Development of a real-time PCR assay for detection and quantification of Francisella tularensis.
The facultative intracellular bacterium, Francisella tularensis, is an etiological agent of tularemia and is also considered to be a potential biological threat agent due to its extreme infectivity. We established a real-time PCR assay using the LightCycler (LC) system to detect a Francisella-specific sequence of the outer membrane protein (fopA) gene. Twenty-five F. tularensis strains including 16 Japanese isolates were subjected to this LC-PCR assay, and were tested positive, whereas Francisella philomiragia and other bacteria species did not show any specific fluorescent signal. A linear response was observed using F. tularensis genomic DNAs of between 20 fg and 2 ng, corresponding to 1.2 to 1.2 x 10(5) bacteria. The newly established real-time PCR allows the detection of the F. tularensis genome specifically, sensitively, and rapidly. This assay may contribute to the standardization of the laboratory diagnosis of tularemia.
Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan. esperanz@nih.go.jp
Osamu
O
Fujita
Masashi
M
Tatsumi
Kiyoshi
K
Tanabayashi
Akio
A
Yamada
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D005604:Francisella tularensis / Q000302:isolation & purification*; D006801:Humans; D007564:Japan; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014406:Tularemia / Q000175:diagnosis* / Q000382:microbiology
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial