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Unit Code 84319:
Ehrlichia/Anaplasma DNA Detection by Rapid PCR, Blood

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Method Description

Nucleic acid is extracted from the pathogens in blood using the

automated MagNA Pure LC system. The extract is then

transferred to individual self-contained capillary cuvettes

for amplification. The LightCycler is an automated instrument

that amplifies and monitors the development of target nucleic

acid (amplicon) after each cycle of PCR. The DNA target for

PCR assay is groEL, the open reading frame gene segment

of the heat-shock protein operon (groESL), 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 (FRET),

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 printers and specific hybridization probes are able to

detect and differentiate among Anaplasma phagocytophilum

(Tm=66.5 degrees C), Ehrlichiosis chaffeensis (Tm=59.2

degrees C), and Ehrlichia ewingii/Ehrlichia canis (Tm=50.2/

49.5 degrees C). Due to close proximity of the melting curves

of Ehrlichia ewingii and Ehrlichia canis, this assay cannot

distinguish between these 2 organisms. (Cockerill FR, Uhl FR:

Applications and challenges of real-time PCR for the clinical

microbiology laboratory. In Rapid Cycle Real-Time PCR. Edited

by U Reischl, C Wittwer, F Cockerill. Springer, NY, 2002)

Performing Laboratory Location

Rochester

Key