Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens

Abstract

Tularemia is the zoonotic disease caused by the gram-negative coccobacillus Francisella tularensis. Its wide distribution in the environment poses a challenge for understanding the transmission, ecology, and epidemiology of the disease. F. tularensis is also considered a potential biological weapon due to its extreme infectivity. We have developed a multitarget real-time TaqMan PCR assay capable of rapidly and accurately detecting F. tularensis in complex specimens. Targeted regions included the ISFtu2 element and the 23kDa, fopA, and tul4 genes. Analysis of the four TaqMan assays demonstrated that three (ISFtu2, 23kDa, and tul4) performed within our established criterion of a detection limit of one organism. The combined use of the three assays was highly specific, displaying no cross-reactivity with the non-Francisella bacteria tested and capable of differentially diagnosing both F. tularensis and Francisella philomiragia. When the multitarget TaqMan assay (ISFtu2, 23kDa, and tul4) was compared to culturing, using environmentally contaminated specimens, the TaqMan PCR assay was significantly more sensitive than culturing (P </= 0.05). The sensitive and specific nature of this rapid multitarget TaqMan assay provides a valuable new tool that with future evaluations can be used for analyzing clinical specimens, field samples during bioterrorism threat assessment, and samples from outbreaks and for improving our understanding of the ecology and environmental prevalence of F. tularensis.

FIG. 1.

Average C_t values for the ISFtu2 (A), 23kDa (B), tul4 (C), and fopA (D) assays. C_t values for the 18 F. tularensis subsp. tularensis strains (F.t.t.), 18 F. tularensis subsp. holarctica strains (F.t.h.), 4 F. tularensis subsp. novicida strains (F.t.n.), and 15 F. philomiragia stains (F.p.) and their respective standard deviations are shown. *, for F. philomiragia, the 23kDa C_t value represents the average for four isolates in which 23kDa was detected.

FIG. 2.

Cell (top) and DNA (bottom) standard curves for F. tularensis subsp. tularensis (strain SchuS4). The relationship between 10-fold serial dilutions of CFU versus C_t for the ISFtu2 (A), 23kDa (B), and tul4 (C) TaqMan assays is shown in the top panels; the calculated square regression coefficients were 0.98, 0.9971, and 0.9967, respectively. The relationship between 10-fold serial dilutions of GEs versus C_t for the ISFtu2 (D), 23kDa (E), and tul4 (F) TaqMan assays is shown in the bottom panels; the calculated square regression coefficients were 0.9853, 0.9997, and 0.9632, respectively. The log-linear regression and standard deviations are indicated.