Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations.
Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations.
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DOI：
10.1007/s13365-014-0249-3
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发表时间：
2014-08
影响因子：
3.2
通讯作者：
Jacobson, Steven
中科院分区：
文献类型：
--
作者：
Brunetto, Giovanna S.;Massoud, Raya;Leibovitch, Emily C.;Caruso, Breanna;Johnson, Kory;Ohayon, Joan;Fenton, Kaylan;Cortese, Irene;Jacobson, Steven
人类 T 细胞嗜淋巴细胞病毒 1 (HTLV)-1 前病毒载量 (PVL) 升高是 HTLV-1 感染者发生 HTLV-1 相关脊髓病/热带痉挛性截瘫 (HAM/TSP) 的主要危险因素，而脑脊液 (CSF) 与外周血单核细胞 (PBMC) PVL 比率较高可能可以诊断该病症。然而，HTLV-1 PVL 定量的标准方法（实时 PCR）具有多种局限性，包括细胞数量较低的区室（例如 CSF）中的测定间变异性增加。因此，在本研究中，我们评估了一种用于 HTVL-1 PVL 定量的新技术，即数字液滴 PCR (ddPCR)。在 ddPCR 中，PCR 样品被分成数千个纳升大小的液滴，在热循环仪上扩增，并查询荧光信号。由于独立事件（液滴）数量较多，泊松算法用于独立于标准曲线确定绝对拷贝数，从而实现高精度定量。该测定法具有较低的测定内变异性，可以在无症状携带者 (AC) 和 HAM/TSP 患者的 PBMC 和 CSF 区室中进行可靠的 PVL 测量。它对于 HTLV-1 相关的临床应用也很有用，例如 PVL 的纵向监测以及引物和探针靶向区域内病毒突变的识别。
An elevated human T cell lymphotropic virus 1 (HTLV)-1 proviral load (PVL) is the main risk factor for developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in HTLV-1 infected subjects, and a high cerebrospinal fluid (CSF) to peripheral blood mononuclear cell (PBMC) PVL ratio may be diagnostic of the condition. However, the standard method for quantification of HTLV-1 PVL—real-time PCR—has multiple limitations, including increased inter-assay variability in compartments with low cell numbers, such as CSF. Therefore, in this study, we evaluated a novel technique for HTVL-1 PVL quantification, digital droplet PCR (ddPCR). In ddPCR, PCR samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, and queried for fluorescent signal. Due to the high number of independent events (droplets), Poisson algorithms are used to determine absolute copy numbers independently of a standard curve, which enables highly precise quantitation. This assay has low intra-assay variability allowing for reliable PVL measurement in PBMC and CSF compartments of both asymptomatic carriers (AC) and HAM/TSP patients. It is also useful for HTLV-1-related clinical applications, such as longitudinal monitoring of PVL and identification of viral mutations within the region targeted by the primers and probe.
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DOI：
10.3390/v3091532
发表时间：
2011-09
期刊：
Viruses
影响因子：
--
作者：
Araya N;Sato T;Yagishita N;Ando H;Utsunomiya A;Jacobson S;Yamano Y
通讯作者：
Yamano Y
影响因子：
3
作者：
Gessain, A.;Mahieux, R.
通讯作者：
Mahieux, R.
影响因子：
1.5
作者：
Demontis, Maria A.;Hilburn, Silva;Taylor, Graham P.
通讯作者：
Taylor, Graham P.
影响因子：
3.7
作者：
Strain MC;Lada SM;Luong T;Rought SE;Gianella S;Terry VH;Spina CA;Woelk CH;Richman DD
通讯作者：
Richman DD
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.