Kierat Szymon, Leś Katarzyna, Przybylski Maciej, Dzieciatkowski Tomasz, Młynarczyk Grazyna
Katedra i Zakład Mikrobiologii Lekarskiej, Warszawski Uniwersytet Medyczny w Warszawie.
Med Dosw Mikrobiol. 2012;64(2):139-49.
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.
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.
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.
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.
水痘带状疱疹病毒（HHV - 3）通过呼吸道传播，扩散至淋巴结，然后经淋巴回流至皮肤，引发水痘皮疹。与其他α疱疹病毒一样，HHV - 3感染背根神经节的神经元，并在其中导致终身潜伏。病毒再激活会引发带状疱疹（缠腰龙）。在严重的HHV - 3感染期间，分子方法，如实时光定量PCR（qPCR）检测，被认为是检测临床标本中病毒DNA的首选方法。本研究的目的是开发一种qPCR检测方法，使用针对HHV - 3 DNA ORF62基因的特异性引物和荧光TaqMan探针，用于检测和定量不同临床样本中的水痘带状疱疹病毒DNA。
使用浓度范围在100至3125000拷贝/毫升之间的病毒DNA系列稀释液测试该检测方法的分析灵敏度。使用概率分析计算检测限（LOD），确定为750拷贝/毫升。在进一步的研究中，使用LightCycler 2.0系统对从一小群5名有水痘症状的儿童采集的18份临床样本（血清、全血、皮肤拭子）进行水痘带状疱疹病毒DNA检测。
所描述的内部qPCR方法在所有检测标本中均检测到病毒DNA。检测到的病毒载量血清样本分别为5750拷贝/毫升，皮肤拭子样本为27300000拷贝/毫升。
这项工作的结果表明，基于TaqMan探针开发的实时PCR检测方法对于检测和定量不同临床样本中的水痘带状疱疹病毒DNA非常可靠且有价值。所开发方法提供的高灵敏度、特异性、准确性和快速性有利于其在实验室实践中用于检测HHV - 3 DNA。
