Method Description

Nucleic acid is extracted from the pathogens in blood using the automated MagNA Pure LC system. The extract is then transferred to a 96-well Lightcycler 480 dish for amplification. The LightCycler 480 is an automated instrument that amplifies and monitors the development of target nucleic acid (amplicon) after each cycle of polymerase chain reaction (PCR). The DNA target for PCR assay is groEL, the open reading frame gene segment of the heat-shock protein operon (groEL), which is present at a frequency of 1 copy per organism in pathogenic species of Anaplasma and Ehrlichia. A specific base pair DNA target sequence is amplified by PCR. The detection of amplicon is based on fluorescence resonance energy transfer, which utilizes a hybridization probe with a donor fluorophore, fluorescein, at the 3' end and a second hybridization probe with an acceptor fluorophore, LC-Red 640, at the 5' end. When the target amplicon is present, the LC-Red 640 emits a measurable and quantifiable light signal at a specific wavelength. Presence of the specific organism nucleic acid may be confirmed by performing a melting curve analysis of the amplicon. Using features of the melting curve analysis, the assay primers and specific hybridization probes are able to detect and differentiate among Anaplasma phagocytophilum, Ehrlichiosis chaffeensis, Ehrlichia muris eauclairensis, and Ehrlichia ewingii/canis. Due to close proximity of the melting curves of Ehrlichia ewingii and Ehrlichia canis, this assay cannot distinguish between these 2 organisms.(Unpublished Mayo method)