C. M, Kratzmeier, Martin, Goldys, Ewa M
Format:	Artikel
Sprache:	eng
Schlagworte:	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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Beschreibung
Zusammenfassung:	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