Review © Future Drugs Ltd. All rights reserved. ISSN 1473-7159 89 CONTENTS Pathogen detection & identification Conventional approaches to nucleic acid signatures Evolution of genetic signatures Bioinformatics & genetic signature development Expert opinion Five-year view Key issues References Affiliations www.future-drugs.com Development of rationally designed nucleic acid signatures for microbial pathogens Catherine A Cleland, P Scott White ‡ , Alina Deshpande, Murray Wolinsky, Jian Song and John P Nolan † The detection and identification of microbial pathogens are critical challenges in clinical medicine and public health surveillance. Advances in genome analysis technology are providing an unprecedented amount of information about bacterial and viral organisms, and hold great potential for pathogen detection and identification. In this paper, a rational approach to the development and application of nucleic acid signatures is described based on phylogenetically informative sequence features, especially single nucleotide polymorphisms. The computational tools that are available to enable the development of the next generation of microbial molecular signatures for clinical diagnostics and infectious disease surveillance are reviewed and the impact on public health and national security will be discussed. Expert Rev. Mol. Diagn. 4(3), (2004) Authors for correspondence † Bioscience Division, MS M888 Los Alamos National Laboratory, Los Alamos, NM 87545, USA Tel.: +1 505 667 1623 Fax: +1 505 665 3024 nolan@telomere.lanl.gov ‡ Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA Tel.: +1 505 667 0053 Fax: +1 505 665 3024 scott_white@lanl.gov. KEYWORDS: assay, bacteria, detection, identification, indel, platform, phylogenetic analysis, SNP, taxonomy, virus Pathogen detection & identification The longstanding need for improved clinical diagnostic tests for pathogenic organisms has recently taken on a new perspective with the increased concerns over bioterrorism. As bio- defense against man-made outbreaks of infec- tious and/or pathogenic organisms represents another dimension of more traditional public health activities, there is a great opportunity for progress that improves our ability to pre- vent, recognize and respond to infectious dis- ease. Much effort is being put into the devel- opment of biosensors for applications ranging from point-of-care detection to epidemiologi- cal and forensic analysis, and these systems vary widely in their mode of operation and general utility [1–4]. Any diagnostic method has two components: an assay chemistry and a detection platform. In general, the assay chemistry determines the sensitivity and specificity of a method, while the detection platform determines features, such as portability, power requirements and sample throughput, as well as sensitivity. For example, immunoassays are widely used on a range of detection platforms including colorimet- ric test strips, microplate-based enzyme-linked immunosorbent assays (ELISAs), as well as a new generation of instruments that promise portability and low cost in an integrated microfabricated package [5]. Often, it is the affinity and specificity of the antibodies used that will ultimately determine assay perform- ance. Nucleic acid-based diagnostics that use PCR technology can specifically amplify targets from just a few organisms in a complex sam- ple. The PCR products can be analyzed by a wide range of detection platforms, but the sen- sitivity and specificity is determined by the PCR step. For example, the popular 5´ nucle- ase assay (also known as Taq-Man ® , real-time PCR or quantitative PCR) employs PCR using two primers combined with a fluorogenic hybridization probe in conjunction with an optical detection-capable thermal cycler. Ide- ally, a single detection platform will be capable of analyzing the results from a variety of assay chemistries. The optimal combination of assay chemistry and detection platform will be determined by the specific features of an application. For example, in a doctor’s office it might be neces- sary to screen a small number of samples for a limited number of pathogenic organisms,