, 2008); however,

such an approach relies on the a priori selection of targets, and therefore suffers from the ‘if you didn’t look for it you won’t find it’ syndrome. When the imminent threat of attack with bioterrorism weapons was realized, the Defense Advanced Research Projects Agency of the US Department of Defense initiated an urgent search for new methods for the broad detection and identification of bacteria. find more Clearly, the existing culture methods were not inclusive of all species and were too slow and cumbersome. Thus, the enemy’s selection of a pathogen that was not detected by our well-known cultural paradigms would result in a disastrous failure to diagnose. In response to this call, David Ecker’s team, at Ibis, developed a novel strategy in which the amplicons produced by PCR would be weighted by mass spectroscopy and their precise weight would be NVP-BKM120 used to calculate their base composition. To provide for the identification of all bacteria, both known and unknown, both pathogen and nonpathogen, multiple sets of primers were designed to detect multiple classes of genes, including those that are highly conserved across entire domains (e.g. 16S and 23S rRNA genes)

as well as sequences that are phylum or class specific, and others that are specific to lower taxonomic groupings. Each set of primers are designed to hybridize to a conserved region of a gene that flanks a variable region. Thus, each species that is amplified by each primer pair will produce a different amplicon that is diagnostic or partially diagnostic for that species. By collectively looking at which primers yielded any product, Org 27569 and then characterizing the weight and ultimately the base composition of all the resulting products, it is possible to precisely determine

all those individual species that were present in the specimen. This approach is extremely flexible, allowing the design of different primer sets for a range of applications such as the broad detection of all bacteria, to the much more specific surveillance of influenza strains. No sequencing is required because the base content of the specific variable regions of each amplicon provides the information necessary for making a diagnosis as the system has a look-up database that uses a complex iterative proprietary algorithm (Eckeret al., 2008) that matches the observed amplicon weights against those of all of the known bacterial pathogens (Fig. 5). If a novel bacterium is present, the system will recognize this because one or more of the amplicon weights will not correspond to any species in the database. In such a case, the system notifies the user that a new species has been identified and what its most closely related relative is.