1. Mohamed Hashem, MD* 2. Marilyn A. Menegus, PhD* 1. *Departments of Pediatrics and Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY Molecular diagnostic tools and detection methods such as nucleic acid amplification are being used increasingly in the clinical microbiology laboratory to enhance the identification of microbial pathogens and to assist physicians in the diagnosis and management of a variety of infectious diseases. The principle of this technique is to detect and amplify a unique gene of the microorganism. This speeds the identification and reporting of microbial pathogens without reliance on their phenotypic characteristics. Effective clinical management of infectious diseases depends primarily on accurate identification of the causative pathogen. The early recognition of an infectious agent allows clinicians to make sound therapeutic decisions and avoid the indiscriminate use of antibiotics, which ultimately favors the development of antimicrobial resistance. To attempt to make nucleic acid amplification techniques (NAATs) understandable, we discuss the limitations of conventional diagnostic methods and how NAATs and polymerase chain reaction (PCR), in particular, can overcome these limitations. Microbiology laboratories have developed many dependable approaches for detecting and characterizing pathogens within a reasonable time frame. These methods include microscopy, culture, antigen detection, immunoserology, and more recently, nucleic acid probes. Conventional identification of microbial pathogens relies on discerning the phenotypic characteristics of the organisms. Bacterial metabolic characteristics, fungal conidiogenesis, parasitic morphology, and viral cytopathic effect are some of the phenotypic characteristics used commonly. Unfortunately, phenotypic characteristics often are not sufficiently discriminatory for strain differentiation. DNA probes, which identify nucleic acid sequences by hybridization, have replaced biochemical reactions and morphology for definitive identification of some infectious agents. Probes are used to identify slowly growing mycobacteria and to detect fastidious organisms such as Chlamydia trachomatis and Neisseria gonorrhoeae. DNA probes offer somewhat greater specificity than do immunoassays, although like immunoassays, they are not sufficiently sensitive to replace conventional culture in most cases. Although conventional diagnostic methods are dependable, most approaches have limitations. …