Method Description

Viral nucleic acid is extracted by the MagNA Pure automated instrument (Roche Applied Science) from clinical specimens. Primers directed to the immediate early gene of human herpesvirus-6 (HHV-6) produce a 195-base pairs amplicon. 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 (1 hour) detect amplicon development through stringent air-controlled temperature cycling and 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 light of a different wavelength that can be measured with a signal that is proportional to the amount of specific PCR product. LightCycler hybridization probes are designed for exact homology to HHV-6.(Cockerill FR III, Uhl JR: Applications and challenges of real-time PCR for the clinical microbiology laboratory. In: Reischel U, Wittwer C, Cockerill F. Rapid Cycle Real-Time PCR - Methods and Applications. Springer-Verlag;2002: 3-30)