title: Sensitive quantification of Escherichia coli O157:H7, Salmonella enterica , and Campylobacter jejuni by combining stopped polymerase chain reaction with chemiluminescence flow-through DNA microarray analysis.
abstract: Rapid analysis of pathogenic bacteria is essential for food and water control to preserve the public health. Therefore, we report on a chemiluminescence (CL) flow-through DNA microarray assay for the rapid and sensitive quantification of the pathogenic bacteria Escherichia coli O157:H7, Salmonella enterica , and Campylobacter jejuni in water. Using the stopped polymerase chain reaction (PCR) strategy, the amount of amplified target DNA was strongly dependent on the applied cell concentration. The amplification was stopped at the logarithmic phase of the PCR to quantify the DNA products on the DNA microarray chip. The generation of single-stranded DNA sequences is essential for DNA hybridization assays on microarrays. Therefore, the DNA strands of the PCR products were separated by streptavidin-conjugated magnetic nanoparticles. This was achieved by introducing a reverse primer labeled with biotin together with a digoxigenin labeled forward primer for CL microarray imaging. A conjugate of an antidigoxigenin antibody and horseradish peroxidase recognized the digoxigenin-labeled antistrands bound to the probes 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 quantification was conducted by a flow-through CL microarray readout system. The DNA microarrays were based on an NHS-activated poly(ethylene glycol)-modified glass substrate. The DNA probes which have the same DNA sequence as the reverse primer were immobilized on this surface. The full assay was characterized by spiking experiments with heat-inactivated bacteria in water. The total assay time was 3.5 h, and the detection limits determined on CL microarrays were for E. coli O157:H7, S. enterica , and C. jejuni 136, 500, and 1 cell/mL, respectively. The results of the DNA microarray assay were comparable to the SYBR green-based assays analyzed with a real-time PCR device. The advantage of the new microarray analysis method is seen in the ability of a high multiplex degree on DNA microarrays, the high specificity of DNA hybridization on DNA microarrays, and the possibility to get quantitative results on an automated CL flow-through microarray analysis system.
authors.affiliation: Chair for Analytical Chemistry and Institute of Hydrochemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München, Germany.
authors.forename: Simon Christian
authors.initials: SC
authors.lastname: Donhauser
authors.forename: Reinhard
authors.initials: R
authors.lastname: Niessner
authors.forename: Michael
authors.initials: M
authors.lastname: Seidel
mesh_terms: D016123:Campylobacter jejuni / Q000235:genetics* / Q000302:isolation & purification*; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification*; D017022:Flow Injection Analysis; D008163:Luminescent Measurements / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019779:Salmonella enterica / Q000235:genetics* / Q000302:isolation & purification*