Department of Obstetrics & Gynecology, University of Washington, Seattle, WA 98109, USA.
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
STAR Protoc. 2021 Oct 11;2(4):100885. doi: 10.1016/j.xpro.2021.100885. eCollection 2021 Dec 17.
Most latent human immunodeficiency virus (HIV) proviruses are defective and cannot produce infectious virions. Thus, the number of HIV proviruses with intact genomes is a relevant clinical parameter to assess therapies for HIV cure. We describe high-molecular-weight DNA isolation, followed by restriction enzyme fragmentation that limits cutting within the HIV genome. Multiplexed droplet digital PCR quantifies five targets spanning the HIV genome to estimate potentially intact proviral copies. A reference assay counts the number of T lymphocytes and assesses the level of DNA shearing. For complete details on the use and execution of this protocol, please refer to Levy et al. (2021).
大多数潜伏的人类免疫缺陷病毒 (HIV) 前病毒是有缺陷的，不能产生感染性病毒粒子。因此，具有完整基因组的 HIV 前病毒数量是评估 HIV 治愈疗法的一个相关临床参数。我们描述了高分子量 DNA 的分离，然后是限制酶片段化，限制了 HIV 基因组内的切割。多重液滴数字 PCR 定量五个跨越 HIV 基因组的靶标，以估计潜在完整的前病毒拷贝数。参考测定法计算 T 淋巴细胞的数量，并评估 DNA 断裂程度。有关该方案使用和执行的完整详细信息，请参阅 Levy 等人（2021 年）。
