Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains.The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimumA group of diverse pathogens has the potential to cause high morbidity and mortality in humans -especially if carried by aerosols- even though they do not pose a major threat to public health under normal circumstances. The most menacing bacterial pathogens of this group are Bacillus anthracis, Francisella tularensis and Yersinia pestis, and these organisms are listed as category A biothreat agents (classification of the CDC, USA, http://www.bt.cdc.gov/agent/agentlist-category.asp webcite) because of the potential danger of their deliberate release. Exposure to aerosolized B. anthracis spores and F. tularensis can lead to inhalational anthrax and tularemia. Y. pestis may cause pneumonic plague, which, unlike the other two diseases, may also spread from person to person.To reduce the public health impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools for their detection are needed. Timely recognition of disease agents will enable appropriate treatment of exposed individuals which will be cr