부산대학교 도서관

This chapter will address some of the many issues associated with the identification of signatures based on genomic DNA/RNA, which can be used to identify and characterize pathogens for biodefense and microbial forensic goals. For the purposes of this chapter, we define a signature as one or more strings of contiguous genomic DNA or RNA bases that are sufficient to identify a pathogenic target of interest at the desired resolution and which could be instantiated with particular detection chemistry on a particular platform. The target may be a whole organism, an individual functional mechanism (e.g., a toxin gene), or simply a nucleic acid indicative of the organism. The desired resolution will vary with each program's goals but could easily range from family to genus to species to strain to isolate. The resolution may not be taxonomically based but rather pan-mechanistic in nature: detecting virulence or antibiotic-resistance genes shared by multiple microbes. Entire industries exist around different detection chemistries and instrument platforms for identification of pathogens, and we will only briefly mention a few of the techniques that we have used at Lawrence Livermore National Laboratory (LLNL) to support our biosecurity-related work since 2000. Most nucleic acid based detection chemistries involve the ability to isolate and amplify the signature target region(s), combined with a technique to detect the amplification. Genomic signature based identification techniques have the advantage of being precise, highly sensitive and relatively fast in comparison to biochemical typing methods and protein signatures. Classical biochemical typing methods were developed long before knowledge of DNA and resulted in dozens of tests (Gram's stain, differential growth characteristics media, etc.) that could be used to roughly characterize the major known pathogens (of course some are uncultivable). These tests could take many days to complete and precise resolution of species and strains is not always possible. In contrast, protein recognition signatures composed of antibodies or synthetic high-affinity ligands offer extremely fast results but require a large quantity of the target to be present. False positives/negatives are also a problem with some protein-based techniques (home pregnancy kits use this basic approach).