Creating standards for absolute quantification of Coxiella burnetii in real-time PCR--a comparative study based on transmission electron microscopy.

Quantitative standards are a prerequisite for quality control and quantification of pathogens. In this study the creation of quantitative standards for use in qPCR is described using the pathogen Coxiella burnetii. Quantification of Coxiella burnetii particles by transmission electron microscopy (TEM) was used as primary standard and compared with data obtained by light microscopy as well as genome equivalents (GE) and plasmid units (recombinant plasmid). Based on pathogen quantification using TEM and light microscopy, pathogen detection limits of 6 and 2 C. burnetii particles could be determined per com1 qPCR reaction, respectively. In comparison, the detection limits were 17 and 13 pathogen units using GE and plasmid units, respectively. The standard generated by TEM can be used as gold standard for universal application due to high accuracy, quantitative control of the producing process and supplying intact pathogen particles.