Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis

Abstract

This report describes the development of in-house real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed as biological threats by the Centers for Disease Control and Prevention.

Assessment of Diagnostic Efficiency of PCR Kits «Gen Francisella tularensis - REF» and «Gen Francisella tularensis - RGF» when Analyzing Biological Samples from Animals with Experimental Tularemia

“…через пять дней заболевания ав-торы детектировали туляремийный микроб дополни-тельно в пробах фекалий и мочи, а его количество в пробах суспензий селезенки и почек увеличилось до 1·10 7 -1·10 9 м.к./г. для выявления днк F. tularensis была использована количественная пцр с праймера-ми на основе гена, кодирующего белок 23 кда, пред-ложенная T.Skottman et al [11].…”

Recent Advancement in the Development of Vaccines Against Y. pestis - A Potential Agent of Bioterrorism

“…Real-time PCR based methods are more specific, and require less time and labor than conventional PCR assays. Real-time PCR methods include SYBR Green (Saikaly et al, 2007), molecular beacons (Varma-Basil et al, 2004), TaqMan probes (Loiez et al, 2003;Chase et al, 2005) and minor groove binding (MGB) probes (Skottman et al, 2007), and target specific sequences on the chromosome and (or) plasmids. However, PCR based diagnosis is expensive compared to immunoassays, which may be useful for mass screening during epidemics.…”

Tularemia: current diagnosis and treatment options

“…This multiplex technology offers the advantage for testing an unknown clinical or environmental sample that may indicate the use of a biologic weapon. Recently published reports have demonstrated the feasibility of this technology [51,52], with further modifications leading to multiplex PCR-coupled liquid-bead array, with a process that is completely automated, utilized primarily for environmental samples [53]. Further testing of clinical samples is still needed.…”