Method Description

Viral nucleic acid is extracted by the MagNA Pure automated instrument (Roche Applied Science) from clinical specimens. Primers directed to target DNA (ss DNA binding proteins: gene 29) produce a 202-base pair amplicon. 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 though stringent air-controlled temperature cycling in capillary cuvettes. The detection of amplified products is based on the fluorescence resonance energy transfer 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° C, the temperature in the thermal chamber is slowly raised to 80° 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.(Dhiman N, Wright PA, Espy MJ, Schneider SK, Smith TF, Pritt BS. Concurrent detection of herpes simplex and varicella-zoster viruses by polymerase chain reaction from the same anatomic location. Diagn Microbiol Infect Dis. 2011;70(4):538-540. doi:10.1016/j.diagmicrobio.2011.03.014; Espy MJ, Teo R, Ross TK, Scien KA, Wold AD, Smith TF. Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol. 2000;38[9]:3187-3189)