[TaqMan fluorescent probe-based real-time PCR assay for detection of varicella-zoster virus].
BACKGROUND
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 alpha-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.


RESULTS
Described in-house qPCR method detected viral DNA in all examined specimens. Detected viral load was between 5750 copies/ml for sera samples and 27 300 000 copies/ ml for skin swabs, respectively.


CONCLUSIONS
The results of this work showed that developed real-time PCR assay based on TaqMan probe was very reliable and valuable for detection and quantification of varicella-zoster virus DNA in different clinical samples. The high level of sensitivity, specificity, accuracy, and rapidity provided by the developed method are favorable for its use for the detection of HHV-3 DNA in laboratory practice.
Katedra i Zakład Mikrobiologii Lekarskiej, Warszawski Uniwersytet Medyczny w Warszawie.
Szymon
S
Kierat
Katarzyna
K
Leś
Maciej
M
Przybylski
Tomasz
T
Dzieciatkowski
Grazyna
G
Młynarczyk
D001483:Base Sequence; D001769:Blood / Q000821:virology*; D002644:Chickenpox / Q000821:virology*; D002648:Child; D004279:DNA, Viral / Q000302:isolation & purification; D014645:Herpesvirus 3, Human / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D010614:Pharynx / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D044967:Serum / Q000821:virology; D012867:Skin / Q000821:virology*
D004279:DNA, Viral