Sensitive quantification of @entity526 O157:H7, @entity11621 , and XXXX by combining stopped polymerase chain reaction with chemiluminescence flow-through DNA microarray analysis.

@entity1885 @entity1859 @entity968 @entity6931 @entity11339 @entity11621 @entity273 @entity5363 @entity526
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 @entity526 O157:H7, @entity11621 , and @entity5363 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 @entity1885 together with a @entity1859 labeled forward primer for CL microarray imaging. A conjugate of an antidigoxigenin antibody and horseradish peroxidase recognized the @entity1859 -labeled antistrands bound to the probes on the microarray surface and catalyzed the reaction of @entity6931 and @entity273 . The generated light emission was recorded by a sensitive @entity11339 ( @entity11339 ) camera. The quantification was conducted by a flow-through CL microarray readout system. The DNA microarrays were based on an NHS-activated @entity968 -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 @entity526 O157:H7, @entity11621 , and @entity5363 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.
