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.
Higgins
J A
JA
Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705, USA.
Hubalek
Z
Z
Halouzka
J
J
Elkins
K L
KL
Sjostedt
A
A
Shipley
M
M
Ibrahim
M S
MS
D000818:Animals; D001096:Arachnid Vectors / Q000382:microbiology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005260:Female; D005604:Francisella tularensis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007124:Immunoenzyme Techniques / Q000379:methods; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction / Q000379:methods; D013987:Ticks / Q000382:microbiology*; D014406:Tularemia / Q000175:diagnosis* / Q000382:microbiology
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial