Whether in clinical or epidemiological settings, there is a continued need in identification of B. anthracis and unambiguous discrimination it from close relatives and organisms with similar characteristics. Genomic analysis provides direct information about tested organism in opposite to indirect, phenotype analysis, however represents in current certified Anthrax tests by complicated (e.g. 16S rRNA gene sequencing), or unreliable (PCR-related) tests and may be performed now at advanced detection laboratories only. Akonni proposes microarray platform that will fill a fundamental technology gap between traditional clinical or disease diagnostics and new biodefense and associated epidemiology research. The objective of this application is to develop a portable clinical oriented diagnostic to assist in the rapid and reliable identification of B. anthracis and relative pathogens from B. cereus group by microarray analysis of pure cultures obtained for sample testing at Laboratory Response Network for Bioterrorism (LRN). Simple, rapid, reliable and low-cost Akonni diagnostic will bring sophisticated and final testing from Level C-D, advanced high-tech laboratories, to Level A, laboratories typical for community hospitals. Akonni will unify benefits of 3D gel drop microarray technology with microfluidic platform, newest 1 minute DNA/RNA labeling-fragmentation chemistry and last developments in phylogenetic classification of B. anthracis and its close relatives. The research of Phase 1 will focus on the method development and validation of the gel-drop microarray microfluidic card components.
Specific aims i nclude integration of sample preparation protocol in microfluidic card, design, produce and test a gel-drop microarray on a glass slide for identification of B. anthracis and testing of gel-drop microarray against samples prepared on microfluidic card from specified isolates. Final card will accept ~50uL of pure bacterial culture suspension; the card will mix the sample represented by 1 bacterial colony grown on agar plate with the buffers and provide rRNA isolation, labeling, fragmentation and purification. A peltier heater will control the temperature conditions within the card. The microfluidic card will handle all mixing, metering and washing steps and will be optically compatible for fluorescence detection and data analysis after integration of microarray into the card on Phase 2 of the project. We will validate our diagnostic in our BSL-2 facilities in Argonne National Laboratory. ? ? ?
| Bavykin, Sergei G; Mikhailovich, Vladimir M; Zakharyev, Vladimir M et al. (2008) Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray. Chem Biol Interact 171:212-35 |
