Counting of viable C. Burnetii cells by quantitative reverse transcription PCR using a recombinant plasmid (PCB-dotA) as a standard

Abstract

Coxiella burnetii is an intracellular pathogenic bacterium and etiological agent of Q fever in humans. Recently, the bacterium has been set free from the strictly intracellular condition by successful cultivation in acidified citrate cysteine medium. Here, we report a bacterial cell counting method that allows rapid quantification of the absolute or relative number of live cells of C. burnetii in a high throughput manner. The method utilizes TaqMan-based quantitative polymerase chain reaction (qPCR) targeting a single dotA gene for determination of genome equivalent (GE) presented either as DNA or complementary DNA (cDNA) synthesized via reverse transcription. The assay was shown to be specific, sensitive and efficiently reproducible. The quantification was linear over a range of 30 to 3x10 8 copies. Since there is only one copy of the dotA gene per Coxiella chromosome, the calculated dotA copy numbers can be compared to the number of bacterial cells. Finally, we demonstrated the potential of the method to assess effects of antibiotic on cell viability and to determine the antibiotic-tolerant fraction within a cell population.