Test ID: LCWNV    
West Nile Virus, Molecular Detection, PCR, Spinal Fluid

This LightCycler PCR assay has been optimized to detect common conserved sequences in the nonstructural protein of West Nile virus (WNV). Viral nucleic acid is extracted by the MagNA Pure automated instrument (Roche Applied Science) from cerebrospinal, or plasma. Primers directed to the nonstructural protein amplify 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, pp3-30)

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