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Unit Code 86197:
West Nile Virus (WNV) RNA Detection by Rapid PCR

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Method Description

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)

Performing Laboratory Location

Rochester

Key