Development of a multi-pathogen oligonucleotide microarray for detection of Bacillus anthracis.

Abstract

An oligonucleotide microarray system has been specifically designed to detect and differentiate Bacillus anthracis from other bacterial species present in clinical samples. The pilot-scale microarray initially incorporated probes to detect six common species of bacteria, which were fully evaluated. The microarray comprised long oligonucleotides (50--70-mer) designed to hybridise with the variable regions of the 16S rRNA genes. Probes which hybridised to virulence genes were also incorporated; for B. anthracis, these initially included the pag, lef, cap and vrrA (for partial genotyping) genes. Hybridisation conditions were initially optimised to be run using 5 x SSC, 0.1% SDS, 50 degrees C for 16 h. The detection limits of the microarray were determined under these conditions by titration of chromosomal DNA and unlabelled amplicons followed by hybridisation to determine the levels of sensitivity that could be obtained with the microarray. Two different amplification methodologies were also compared-specific-primer based PCR and random PCR (with the labelling stage incorporated). Higher sensitivity was obtained using specific PCR primers, however, since one of the desired outcomes of a microarray-based detection system was the high discrimination that it offered, random amplification and labelling was used as the amplification method of choice. The length of hybridisation was investigated using a time-course, and 1--2h was found to give optimal and higher signals than 16 h incubation. These results indicate that microarray technology can be employed in a diagnostic environment and moreover, results may be obtained in a similar time-scale to a standard PCR reaction, but with the advantage that no a priori knowledge of the infectious agent is required for detection.