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 (GEs) 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 x 10(4) colony forming units per 0.5 g of soil (similar to 3 GEs 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.

Automated summary

This paper presents a specific real-time polymerase chain reaction (PCR) assay for the detection of Coxiella burnetii, which is highly infectious and considered a biological threat. The assay is based on amplification of a specific gene fragment and has high sensitivity, reproducibility, and specificity. It can differentiate strains based on a point mutation and has been successfully applied to environmental soil samples.