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一种高度多重化的液滴数字 PCR 检测方法,用于测量完整的 HIV-1 前病毒库。

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

机构信息

Department of Obstetrics & Gynecology, University of Washington, Seattle, WA, USA.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

出版信息

Cell Rep Med. 2021 Apr 12;2(4):100243. doi: 10.1016/j.xcrm.2021.100243. eCollection 2021 Apr 20.

Abstract

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4 T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

摘要

量化具有复制能力的 HIV 储存库对于评估治愈策略至关重要。病毒生长测定(VOA)会低估储存库,因为它们不能诱导所有具有复制能力的前病毒。单区或双区 HIV DNA 测定会高估它,因为它们不能排除许多有缺陷的前病毒。我们设计了两种三联体液滴数字 PCR 测定法,每个测定法都有 2 个独特的靶标和 1 个共同的靶标,并将结果归一化为基于 PCR 的 T 细胞计数。这两种 HIV 测定法均具有特异性、敏感性和可重复性。它们共同估计包含所有五个引物-探针区域的前病毒数量。我们的 5 个靶标结果平均比配对定量 VOA 高 12.1 倍(Spearman ρ=0.48),但与之前的 DNA 测定法相比,估计的储存库要小得多。在接受抗逆转录病毒治疗的患者中,完整前病毒的血液 CD4 T 细胞衰减速度比缺陷前病毒快,黏膜和循环 T 细胞中的完整前病毒频率相似。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c0e3/8080125/eac8d074b5ff/fx1.jpg

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