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 agains t the Francisella tularensis outer membrane protein (Fop) gene and a polyme rase 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 formu lated filter paper (FTA(TM)) for rapid sample preparation. The 5NA had a de tection 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 genomi c DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. t ularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA pa per provided inexpensive, rapid, template preparation for the tick extracts , mouse tissues, and DNA obtained from clinical specimens. These probe-base d assays have the potential to provide rapid, real-time/high-throughput mol ecular diagnostics in field situations.