However, it is noted that as the number of targets grows, artifacts generated by unrelated primer pairs must be designed out of the assay. Each of the 3 10-plex PCR biothreat agent panels was validated by analyzing genomic DNA isolated from 20 Ba, 30 Ft and 34 Yp strains as well as DNA from 10 representatives each of their respective near neighbors strains or closely-related species. The choice of loci and the primer design warrants correct discrimination of non-pathogenic near neighbors from biothreat strains and allows identification of species and characterization into subtypes, biovars and potentially attenuated strains. In contrast to MLST methods, the Rapid Dasatinib Src-bcr-Abl inhibitor Focused Sequencing approach is not limited to selectively neutral housekeeping genes but instead also utilizes virulence factor, VNTR-like, non-coding, and other targets. The development of an appropriate set of targets allows powerful identification and genotyping of pathogens of interest. The total sequence information analyzed via the 10-plex PCR Ba panel represents approximately 0.07% of the,5.8 Mbp Ba genome, 0.19% of the,2 Mbp Ft genome, and 0.09% of the,4.6 Mbp Yp genome. Rapid Focused Sequencing of this small fraction of the Ba genome, for example, is sufficient to determine presence or absence of the pathogen itself, to distinguish and characterize the majority of Ba strains by virulence, and to unambiguously distinguish Ba strains from near neighbors. Gefitinib Similarly, Rapid Focused Sequencing of the Ft genome is sufficient to determine presence or absence of Ft-specific DNA, to distinguish between subspecies, and between subsp. tularensis type A1 and A2 strains. The approach also unambiguously distinguishes the near neighbors, subsp. novicida, from from pathogenic Ft subspp. tularensis and holarctica strains. The number and nature of the biothreat targets can be modified as the understanding of the genetics and biology of the pathogens advances over time. The ability to perform the highly multiplexed amplification and Sanger sequencing reactions in microfluidic biochips offers the potential to perform these assays in the field, on both environmental and clinical samples. The choice of assay is dependent on the given concept of operation. For example, in certain clinical settings, fewer near neighbors may be present, and the multiplexed PCR assay may provide sufficient diagnostic information. In most environmental settings, however, the potential for an enormous variety of near neighbors and the need for forensic analysis may lead to the preferential use of the sequencing assay. We have recently developed a ruggedized system that incorporates nucleic acid purification, multiplexed amplification, and electrophoretic separation in a single microfluidic biochip. We believe that this foundational system can be adapted to perform both the multiplexed PCR sizing and Rapid Focused Sequencing assays. A fully integrated microfluidic platform that enables rapid pathogen analysis in the field has the potential to dramatically improve biothreat detection capabilities. Individual primer pairs designed for each target loci were initially tested for performance by microfluidic singleplex PCR using 100 genome equivalents of reference strain template DNAs representing each agent and using only half of the total PCR reaction for separation and detection.