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Unit Code 800168:
Varicella-Zoster Virus (VZV) by Rapid PCR

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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 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 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 (FRET)

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, Teo R, Ross TK,

et al:  Diagnosis of varicella-zoster virus infections in the clinical

laboratory by LightCycler PCR. J Clin Microbiol 2000;38(9):3187-3189)

Performing Laboratory Location

Jacksonville

Key