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This LightCycler PCR assay has been optimized to detect common
conserved sequences in the nonstructural protein of WNV.
Viral nucleic acid is extracted by the MagNA Pure automated
instrument (Roche Applied Science) from CSF, plasma, or serum.
Primers directed to the nonstructural protein amplifies a specific
sequence of the virus. For the test, WNV genomic RNA is transcribed
to cDNA. The LightCycler instrument amplifies and monitors the
development of target nucleic acid sequences after the annealing
step during PCR cycling by fluorescence assay. This automated
PCR system utilizes stringent air-controlled temperature cycling
and capillary cuvettes to rapidly detect (30-40 minutes) amplicon
development. The detection of amplified products is based on the
fluorescence resonance energy transfer (FRET) principle. For
FRET product detection, a hybridization probe with a donor fluorophore,
fluorescein, on the 3'-end is excited by an external light source and
emits light that is absorbed by a second hybridization probe with an
acceptor fluorophore, LC-Red 640, at the 5'-end. The acceptor
fluorophore then emits a light of a different wavelength that can be
measured with a signal that is proportional to the amount of specific
PCR product. Analysis of the PCR amplification and probe melting
curves are accomplished through the use of LightCycler software.
(Cockerill FR III, Uhl JR: Applications and challenges of real-time
PCR for the clinical microbiology laboratory. In Rapid Cycle Real-
Time PCR Methods and Applications. Edited by U Reischel, C
Wittwer, F Cockerill. Berlin, Germany, Springer-Verlag; 2002, pp 3-30)