用核酸外切酶V-qPCR分析人乳头瘤病毒16在细胞系和组织中的基因组状态

Current Protocols in Microbiology Pub Date : 2020-10-16 DOI:10.1002/cpmc.119

Julia E. Myers, Katarzyna Zwolinska, Martin J. Sapp, Rona S. Scott

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引用次数: 8

摘要

人类乳头瘤病毒(HPV)基因组整合到宿主细胞染色体中，已经在大多数HPV阳性的宫颈癌和一部分口腔HPV相关癌症中观察到。HPV整合也发生在长期细胞培养中。HPV整合筛查可能是劳动密集型的，并且产生难以解释的结果。在这里，我们描述了一种基于外切酶V (ExoV/RecBCD)和定量聚合酶链反应(qPCR)的检测方法，以确定来自细胞系和组织的样本是否含有外生或整合的HPV。该方法可用于筛选病毒生命周期中具有外泌体/线性基因组结构的其他小DNA病毒，并具有在临床环境中用于确定与疾病相关的病毒基因组构象的潜力。©2020 Wiley期刊有限公司基本方案:细胞中HPV16基因组结构的外切酶V基因组DNA酶切和qPCR检测支持方案:组织中HPV16基因组结构的外切酶V分析替代方案:确定HPV整合类型或HPV片段的完整性

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An Exonuclease V–qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues

Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC.

Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells

Support Protocol: Exonuclease V analysis of HPV16 genome configuration in tissues

Alternate Protocol: Determining HPV integration type or integrity of HPV episome

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来源期刊

Current Protocols in Microbiology Immunology and Microbiology-Parasitology

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期刊介绍： Current Protocols in Microbiology provides detailed, step-by-step instructions for analyzing bacteria, animal and plant viruses, fungi, protozoans and other microbes. It offers updated coverage of emerging technologies and concepts, such as biofilms, quorum sensing and quantitative PCR, as well as proteomic and genomic methods. It is the first comprehensive source of high-quality microbiology protocols that reflects and incorporates the new mandates and capabilities of this robust and rapidly evolving discipline.