Application of long PCR method of identification of variations in nucleotide sequences among varicella-zoster virus isolates
Application of long PCR method of identification of variations in nucleotide sequences among varicella-zoster virus isolates
复制标题
DOI：
10.1128/jcm.34.12.2869-2874.1996
复制
发表时间：
1996-12
影响因子：
9.4
通讯作者：
M. Takayama;N. Takayama;N. Inoue;Y. Kameoka
中科院分区：
文献类型：
--
作者：
M. Takayama;N. Takayama;N. Inoue;Y. Kameoka
水痘-带状疱疹病毒（VZV）全病毒DNA的限制性片段长度多态性（RFLP）分析需要制备大量纯化的病毒DNA，费时费力。PCR扩增的小DNA片段的RFLP分析被开发作为一种替代方法。然而，由于VZV的变化数量很少，其使用受到限制。为了克服这些缺点，并确定在VZV的变化，我们开发了一种RFLP分析方法结合长PCR方法，最近已经开发了5和35 kb的长度之间的DNA片段的扩增。我们扩增了三个DNA区域，从6.8到11.4 kb，并证明这些区域的RFLP分析允许40 VZV分离株在日本分为17组的分类。四分之一的分离株含有C与T的核苷酸差异，这消除了76530位的StyI位点;这种改变与报告的69349位的Pst I位点多态性有关（核苷酸位置基于菌株大仲马的核苷酸位置）。在日本的VZV分离株之间的研究区域的核苷酸序列变异估计约小于0.05%，证实了先前提出的想法，即VZV是遗传稳定的，而不是高度多样化。该方法可用于VZV的分子流行病学研究。
Restriction fragment length polymorphism (RFLP) analysis of whole viral DNA of varicella-zoster virus (VZV) requires the time-consuming and laborious preparation of a large amount of purified viral DNA. RFLP analysis of small DNA fragments amplified by PCR was developed as an alternative method. However, its use has been limited because of the small number of variations in VZV. To overcome these drawbacks and to identify variations in VZV, we developed an RFLP analysis method combined with the long PCR method which has recently been developed for the amplification of DNA fragments between 5 and 35 kb in length. We amplified three DNA regions ranging from 6.8 to 11.4 kb and demonstrated that RFLP analyses of these regions allowed for the classification of 40 VZV isolates in Japan into 17 groups. One-fourth of the isolates contained a nucleotide difference of C versus T, which abolished the StyI site at position 76530; this alteration was linked to the reported PstI site polymorphism at position 69349 (nucleotide positions are based on those of strain Dumas). Nucleotide sequence variation in the examined regions among VZV isolates in Japan was estimated at roughly less than 0.05%, confirming the previously proposed idea that VZV is genetically stable and not highly diversified. Our method will be useful for studies of the molecular epidemiology of VZV.