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首页> 外文期刊>感染症学雑誌 >Long PCR amplification of varicella-zoster virus DNA in clinical specimens from the patients with varicella and herpes zoster
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Long PCR amplification of varicella-zoster virus DNA in clinical specimens from the patients with varicella and herpes zoster

机译：水痘和带状疱疹患者临床标本中水痘-带状疱疹病毒DNA的长PCR扩增

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摘要

Long PCR amplification of the 7.7 to 33.5 Kbp regions of varicella-zoster virus (VZV) genomic DNA was performed using template DNAs extracted from clinical specimens such as vesicle fluid and crusts which had been obtained from varicella or herpes zoster patients. PCR products of 7.7-14.4 Kbp in length were efficiently amplified from all of the 14 template DNAs of crust specimens. Targets of 18.6-20.0 Kbp DNA could be also amplified from 14 crust samples except one. From all of the 7 samples derived from infected cells, the DNA targets up to 27.2 Kbp in length could be amplified. Whereas, the efficiency of amplification of 27.2 Kbp DNAs from crust samples was somewhat lower (9/14,64%) than that of DNAs from infected cells. In 83% (5/6) of target DNAs from infected cells, amplification of DNA as long as 33.5 Kbp was possible, while only in 40% (2/5) of these from crust specimens. From crust samples, the efficiency of amplification of DNA longer than 20 Kbp tended to decline. We also confirmed that long target DNA was amplifiable directly from vesicle fluid specimens as effective as from crust specimens. Restriction fragment length polymorphism (RFLP) analyses combined with R2-nested PCR of the long PCR products allowed classification of the 14 clinical specimens into 9 groups. Long PCR derived from clinical specimens was demonstrated to be applicable to RFLP analyses and sequencing without laborious test of virus isolation. Furthermore, the long PCR method described here will be useful for studies of the molecular epidemiology of VZV and for investigating variations among VZV isolates.
机译：使用从临床标本中提取的模板DNA进行水痘-带状疱疹病毒（VZV）基因组DNA的7.7至33.5 Kbp区域的长PCR扩增，这些模板DNA是从水痘或带状疱疹患者身上获得的囊泡液和结壳。从地壳样本的所有14个模板DNA中有效扩增了长度为7.7-14.4 Kbp的PCR产物。除一个外，还可以从14个地壳样品中扩增18.6-20.0 Kbp DNA的靶标。从所有受感染细胞中提取的7个样品中，可以扩增长度达27.2 Kbp的DNA靶标。而从外壳样品中扩增出27.2 Kbp DNA的效率要比从感染细胞中扩增出的DNA效率低（9 / 14,64％）。在感染细胞的目标DNA的83％（5/6）中，可以扩增长达33.5 Kbp的DNA，而在外壳样品中只有40％（2/5）。从外壳样品中，长于20 Kbp的DNA扩增效率趋于下降。我们还证实，长靶DNA可以直接从囊泡样品中扩增，就像从壳样品中扩增一样。限制性片段长度多态性（RFLP）分析与长PCR产物的R2巢式PCR相结合，可将14个临床标本分为9组。经证明，源自临床标本的长PCR可用于RFLP分析和测序，而无需费力地进行病毒分离测试。此外，此处描述的长PCR方法对于研究VZV的分子流行病学以及研究VZV分离株之间的变异将很有用。

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