Test ID: LEBV    
Epstein-Barr Virus (EBV), Molecular Detection, PCR

Viral nucleic acid is extracted by the MagNA Pure automated instrument (Roche Applied Science) from clinical specimens. Primers are directed to the target gene (latent membrane protein). The LightCycler instrument amplifies and monitors by fluorescence the development of target nucleic acid sequences after the annealing step during PCR cycling. This is an automated PCR system that can rapidly detect (30-40 minutes) 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. Melting curve analysis is performed following PCR amplification. Starting at 45 degrees C, the temperature in the thermal chamber is slowly raised to 80 degrees C and the fluorescence is measured at frequent intervals. Analysis of the PCR amplification and probe melting curves is accomplished through the use of LightCycler software. (Espy MJ, Patel R, Paya C, Smith TF: Quantification of Epstein-Barr virus viral load in transplant patients by LightCycler PCR. Abstr Gen Meet Am Soc Microbiol 2001;May:20-24)

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