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首页> 外文期刊>Journal of Clinical Microbiology >Rapid Typing of Human Adenoviruses by a General PCR Combined with Restriction Endonuclease Analysis
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Rapid Typing of Human Adenoviruses by a General PCR Combined with Restriction Endonuclease Analysis

机译：通用PCR结合限制性内切核酸酶分析技术快速分型人类腺病毒

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We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE 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.
机译：我们已经开发了一种基于PCR和限制性内切核酸酶（RE）消化（PCR-RE消化）结合的腺病毒（Ads）快速分型系统。设计了退化的共有引物，可从所有51种人类Ad原型菌株和总共44种不同的Ad血清型1、3、4、5、7、11、19、40和41的基因组变异体中扩增DNA。301 bp的扩增子代表全部六个亚属的22种原型菌株中，选择基因组变异体作为测序的目标，以寻找亚属和基因组类型的变异性。通过遵循301 bp扩增子的切割或不切割的概念，将获得的序列用于在诊断分析中用于区分目的的特定RE的选择。基于这些结果，构建了流程图，可以识别B：2和D亚型血清型，并几乎完全区分A，B：1，C，E和F亚型血清型。将PCR-RE消化系统应用于临床样品后，可以输入40个Ad阳性的临床样品中的34个。通过这种方法确定的基因组类型与通过全长RE DNA的传统RE分型获得的基因组类型相同。其余六个样品仅在巢式PCR之后才呈阳性。因此，为减少假阴性结果的风险，应通过上述嵌套式PCR对PCR-RE消化系统评分为阴性的样品进行评估。 PCR-RE消化方法与嵌套式PCR结合使用，可提供一种可靠且灵敏的系统，当需要快速鉴定腺病毒时，可轻松应用于各种临床样品。

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