Development of a Specific Real-Time PCR Assay for Simultaneous Detection and Differentiation of Coxiella burnetii Strains from Environmental Soil Samples.

Coxiella burnetii, the causative agent of Q fever, is a small, coccoid, Gram-negative strict intracellular pathogen. One of the most common ways of acquiring Q fever is through inhalation of aerosols containing the bacteria. Because C. burnetii is highly infectious, spreads easily through the air, and is very resistant to environmental conditions, it is considered a biological threat. This paper presents the development and validation of a specific real-time polymerase chain reaction (real-time PCR or qPCR) assay for the detection of C. burnetii, based on the amplification of a fragment of the isocitrate dehydrogenase (icd) encoding gene. This real-time PCR is highly specific, reproducible and sensitive, allowing the detection of as few as 5 genome equivalents (GE) of C. burnetii per reaction. The method enables a rapid preliminary differentiation among strains, based on a point mutation at nucleotide 745 of the icd gene. The assay was successfully evaluated in environmental soil samples; a limit of detection of 3×104 colony forming units (CFU) per 0.5 g of soil (approx. 3 GE per reaction) was achieved. The newly developed real-time PCR offers a valuable tool for differential detection of C. burnetii strains in environmental soil samples.