Sequence-specific identification of 18 pathogenic microorganisms using microarray technology.

Abstract

We have developed a Multi-Pathogen Identification (MPID) microarray for high confidence identification of eighteen pathogenic prokaryotes, eukaryotes and viruses. Analysis of amplified products from pathogen genomic DNA using microarray hybridization allows for highly specific and sensitive detection, and allows the discrimination between true amplification products and false positive amplification products that might be derived from primers annealing to non-target sequences. Species-specific primer sets were used to amplify multiple diagnostic regions unique to each individual pathogen. Amplified products were washed over the surface of the microarray, and labelled with phycoerythrin-streptavidin for fluorescence detection. A series of overlapping 20-mer oligonucleotide probes hybridize to the entire diagnostic region, while parallel hybridizations on the same surface allow simultaneous screening for all organisms. Comparison to probes that differ by a single mismatch at the central position reduced the contribution of non-specific hybridization. Samples containing individual pathogens were analyzed in separate experiments and the corresponding species-specific diagnostic regions were identified by fluorescence among their highly redundant probe sets. On average, 91% of the 53 660 pathogen probes on the MPID microarray performed as predicted. The limit of detection was found to be as little as 10 fg of B. anthracis DNA in samples that were amplified with six diagnostic primer-pairs. In contrast, PCR products were not observed at this concentration when identical samples were prepared and visualized by agarose gel electrophoresis.