Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

We investigated the use of a TaqMan 5 nuclease assay (5NA) directed against the Francisella tularensis outer membrane protein (Fop) gene and a polymerase chain reaction-enzyme immunoassay (PCR-EIA) directed against the tul 4 gene for detection of this organism in experimentally infected mice and in field-collected tick vectors. We also evaluated the use of specially formulated filter paper (FTA ) for rapid sample preparation. The 5NA had a detection limit of 1 pg of genomic DNA (100 colony-forming units) and could be completed within several hours. The PCR-EIA could detect 1 pg of genomic DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. tularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA paper provided inexpensive, rapid, template preparation for the tick extracts, mouse tissues, and DNA obtained from clinical specimens. These probe-based assays have the potential to provide rapid, real-time/high-throughput molecular diagnostics in field situations. Tularemia is a zoonosis caused by the gram-negative, pleomorphic, nonmotile bacterium Francisella tularensis. Rodents and lagomorphs are principle natural hosts. In the United States, infection is usually acquired from handling animal skins or carcasses, and much less frequently from tick (Dermacentor spp.) or deer fly (Chrysops spp.) bites. It is also possible to acquire the disease from drinking water that has been contaminated with animal feces and urine, or by eating inadequately cooked meat. Francisella tularensis can be infectious via aerosol route, and laboratory-acquired cases are not uncommon. Because of the relative ease with which this agent can be cultivated, and its highly infectious nature, there is some concern that it may be used for bio- logical warfare or bioterrorism.