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Unit Code 800170:
JC Virus Detection by PCR, Spinal Fluid

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Method Description

Viral nucleic acid is extracted from the specimen using the MagNA

Pure automated instrument (Roche Applied Science) from clinical

specimens. Primers are directed to the VP2 gene, which is a

conserved sequence specific for JCV. The LightCycler instrument

(Roche Applied Science) amplifies and monitors the development

of target nucleic acid sequences after the annealing step during

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. (Whiley D, Mackay IM, Sloots TP: Detection

and differentiation of human polyomaviruses JC and BK by LightCycler

PCR. J Clin Microbiol 2001;39:4357-4361)

Performing Laboratory Location

Jacksonville

Key