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Medycyna Doświadczalna i Mikrobiologia 2012, 64(2): 139-149

Opracowanie metody PCR w czasie rzeczywistym do wykrywania wirusa ospy wietrznej i półpaśca z wykorzystaniem fluorescencyjnej sondy typu TaqMan
[TaqMan fluorescent probe-based real-time PCR assay for detection of varicella-zoster virus]

Szymon Kierat, Katarzyna Leś, Maciej Przybylski, Tomasz Dzieciątkowski, Grażyna Młynarczyk

Streszczenie

Celem pracy było opracowanie, optymalizacja oraz zbadanie użyteczności laboratoryjnej metody real-time PCR, z wykorzystaniem sondy hydrolizu­jącej typu TaqMan, do badania zakażeń wirusem ospy wietrznej i półpaśca (VZV, HHV-3).

Abstract

Introduction. Varicella-zoster virus (HHV-3) is spread by the respiratory route and dis­seminates to lymph nodes and then via lymph back to the skin, resulting with the rash of chickenpox. Like other α-herpesviruses, HHV-3 infects the neurons of the dorsal root gan­glia, where it causes lifelong latency. Virus reactivation causes episode of herpes zoster (shingles). During severe HHV-3 infections molecular methods, such as real-time PCR (qPCR) assay, are recognized as a method-of-choice for detecting viral DNA in clinical specimens. The aim of this study was to develop the qPCR assay for detection and quantification of varicella-zoster virus DNA in different clinical samples, using specific primers targeting a HHV-3 DNA ORF62 gene and a fluorescent TaqMan probe. Methods. The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 100 and 3 125 000 copies/ml. Limit of detection (LOD) was calculated using probit analysis, and was determined as 750 copies/ml. In further studies 18 clinical samples (sera, whole blood, skin swabs), taken from a small group of 5 children with symptoms of chickenpox were tested for the presence of varicella-zoster virus DNA, using LightCycler 2.0 system.

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