Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay
Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay
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DOI：
10.1016/j.virol.2019.08.021
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发表时间：
2019-11-01
期刊：
影响因子：
3.7
通讯作者：
Scott, R. S.
中科院分区：
文献类型：
--
作者：
Myers, J. E.;Guidry, J. T.;Scott, R. S.
筛查人乳头瘤病毒 (HPV) 是否整合到宿主细胞染色体中通常需要大量时间和试剂。我们开发了一种基于核酸外切酶 V (ExoV) 和定量聚合酶链式反应 (qPCR) 的快速、灵敏的检测方法，以确定细胞系和组织中的 HPV 基因组配置。我们使用来自已知携带整合型或附加型 HPV16 的细胞系的基因组 DNA 建立了该检测方法。将 DNA 与线性 DNA 特异性的 ExoV 一起孵育，并通过 qPCR 测量抗消化的 DNA 部分。相对于未消化的 DNA 计算对 ExoV 消化具有抗性的 DNA 百分比，以测定附加型或整合型 HPV16。 ExoV 检测准确，能够区分细胞系和组织中的附加型 HPV16 和整合型 HPV16。 ExoV 检测的未来应用可能包括筛查 HPV 相关癌症进展中的 HPV 基因组配置。
Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.