Diagnostic system for rapid and sensitive differential detection of pathogens. Emerg. Infect. Dis

Naturally emerging and deliberately released pathogens demand new detection strategies to allow early recognition and containment. We describe a diagnostic system for rapid, sensitive, multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples. Efficient laboratory diagnosis of infectious diseases isincreasingly important to clinical management and public health. Methods to directly detect nucleic acids of microbial pathogens in clinical specimens are rapid, sen-sitive, and may succeed when culturing the organism fails. Clinical syndromes are infrequently specific for single pathogens; thus, assays are needed that allow multiple agents to be simultaneously considered. Current multiplex assays employ gel-based formats in which products are distinguished by size, fluorescent reporter dyes that vary in color, or secondary enzyme hybridization assays. Gel-based assays are reported that detect 2–8 different targets with sensitivities of 2–100 PFU or <1–5 PFU, depending on whether amplification is carried out in a single or nest-ed format, respectively (1–4). Fluorescence reporter sys-tems achieve quantitative detection with sensitivity similar to that of nested amplification; however, their capacity to simultaneously query multiple targets is limit-ed to the number of fluorescent emission peaks that can be unequivocally resolved. At present, up to 4 fluorescent reporter dyes can be detected simultaneously (5,6). Multiplex detection of up to 9 pathogens has been achieved in hybridization enzyme systems; however, the method requires cumbersome postamplification process-ing (7). The Study To address the need for sensitive multiplex assays in diagnostic molecular microbiology, we created a poly-merase chain reaction (PCR) platform in which microbial gene targets are coded by a library of 64 distinct Masscod