A novel DNA microarray for rapid diagnosis of enteropathogenic bacteria in stool specimens of patients with diarrhea

Abstract

A microarray technique for the detection and identification of enteropathogenic bacteria at the species and subspecies levels was developed in this study, and the target bacteria included pathogenic Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella enterica, Campylobacter jejuni, Shigellae, Yersinia enterocolitica, and Listeria monocytogenes. The virulence gene of each pathogen was chosen as the amplification target, labeled with a fluorescence dye by multiplex polymerase chain reaction (PCR), and hybridized to the specific virulence gene probes that had been immobilized on a microchip. Stool specimens from 34 patients with diarrhea were tested in this study. Five were positive for multiple genera. Nested PCRs and sequencing were used to amplify and identify the related genes, which were found to share 95.8% to 100% of the nucleotide identity with the corresponding regions in the Genbank database. Real-time PCR was used to determine the number of gene copies to determine the sensitivity of this technique, which was shown to be 58 copies/microl. The results indicated that the microarray technique which targets multiple virulence genes of enteropathogenic bacteria at the species and subspecies levels is an attractive diagnostic tool for rapidly and simultaneously identifying multiple enteropathogenic pathogens in clinical practice, especially in patients with infectious diarrhea.