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 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.
Macquarie University , North Ryde, NSW, Australia.
Philip
P
Butterworth
Henrique T M C M
HT
Baltar
Martin
M
Kratzmeier
Ewa M
EM
Goldys
D001483:Base Sequence; D048168:Escherichia coli K12 / Q000235:genetics / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000379:methods*; D050296:Microbial Viability; D008863:Microspheres; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D011137:Polystyrenes / Q000737:chemistry; D012335:RNA, Ribosomal / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence
D015345:Oligonucleotide Probes; D011137:Polystyrenes; D012335:RNA, Ribosomal