Method Description

Viral nucleic acid is extracted from the specimen using the MagNA Pure automated instrument (Roche Applied Science). Primers are directed to the large T antigen gene, which is a conserved sequence specific for JC virus (JCV). This assay detects only JCV; it does not detect BK virus or Simian virus 40 (SV40) (other polyomaviruses). The LightCycler instrument (Roche Applied Science) amplifies and monitors the development of target nucleic acid sequences after the annealing step during polymerase chain reaction (PCR) cycling. This automated PCR system can rapidly detect amplicon development through stringent air-controlled temperature cycling in capillary cuvettes. 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a different wavelength that can be measured with a signal that is proportional to the amount of specific PCR product.(Unpublished Mayo method)