Testing and Validation of High Density Resequencing Microarray for Broad Range Biothreat Agents Detection
NAVAL RESEARCH LAB WASHINGTON DC CENTER FOR BIOMOLECULAR SCIENCE AND ENGINEERING
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Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription andor polymerase chain reaction RT-PCRPCR provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray RPM for detection of tropical and emerging infectious agents TEI including biothreat agents RPM-TEI v 1.0 RPM-TEI. The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention CDC, Atlanta, GA. Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assays limit of detection LOD. Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 104 copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses.
- Medicine and Medical Research