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Molecular Beacons for Multiplex Detection of Four Bacterial Bioterrorism Agents

¹ Department of Medicine, Division of Infectious Disease, New Jersey Medical School, The University of Medicine and Dentistry of New Jersey, Newark, NJ;² Department of Molecular Genetics, The Public Health Research Institute, Newark, NJ

^aaddress correspondence to this author at: Division of Infectious Disease, New Jersey Medical School, 185 South Orange Ave., MSB A920C, Newark, NJ 07103; fax 973-972-0713, e-mail allandda@umdnj.edu

The first 300 words of the full text of this article appear below.

The advent of bioterrorism has highlighted the need for rapid, simple, and robust diagnostic assays to detect select agents. Mortality from select agents may be greatly reduced by prompt treatment (1); however, treatment may be delayed if diagnostic assays are outsourced to reference laboratories. Most bacterial species that would likely be used as bioterrorism agents infect the blood stream during the course of life-threatening disease. Furthermore, even "nonseptic" syndromes may produce hematogenous bacterial DNA that could be detected by a sensitive assay (2). This means that a rapid "molecular" version of a blood culture would fulfill many of the rapid diagnostic needs for biodefense.

Bacteria can be detected in blood and other sterile body sites by the identification of species-specific DNA sequences in their 16S rRNA genes. These species-specific sequences are flanked by conserved sequences, permitting most rRNA targets to be amplified by PCR using a limited set of "universal" primers (3). Real-time PCR is well suited for sensitive and specific pathogen detection because it is performed in hermetically sealed wells, which greatly reduces the risk of cross-contamination, and it does not require post-PCR analysis (4). Real-time PCR assays have been developed for some select agents, most of which use fluorogenic 5'-nuclease (TaqMan) probes (5)(6)(7). However, TaqMan probes are difficult to use in multiplex PCR assays (8)(9). In contrast, molecular beacons are real-time PCR probes that are particularly amenable to multiplexing (10). They can be labeled with differently colored fluorophores (11), use a common nonfluorescent quenching moiety (9), and have thermodynamic properties that favor highly specific detection of nucleic acid sequences (12).

Here we describe a real-time PCR assay that simultaneously detects four bacterial . . . [Full Text of this Article]

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