|
Target Organism
|
Number of Signatures
|
Sensitivity of Pure Template
|
Number of Signatures
|
Sensitivity of template DNA + 10,000x human DNA
|
|
|
Bacillus anthracis
|
Chromosomal
|
2
|
0.01 pg
|
2
|
0.01 pg
|
|
pXO1
|
2
|
0.01 pg
|
2
|
0.01 pg
|
|
|
pXO2
|
1
|
0.1 pg
|
1
|
0.1 pg
|
|
|
Yersinia Pestis
|
CO92
|
5
|
0.1 pg
|
4
|
0.1 pg
|
|
Francisella tularensis
|
subsp. tularensis SCHU S4
|
5
|
0.01 pg
|
4
|
0.01 pg
|
|
subsp. novicida
|
4
|
0.01 pg
|
3
|
0.01 pg
|
|
|
Burkholderia
|
mallei
|
2
|
0.01 pg
|
2
|
0.01 pg
|
|
pseudomallei
|
3
|
0.1 pg
|
3
|
0.1 pg
|
|
|
Clostridium
|
botulinum
|
3
|
1 pg
|
3
|
1 pg
|
|
difficile
|
1
|
0.1 pg
|
1
|
0.1 pg
|
|
|
perfringens
|
3
|
10 pg
|
2
|
1 pg
|
|
|
Vaccinia
|
3
|
0.01 pg
|
3
|
0.01 pg
|
|
|
Salmonella
|
enterica
|
3
|
0.1 pg
|
ND
|
ND
|
|
Shigella
|
flexneri
|
3
|
0.01 pg
|
ND
|
ND
|
|
Escherichia coli
|
O157:H7
|
2
|
0.01 pg
|
ND
|
ND
|
Our assay development strategy combines sophisticated bioinformatic characterization of a genetic sequence resulting in the definition of sequences unique to the target organism (or gene) as compared with GenBank published sequences. Typically we select oligonucleotides between 35 to 50 bases in length for laboratory microarray validation. Validation microarrays contain hundreds to thousands of candidate signature sequences for each target organism. Frequently fifteen to twenty organisms are represented on a single array. Purified genomic DNA or RNA from reference stocks are whole genome amplified (WGA) with the incorporation of a visualization marker, such as biotin. The product of the WGA is a fragmented and labeled representation of the starting material with size ranges of 200-500 base pairs. This material is subsequently hybridized to the sequences on the microarray to determine hybridization efficiencies. WGA labeled material from near neighbor organisms is also employed to demonstrate lack of cross-reactivity. We have conducted laboratory validation of signature molecular sequences from dozens of organisms and have a reference data base of over 40,000 signature molecular markers. These laboratory validated molecular signatures become the target for molecular assays. These molecular assays can be either classical PCR or qPCR as well as bead based assays.
Our bead based multiplexed molecular assays are developed using the proven Luminex technology and their magnetic beads. The oligonucleotides validated on microarrays become the capture oligonucleotide on the magnetic beads. We then identify candidate forward and reverse primers from flanking regions within the genome. The primers are all within unique genome regions from which the capture oligonucleotide was selected. Assay optimization includes evaluation of both symmetric and asymmetric PCR and the determination of limits of detection in the presence and absence of a 10,000 fold excess of human DNA.
Our bead based multiplexed molecular assays are developed using the proven Luminex technology and their magnetic beads. The oligonucleotides validated on microarrays become the capture oligonucleotide on the magnetic beads. We then identify candidate forward and reverse primers from flanking regions within the genome. The primers are all within unique genome regions from which the capture oligonucleotide was selected. Assay optimization includes evaluation of both symmetric and asymmetric PCR and the determination of limits of detection in the presence and absence of a 10,000 fold excess of human DNA.
Rapid Development of Multiplexed Molecular Assays for Biothreat Organisms
© 2008 GenArraytion, Inc. All Rights Reserved.
Assay Development
