Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR.
Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR.
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DOI：
10.1038/s41598-017-01221-5
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发表时间：
2017-04-26
影响因子：
4.6
通讯作者：
Garson JA
中科院分区：
文献类型：
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作者：
Busby E;Whale AS;Ferns RB;Grant PR;Morley G;Campbell J;Foy CA;Nastouli E;Huggett JF;Garson JA
潜伏感染细胞的持续存在阻碍了艾滋病毒的治愈，而这些细胞构成了病毒库。实时定量聚合酶链式反应用于通过测量HIV DNA来量化这个储存库，需要外部校准；校准剂的一个常见选择是8E5细胞系，它被认为是稳定的，每个细胞含有一个HIV前病毒。相比之下，数字聚合酶链式反应不需要外部校准，有可能提供“绝对”定量。我们比较了定量聚合酶链式反应和直接聚合酶链式反应在18例患者样本中检测HIV DNA的效果。有18例用qPCR检测到HIV DNA，15例用dPCR检测到HIV DNA，这是由于dPCR分析的样本量较小所致。两种技术之间有很好的数量相关性(R2、 = 、0.86)，但平均dPCR值仅为qPCR值的60%。令人惊讶的是，调查显示，这种差异是由于8E5细胞校准中艾滋病毒DNA的丢失造成的。另外两个来源的8E5提取物每个细胞的HIV DNA拷贝数也明显少于一个，8E5细胞在培养过程中HIV逐渐丢失。因此，我们建议8E5提取液中HIV的拷贝数在用作校准物之前通过dPCR来确定。
Establishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides ‘absolute’ quantification. We compared the performance of qPCR and dPCR in quantifying HIV DNA in 18 patient samples. HIV DNA was detected in 18 by qPCR and in 15 by dPCR, the difference being due to the smaller sample volume analysed by dPCR. There was good quantitative correlation (R2 = 0.86) between the techniques but on average dPCR values were only 60% of qPCR values. Surprisingly, investigation revealed that this discrepancy was due to loss of HIV DNA from the 8E5 cell calibrant. 8E5 extracts from two other sources were also shown to have significantly less than one HIV DNA copy per cell and progressive loss of HIV from 8E5 cells during culture was demonstrated. We therefore suggest that the copy number of HIV in 8E5 extracts be established by dPCR prior to use as calibrator.