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.
Fujita
Osamu
O
Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan. esperanz@nih.go.jp
Tatsumi
Masashi
M
Tanabayashi
Kiyoshi
K
Yamada
Akio
A
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