Quantification of E. coli DNA on a flow-through chemiluminescence microarray readout system after PCR amplification.
We report on a hybridization assay using DNA microarrays for the quantification of amplification products of the uidA gene of E. coli. Using the stopped-PCR strategy, the amplified target DNA was strongly dependent on the applied gene copies. The quantification was carried out by a flow-through chemiluminescence microarray readout system. The DNA microarrays were based on a poly(ethylene glycol)-modified glass substrate. The probes on the surface were 18 or 25 nucleotides long and the quantified PCR product was 60 nucleotides. The amplification was stopped after 25 cycles; at this point amplification was in the middle of the logarithmical phase, and the spread between different DNA starting concentrations reached the maximum. A conjugate of streptavidin and horseradish peroxidase (HRP) bound to the biotinylated strands on the microarray surface and catalyzed the reaction of luminol and hydrogen peroxide. The generated light emission was recorded by a sensitive charge-coupled device (CCD) camera. The detection limit for the gene uidA (beta-galactosidase) of E. coli was 1.1 x 10(5) copies/mL. This system allowed for a sensitive detection and quantification of E. coli in a concentration range from 10(6) to 10(9) copies/mL.
Donhauser
Simon C
SC
Institute of Hydrochemistry, Technische Universität München, Germany.
Niessner
Reinhard
R
Seidel
Michael
M
D002138:Calibration; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D005898:Glass / Q000737:chemistry; D049449:Luminescence; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000138:chemical synthesis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011092:Polyethylene Glycols