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

Abstract

Abstract. We investigated the use of a TaqMan 5Ј 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 (Ͻ100 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.

Comparison of four methods of extracting DNA fromD. gallinae(Acari: Dermanyssidae)

“…Another attractive method, the FTA technology, was also tested, and compared to the others. It has been reported for detection of pathogenic DNA [19] and particularly for the detection of microsporidia in fire ants and of Francisella tularensis in pools of ticks, with satisfying results [12,24].…”

Histologic and molecular diagnosis of tularemia: a potential bioterrorism agent endemic to North America

“…Although previous studies have demonstrated the utility of PCR assays in analyzing blood samples, swabs from ulcerative lesions, and pus for the presence of FT DNA, 8,10,11,14,[17][18][19][20][21] we describe the first PCR assay designed for use in formalin fixed, routinely processed, archival tissues. Our findings indicate that PCR of processed tissues is a safe, rapid, sensitive, and specific method for the detection of FT in both human and animal tissues.…”

“…10,14,[17][18][19][20][21] Our goal was to develop a PCR assay that could be used in formalin-fixed, routinely processed tissues, which are (1) safer to handle and (2) allow diagnostic testing even after fixation and processing. We then compared the clinical and pathologic findings with the molecular data.…”

Detection ofFrancisella tularensiswithin Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

“…In that study, the extraction of the pathogen DNA used chaotropic disruption, as did a study by Fulop et al in 1996 andJunhui et al in 1996 (6, 11). The use of Whatman FTA paper for spleen and blood samples, carried out in a study by Higgins et al, is a method worth further investigation since the recovery is good and the samples can be preserved for significant periods of time (8).…”

“…A number of studies identified gene targets within F. tularensis affording suitable PCR targets for identification (3,5,11,16,18). While some studies targeted 16S rRNA or repetitive sequences, others targeted the tul4 and fopA genes to produce gel-based assays for identification of F. tularensis from blood, wound scabs, environmental specimens, and tissues of infected animals (3,7,8,10,11,17). We developed a TaqMan assay for use on fluorescence-based thermocyclers being used within the biological detection community.…”