Rapid typing of human adenoviruses by a general PCR combined with restriction endonuclease analysis
Rapid typing of human adenoviruses by a general PCR combined with restriction endonuclease analysis
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DOI：
10.1128/jcm.39.2.498-505.2001
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发表时间：
2001-02-01
影响因子：
9.4
通讯作者：
Wadell, G
中科院分区：
文献类型：
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作者：
Allard, A;Albinsson, B;Wadell, G
我们建立了一种基于PCR和限制性内切酶（RE）消化（PCR-RF：digestion）相结合的腺病毒（Ad）快速分型系统。设计了简并的共有引物，允许扩增来自所有51个人Ad原型株和总共44个不同的Ad血清型1、3、4、5、7、11、19、40和41的基因组变体的DNA。选择代表所有六个亚属和基因组变体的22个原型菌株的301-bp扩增物作为测序的靶，以寻找亚属和基因组类型变异性。根据301-bp扩增物的切割或非切割的概念，将获得的序列用于促进特异性RE的选择以用于诊断测定中的区分目的。在这些结果的基础上，构建了流程图，允许鉴定亚属B：2和D血清型，并且几乎完全区分亚属A、B：1、C、E和F血清型。用该方法确定的基因组型与传统的全长Ad DNA RE分型结果一致。其余6份样本仅在巢式PCR后呈阳性，因此，为了降低假阴性结果的风险，应通过所述巢式PCR对PCR-RE消化系统评分为阴性的样本进行评价。当需要快速鉴定腺病毒时，PCR-RE消化法和巢式PCR联合使用提供了可靠和灵敏的系统，可以容易地应用于各种临床样品。
We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RF: digestion), Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41., The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer, On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes, Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR, Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.