Microarray analysis of microbial virulence factors

Abstract

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.

FIG. 1

Identification of virulence factor genes by multiplex PCR. The PCR (annealing temperature, 59°C) was performed in the presence of primers designed to detect the rfbE, fliC, eaeA, slt-I, and slt-II genes (A) or the ipaH gene (B). Lane numbers correspond to the bacterial sample numbers in Table 1. Lanes M, 100-bp molecular size DNA marker.

FIG. 2

Synthesis of fluorescent probes using multiplex PCR. The Cy5-labeled PCR products were generated in the presence of six pairs of primers required for the simultaneous amplification of all target microbial virulence factor genes. The annealing temperature of the PCR was 45°C. Lane numbers correspond to the bacterial sample numbers in Table 1. Lanes M, 100-bp molecular size DNA marker.

FIG. 3

Detection of the presence of virulence factor genes in the bacterial DNAs by using microarray technology. Yellow numbers correspond to the sample numbers of the bacterial strains listed in Table 1. Shown are nonbacterial marker spots (lanes 1) and oligonucleotide targets for specific detection of the rfbE (lanes 2), fliC (lanes 3), eaeA (lanes 4), sltI (lanes 5), sltII (lanes 6), and ipaH (lanes 7) genes. Each spot is one of two independent sequences chosen from the gene. The horizontal bar at the bottom is the scale for the color representation of fluorescent-signal intensity.