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

摘要

ABSTRACT: Rapid analysis of pathogenic bacteria is essential for food andnwater control to preserve the public health. Therefore, we report on anchemiluminescence (CL) flow-through DNA microarray assay for thenrapid and sensitive quantification of the pathogenic bacteria Escherichia colinO157:H7, Salmonella enterica, and Campylobacter jejuni in water. Using thenstopped polymerase chain reaction (PCR) strategy, the amount ofnamplified target DNA was strongly dependent on the applied cell con-ncentration. The amplification was stopped at the logarithmic phase of thenPCR to quantify the DNA products on the DNA microarray chip. Thengeneration of single-stranded DNA sequences is essential for DNAnhybridization assays on microarrays. Therefore, the DNA strands of the PCR products were separated by streptavidin-conjugatednmagnetic nanoparticles. This was achieved by introducing a reverse primer labeled with biotin together with a digoxigenin labelednforward primer for CL microarray imaging. A conjugate of an antidigoxigenin antibody and horseradish peroxidase recognized thendigoxigenin-labeled antistrands bound to the probes on the microarray surface and catalyzed the reaction of luminol and hydrogennperoxide. The generated light emission was recorded by a sensitive charge-coupled device (CCD) camera. The quantification wasnconducted by a flow-through CLmicroarray readout system. The DNAmicroarrays were based on anNHS-activated poly(ethylenenglycol)-modified glass substrate. The DNA probes which have the same DNA sequence as the reverse primer were immobilized onnthis surface. The full assay was characterized by spiking experiments with heat-inactivated bacteria in water. The total assay time wasn3.5 h, and the detection limits determined on CL microarrays were for E. coli O157:H7, S. enterica, and C. jejuni 136, 500, and 1 cellmL, respectively. The results of the DNA microarray assay were comparable to the SYBR green-based assays analyzed with a real-ntime PCR device. The advantage of the new microarray analysis method is seen in the ability of a high multiplex degree on DNAnmicroarrays, the high specificity of DNA hybridization on DNA microarrays, and the possibility to get quantitative results on annautomated CL flow-through microarray analysis system.