Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.
Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.
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DOI：
10.1016/j.jviromet.2012.08.019
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发表时间：
2012-12
影响因子：
3.1
通讯作者：
Kuritzkes DR
中科院分区：
文献类型：
--
作者：
Henrich TJ;Gallien S;Li JZ;Pereyra F;Kuritzkes DR
微滴数字PCR（ddPCR）是一种新兴的核酸检测方法，它无需依赖标准曲线就能对靶序列进行绝对定量。我们评估了ddPCR相对于已建立的实时（RT）-PCR方法，从一组接受和未接受抗病毒治疗的患者中检测和定量总HIV - 1 DNA和2 - LTR环的能力。为了计算ddPCR对HIV - 1 DNA和2 - LTR环的动态范围，我们通过ddPCR以及RT - PCR对DNA扩增子或游离型DNA进行了系列稀释测定。我们还对3名病毒血症患者和4名接受抑制性抗逆转录病毒治疗患者的HIV - 1 DNA，以及3名低水平病毒血症患者的2 - LTR环进行了定量。通过ddPCR测定的HIV - 1或人CCR5 DNA扩增子标准品系列稀释的拷贝数与标称输入拷贝数相当。ddPCR检测HIV - 1或CCR5 DNA的灵敏度与RT - PCR相似。通过ddPCR和RT - PCR在所有3名患者的样本中都检测到了低水平的2 - LTR环。ddPCR是一种用于研究HIV - 1储存库和持续性的有前景的新技术，但对这种新技术的进一步优化将提高对极低水平病毒基因靶标的检测能力。
Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia was also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets.
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影响因子：
7.4
作者：
Hindson, Benjamin J.;Ness, Kevin D.;Masquelier, Donald A.;Belgrader, Phillip;Heredia, Nicholas J.;Makarewicz, Anthony J.;Bright, Isaac J.;Lucero, Michael Y.;Hiddessen, Amy L.;Legler, Tina C.;Kitano, Tyler K.;Hodel, Michael R.;Petersen, Jonathan F.;Wyatt, Paul W.;Steenblock, Erin R.;Shah, Pallavi H.;Bousse, Luc J.;Troup, Camille B.;Mellen, Jeffrey C.;Wittmann, Dean K.;Erndt, Nicholas G.;Cauley, Thomas H.;Koehler, Ryan T.;So, Austin P.;Dube, Simant;Rose, Klint A.;Montesclaros, Luz;Wang, Shenglong;Stumbo, David P.;Hodges, Shawn P.;Romine, Steven;Milanovich, Fred P.;White, Helen E.;Regan, John F.;Karlin-Neumann, George A.;Hindson, Christopher M.;Saxonov, Serge;Colston, Bill W.
通讯作者：
Colston, Bill W.
影响因子：
14.8
作者：
Malnati, Mauro S.;Scarlatti, Gabriella;Lusso, Paolo
通讯作者：
Lusso, Paolo
影响因子：
5.4
作者：
Butler, SL;Johnson, EP;Bushman, FD
通讯作者：
Bushman, FD
影响因子：
3.8
作者：
Morlese, J;Teo, IA;Shaunak, S
通讯作者：
Shaunak, S
影响因子：
82.9
作者：
Siliciano, JD;Kajdas, J;Siliciano, RF
通讯作者：
Siliciano, RF