DNA Microarray Chip

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.

Abstract: Background A rapid, accurate, flexible and reliable diagnostic method may significantly decrease the costs of diagnosis and treatment. Designing an appropriate microarray chip reduces noises and probable biases in the final result. Objective The aim of this study was to design and construct a DNA Microarray Chip for a rapid detection and identification of 10 important bacterial agents. Method In the present survey, 10 unique genomic regions relating to 10 pathogenic bacterial agents including Escherichia coli (E.coli), Shigella boydii, Sh.dysenteriae, Sh.flexneri, Sh.sonnei, Salmonella typhi, S.typhimurium, Brucella sp., Legionella pneumophila, and Vibrio cholera were selected for designing specific long oligo microarray probes. For this reason, the in-silico operations including utilization of the NCBI RefSeq database, Servers of PanSeq and Gview, AlleleID 7.7 and Oligo Analyzer 3.1 was done. On the other hand, the in-vitro part of the study comprised stages of robotic microarray chip probe spotting, bacterial DNAs extraction and DNA labeling, hybridization and microarray chip scanning. In wet lab section, different tools and apparatus such as Nexterion® Slide E, Qarraymini spotter, NimbleGen kit, TrayMixTM S4, and Innoscan 710 were used. Results A DNA microarray chip including 10 long oligo microarray probes was designed and constructed for detection and identification of 10 pathogenic bacteria. Conclusion The DNA microarray chip was capable to identify all 10 bacterial agents tested simultaneously. The presence of a professional bioinformatician as a probe designer is needed to design appropriate multifunctional microarray probes to increase the accuracy of the outcomes.