Rapid simultaneous screening of seven clinically important enteric pathogens using a magnetic bead based DNA microarray

In this study, we describe a DNA microarray assay by using bead-mediated visible light-assisted signal detection for simultaneous screening of seven clinically important enteric pathogens, including Escherichia coli O157:H7, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella spp., Staphylococcus aureus, Rotavirus, and Norwalk virus (including genogroup I and II). Seven pairs of primers, in which the forward primers were labeled with biotin at the 5′ end, were designed and two sets of multiplex asymmetric PCR system were established to amplify the target genes of the seven pathogens. Twelve type specific oligonucleotides were designed and immobilized onto the aldehyde radical modified glass slide to function as target capture probes. After hybridization and stringency washes, the hybridized biotinylated PCR products were detected by the streptavidin-coated magnetic beads. The final hybridization results were visible to the naked eyes and can be imaged by CCD or digital camera. A total of 86 samples previously identified by conventional microbiological methods and/or PCR method were randomly selected to assess the specificity of this assay by a blind study. A coincidence rate of 100% was obtained. Due to the simplicity and specificity of the magnetic bead based DNA microarray, it is especially appropriate for the diagnosis and monitoring of enteric infectious diseases in the community and seaport.