Rapid and sensitive PCR-based detection and differentiation of aetiologic agents of human granulocytotropic and monocytotropic ehrlichiosis

Abstract

The potential of fatal outcome for patients afflicted with human ehrlichioses (HME and HGE) necessitates fast and accurate detection of the aetiologic agents and timely antibiotic treatment. A polymerase chain reaction (PCR)-based protocol is described that can detect as little as 10 copies of ehrlichial 16S rDNA and as few as 0.3 HGE-infected neutrophils. The method employs DNAzol for rapid DNA extraction from unfractionated whole blood in less than 1 h. For DNA amplification, highly specific oligonucleotide primers are designed that efficiently detect and distinguish between Ehrlichia chaffeensis and the HGE agent. These primers do not prime DNA extracted from closely related ehrlichial and rickettsial species. Although total DNA extracted from human blood contains nucleic acids that can be non-specifically amplified at moderate to high MgCI2 concentrations, such non-specific priming of non-ehrlichial DNA can be completely eliminated by lowering the MgCI2 concentration to 1 mM. Thus, this PCR-based procedure can detect and differentiate HGE and HME with speed, simplicity, specificity and sensitivity.