Development of a PCR Kit for Detection of Coxiella burnetii in Ukraine

Coxiella burnetii is an obligate intracellular pathogen and the causative agent of Q fever. In Ukraine, 28 human cases of Q fever were reported between 1997 and 2006; however, there are no state-approved, standardized molecular diagnostic assays that can be used systematically to investigate C. burnetii transmission to humans and its distribution throughout Ukraine. To address this deficiency, we followed the recommendation of the World Organization for Animal Health (OIE) and developed a confirmatory PCR for C. burnetii for veterinary diagnosis in Ukraine. The PCR assay targeted the outer membrane-associated gene com1 in C. burnetii. Oligonucleotide primers were selected that amplify a 689-bp DNA fragment of the com1 gene (primers: CoxF2 = 5′-ACYGCAGGCGTGGCGATAG-3′ and CoxR4 = 5′-TGAAGGTTTTGTTGTGAGGTGGC-3′). The assay proved highly sensitive and specific to C. burnetii DNA detection (LOD = 0.37 pg/μL). Reproducibility of the test was verified by comparing the PCR results with those of a different PCR protocol and qPCR. Using the CoxF2/CoxR4 primer set and reaction conditions described here, the PCR Diagnostic Kit C. burnetii-PCR-TEST was developed and officially registered for use in Ukraine by the State Scientific Control Institute of Biotechnology and Strains (Kyiv, Ukraine) for diagnostic purposes.