Rapid, accurate and sensitive detection of bio-threat agents requires a broad-spectrum assay capable of discriminating between closely related microbial or viral pathogens. In cases where a biological agent release has been identified, forensic analysis demands detailed genetic signature data for accurate strain identification and attribution. Identification of genetic signatures for detection coupled with identification of pathogenic phenotypes would provide a robust means of discriminating pathogens from closely related but benign species .
Current forensics methods based on bacteriological, serological, biochemical and genomic strategies have been used to detect pathogens using serological methods , PCR , real time PCR [4, 5] and Multi-loci VNTR (variable-number tandem repeats) or MLVA [6–9]. Although bacteriological culture of Brucella spp. from blood, milk, fetal fluids and tissues, or other host tissues remain the 'gold standard' for diagnosis, bacteriologic culture has reduced sensitivity, is labour intensive, time consuming, typically requiring two weeks, and is a risk for laboratory personnel . Serological assays, such as Rose Bengal, a rapid plate agglutination diagnostic test, is currently used for diagnosing infection with Brucella species in the field , however serological tests frequently have reduced specificity due to cross reactivity with other bacteria. Specific antibodies are required to be present at sufficiently high level and may require several weeks to develop before they are detectable. PCR based methods are used for epidemiological trace back and strain specific identification . Although rapid in nature, specific primers are required for specific genes from these genomes or 16S rRNA genes or VNTR (variable-number tandem repeats) in a given genome. Real time PCR based methods have been used to identify Brucella species using IS711, bcsp31 and per target genes [4, 5]. In addition, assays based on single-nucleotide polymorphisms have been developed for identification of Brucella isolates at the species level. These SNPs have been used to classify isolates into known Brucella species . Recently MLVA or multi-loci VNTR (Variable-number tandem repeats) a genotype-based typing method and has been used as an epidemiological classification and SNP identification method for Brucella isolates in a field population [6–9]. MLVA method is used to understand the genetic diversity in polymorphic loci and to establish taxonomic relationships between different biovars of Brucella. It is used for microbial typing and epidemiologic studies by amplifying loci which are specific to a given genome and sequencing these regions. This is a powerful approach and is being used to create phylogenetic relationships and discovery of single nucleotide polymorphisms in independent loci from different Brucella isolates .
Array based approaches for forensic detection utilizes genome specific ribosomal RNA genes, genome specific PCR markers or oligonucleotide probes. Arrays from rRNA are derived from a combination of rRNA genes from a given set of organisms of high priority. Universal PCR is used to amplify one or more universal genes, including 16S, 18S and 23S as well as screen for pathogen-specific polymorphisms . One of the challenges of this approach is the frequent and unexpected amplification of contaminating template DNA, as observed in control reactions. Another potential problem with targeting 16S rRNA pathogen specific sequences is unexpected polymorphisms. Hence, naturally occurring variants may not be represented on the microarray, and failure to detect the variants would represent false negatives . Another common PCR based approach detects pathogen type by amplification of a specific set of genetic markers that are measured on an array that has several probes for genes from a set of organisms. Such tests have been used for food-borne bacteria such as E. coli O157:H7 , viruses  and mixtures of pathogens . The drawback of using this approach with multiplex PCR primers sets is the generation of spurious products . Array based technologies using 70-mer oligonucleotide probes derived from pathogen specific genes have similar factors that require consideration. For instance, viral detection using a microarray composed of 1,600 unique viral oligonucleotides (70-mers) derived from 140 distinct viral genomes has been previously demonstrated  as a powerful viral detection mechanism, but the drawback of this strategy is that only the group of known pathogen-specific genes will be queried.
Given the enormous spectrum of genetic possibilities, only a highly parallel field deployable technology that is universal in nature has near-term potential to address these needs. The initial vision for a universal DNA microarray was a matrix of oligonucleotide containing features with unique n-mer probes . This matrix could in theory be used to query a biological sample for the presence of any nucleic acid sequence. This technique requires constructing an array that contains 4n features. Larger values of n infuse greater specificity into the arrayed probes, but as n increases the number of required features grows rapidly. This universality is obtained by synthesizing a combinatorial n-mer array containing all 4n possible sequences of length n . The key issue is to find a value of n that is large enough to afford sufficient hybridization specificity, yet small enough to be practically fabricated and analyzed.
We have previously demonstrated the utility of a genome sequence-independent microarray for identifying genetic differences [18, 19]. The initial prototype of universal arrays used oligonucleotide probe lengths of 12 and 13 bases. From 412 possible probes, a subset of 14,283 probes was synthesized using in situ synthesis technology and digital optical chemistry (DOC) [20–22]. Fluorescently labelled genomic DNA was hybridized to produce unique informative patterns (i.e. bio-signatures) on a test set of pathogens and host (Bacillus subtilis, Yersinia pestis, Streptococcus peumoniae, Bacillus anthracis, and Homo sapiens). In addition, we have shown that a custom microsatellite microarray can be used to demonstrate global differences between species by measuring hybridization intensities for every possible repetitive nucleotide motif from 1-mers to 6-mers . Further we have used genome sequence independent microsatellites to identify global differences in the genomes of 93 cancer, cancer-free and high risk patient cell line samples . This paper describes a larger high density oligonucleotide microarray with 370,000 elements, called Universal Bio-signature Detection Array (UBDA), designed by our laboratory and commercially produced by Roche-Nimblegen (Madison, WI) using light-directed photolithography [16, 24]. The platform design which consists mainly of probes, that are tailored to be genome independent, is mathematically derived and therefore unbiased (Additional file 1, Table S1).
This strategy exploits the unique signature of a sample in the form of a pattern generated from hybridization of any unknown genome (DNA or cDNA) to a very high-density species-independent oligonucleotide microarray. Brucella species and several other pathogens were used as examples to demonstrate this forensics technology platform. Hybridization patterns are unique to a genome, and potentially to different isolates or a mixture of organisms. These techniques may be especially useful in evaluating and differentiating species whose genome has not yet been sequenced.