Test ID: VDER    
Herpes Simplex Virus (HSV) and Varicella-Zoster Virus (VZV), Molecular Detection, PCR, Dermal

Herpes Simplex Virus (HSV):

Viral nucleic acid is extracted from genital, dermal, and ocular specimens. Primers directed to the DNA polymerase gene of HSV produce 215-bp and 239-bp amplicons, respectively. 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 (30-40 minutes) 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 2nd 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 were 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 54 degrees C, the temperature in the thermal chamber is slowly raised to 95 degrees C, and the fluorescence is measured at frequent intervals. Sequence differences between the PCR product and hybridization probes result in shifts in the melting temperatures. (66.7 degrees C fro HSV type 1 and 74.7 degrees C for HSV type 2) that are detected. Analysis of the PCR amplification and probe melting curves is accomplished through the use of LightCycler software. (Espy MJ, Uhl JR, Svien KA, et al. Laboratory diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol 2000;38:795-799)

Varicalla-Zoster Virus (VZV):

Viral nucleic acid is extracted from dermal specimens. Primers directed to gene 28 of VZV produce a 287 bp 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 (30-40 minutes) 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 a light of a different wavelength that can be measured with a signal that is proportional to the amount of specific PCR product. Analysis of the PCR amplification is accomplished through the use of LightCycler software. (Espy MJ, Uhl JR, Svien KA, et al: Laboratory diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR. J Clin Microbiol 2000;38:795-799)

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