|
Several Levels of Identification
To enhance their detection development effort, researchers are exploring advanced methods that distinguish slight differences in DNA. They are using the multidisciplinary approach that characterizes Livermore research programs. In this case, DNA signature development involves a team of microbiologists, molecular biologists, biochemists, geneticists, and computer experts. In addition, the Livermore work benefits from collaborations with experts worldwide, extensive experience with DNA sequencing, and affiliation with DOE's Joint Genome Institute (see S&TR, April 2000, The Joint Genome Institute: Decoding the Human Genome).
Much of the work is focused on screening the two to five million bases that comprise a typical microbial genome to design unique DNA markers that will identify the microbe. The markers, called primer pairs, typically contain about 30 base segments and bracket specific regions of DNA that are a few hundred bases long. The bracketed regions are replicated many thousands of times with a detector that uses polymerase chain reaction (PCR) technology. Then they are processed to unambiguously identify and characterize the organism of interest.
Weinstein notes that different signatures will be needed for different levels of resolution. For example, authorities trying to characterize an unknown material or respond to a suspected act of bioterrorism will begin with fairly simple signatures that flag potentially harmful pathogens within a few minutes. Typically, such a signature would encompass one or two primer pairs and be sufficient for identification at the genus level (Yersinia or Bacillus, for example) or below.
A signature in the next level of resolution is needed for unambiguously identifying a pathogen at the species level (Yersinia pestis, for example). This signature involves about 10 primer pairs. Currently, it takes several days to obtain conclusive data for a species-level signature. The goal is to reduce that time to less than 30 minutes.
The third signature level is used in pathogen characterization, identifying any features that could affect medical response (for example, harmless vaccine materials versus highly virulent or antibiotic resistance pathogens). This signature level involves some 20 to 30 primer pairs. Together, the primer pairs offer a certainty of correct identification. Currently, providing such a high level of confidence requires several days; the goal again is to reduce the time to less than 30 minutes.
The final signature level, intended primarily for law enforcement use, will permit detailed identification of a specific strain of a pathogen (for example, Yersinia pestis KIM) and correlate that strain with other forensic evidence. Such data will help to identify and prosecute attackers. The present typical time lag for results is currently a few weeks, and the goal is to reduce that to a few days.
Biological foundations program scientists have worked with DOE and other agencies to assemble a list of natural pathogens most likely to be used in a domestic attack. The list includes bacteria, viruses, and other classes of threats, such as agricultural pathogens. Two extremely virulent pathogens head the list: B. anthracis and Y. pestis, which cause anthrax and plague in humans, respectively. Bacillus anthracis has few detectable differences among its strains, whereas Y. pestis strains can vary considerably in genetic makeup. Unraveling the significant differences between the two organisms will give national laboratory researchers experience vital for facing the challenges of the next few years, as they develop signatures for a wide spectrum of microbes. |