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Am. J. Trop. Med. Hyg., 62(2), 2000, pp. 310-318
Copyright © 2000 by The American Society of Tropical Medicine and Hygiene

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American Journal of Tropical Medicine and Hygiene, Vol 62, Issue 2, 310-318
Copyright © 2000 by American Society of Tropical Medicine and Hygiene

Research Articles


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

JA Higgins, Z Hubalek, J Halouzka, KL Elkins, A Sjostedt, M Shipley, and MS Ibrahim

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.


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