Test ID: LHSV    
Herpes Simplex Virus (HSV), Molecular Detection, PCR

Viral nucleic acid is extracted by the MagNA Pure automated instrument (Roche Applied Science) from genital, dermal, tissue, or cerebrospinal fluid specimens. Primers directed to the DNA polymerase of herpes simplex virus produce a 215 base pair amplicon. The LightCycler instrument (Roche Applied Science), 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 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 a 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 HSV-type 2 and sequence differences between HSV-type 2- and HSV-type 1 are detected by melting curve analysis. 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. Sequence differences between the PCR amplification and probe melting curves are accomplished through the use of LightCycler software. (Espy MJ, Uhl JR, Svien KA: Laboratory diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol 2000;38[2]:795-799)

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