Sergeev Nikolay, Rubtcova Elena, Chizikov Vladimir, Schmid D Scott, Loparev Vladimir N
Food and Drug Administration, Center for Devices and Radiological Health, Office of Science and Technology, Division of Life Sciences, Silver Spring, MD 20993, USA.
J Virol Methods. 2006 Sep;136(1-2):8-16. doi: 10.1016/j.jviromet.2006.03.021. Epub 2006 May 3.
A rapid and sensitive microarray-based method was used to distinguish the three major circulating genotypes of varicella-zoster virus (VZV). The method analyzes five variable positions located in a 447-nucleotide variable region 1 of open reading frame 22 (ORF 22r1); these single nucleotide polymorphisms (SNP) display in stably occurring patterns specific to each of the VZV genotypes established in previously published studies. Pairs of short oligonucleotide probes (oligoprobes) with sequences corresponding to all of the observed SNP were used to detect specific sequences. Fluorescently labeled ssRNA samples for hybridization with a chip were prepared by in vitro T7 polymerase driven transcription of the amplicons of ORF 22r1, followed by chemical labeling with Cy5 into RNA sample. Ratios between fluorescent hybridization signals from each pair of oligoprobes were used to assess the sequence at each SNP. We evaluated six reference VZV strains and 130 VZV clinical specimens to validate the method. The microarray method accurately identified strains isolated in the US in 2001-2002, representing all major genotypes as determined using more extensive sequence analysis, correctly assigning strains to genotypes E (81.5%), J (3%) and M (15.5%). In addition, a new M variant (M3) was identified.
一种基于微阵列的快速灵敏方法被用于区分水痘带状疱疹病毒（VZV）的三种主要循环基因型。该方法分析位于开放阅读框22（ORF 22r1）的447个核苷酸可变区1中的五个可变位置；这些单核苷酸多态性（SNP）以先前发表研究中确定的每种VZV基因型特有的稳定出现模式显示。使用与所有观察到的SNP序列相对应的短寡核苷酸探针（寡探针）对来检测特定序列。通过体外T7聚合酶驱动ORF 22r1扩增子的转录，然后用Cy5对RNA样品进行化学标记，制备用于与芯片杂交的荧光标记单链RNA样品。每对寡探针的荧光杂交信号之间的比率用于评估每个SNP处的序列。我们评估了六种参考VZV菌株和130份VZV临床标本以验证该方法。微阵列方法准确鉴定了2001 - 2002年在美国分离的菌株，这些菌株代表了使用更广泛序列分析确定的所有主要基因型，正确地将菌株归类为E基因型（81.5%）、J基因型（3%）和M基因型（15.5%）。此外，还鉴定出一种新的M变异体（M3）。
