Highly precise measurement of HIV DNA by droplet digital PCR.
Highly precise measurement of HIV DNA by droplet digital PCR.
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DOI：
10.1371/journal.pone.0055943
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Richman DD
中科院分区：
文献类型：
--
作者：
Strain MC;Lada SM;Luong T;Rought SE;Gianella S;Terry VH;Spina CA;Woelk CH;Richman DD
人类免疫缺陷病毒（HIV）的脱氧核糖核酸（DNA）是对接受有效联合抗逆转录病毒治疗（cART）的患者残留感染的最敏感测量方法。液滴数字PCR（ddPCR）最近已被证明可以提供高度准确的DNA拷贝数定量，但其应用于HIV DNA或其他同样罕见的靶点的定量尚未报道。本文展示并分析了ddPCR在测量从感染患者分离的细胞中的总HIV DNA（每百万个细胞的pol拷贝数）和附加型2-LTR（长末端重复）环的频率中的应用。对超过300个临床样品的分析，包括通过ddPCR和通过实时PCR（qPCR）一式三份测定的超过150个临床样品，证明了精确度的显著增加，pol拷贝数的变异系数平均降低5倍，2-LTR环的准确度提高>20倍。ddPCR检测相对于qPCR的其他优势包括不依赖外部标准品的绝对定量以及对引物和探针序列中的错配相对不敏感。这些特点使数字PCR成为一种有吸引力的替代方案，用于测量临床标本中的HIV DNA。提高这些罕见事件的测量灵敏度和精确度，应有助于测量潜伏的艾滋病毒储存库和干预措施，以消除它的特点。
Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it.
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DOI：
10.1073/pnas.0736332100
发表时间：
2003-04-15
影响因子：
11.1
作者：
Strain, MC;G端nthard, HF;Wong, JK
通讯作者：
Wong, JK
影响因子：
11.4
作者：
WUBAER, F;LANE, WS;GAYNOR, RB
通讯作者：
GAYNOR, RB
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：
9.8
作者：
Fan, H. Christina;Blumenfeld, Yair J.;El-Sayed, Yasser Y.;Chueh, Jane;Quake, Stephen R.
通讯作者：
Quake, Stephen R.
影响因子：
39.2
作者：
SAKSELA, K;STEVENS, CE;BALTIMORE, D
通讯作者：
BALTIMORE, D