Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA.
Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA.
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DOI：
10.1371/journal.pone.0085999
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Vandekerckhove L
中科院分区：
文献类型：
--
作者：
Kiselinova M;Pasternak AO;De Spiegelaere W;Vogelaers D;Berkhout B;Vandekerckhove L
细胞相关（CA）的HIV - 1 RNA被认为是评估HIV感染患者病毒储存库动态和抗逆转录病毒治疗（ART）反应的一个潜在标志物。近期研究采用灵敏的半巢式实时定量（q）PCR来定量CA HIV - 1 RNA。数字PCR最近被描述为一种基于PCR的替代技术，与qPCR相比具有更高的准确性用于绝对定量。在此，对液滴数字PCR（ddPCR）和半巢式qPCR在定量未剪接（us）和多重剪接（ms）的CA HIV - 1 RNA方面进行了比较。用这两种方法对合成RNA标准品以及接受和未接受抗逆转录病毒治疗的感染患者的CA HIV - 1 RNA（N = 34）进行定量。对于连续稀释的合成标准品（usRNA：R² = 0.97，msRNA：R² = 0.92）以及患者来源的样本（usRNA：R² = 0.51，msRNA：R² = 0.87），两种方法之间均观察到相关性。半巢式qPCR在合成标准品定量方面比ddPCR表现出更好的定量线性、准确性和灵敏度，尤其是在较低定量范围。两种方法在接受和未接受抗逆转录病毒治疗的患者样本中对usRNA的检测率相同且都较高（91%），而ddPCR在更大比例的接受抗逆转录病毒治疗患者的样本中检测到msRNA（p = 0.13）。我们观察到在患者样本中usRNA定量方面两种方法之间平均一致性较好，尽管标准差较大（偏差 = 0.05±0.75 log₁₀）。然而，对于msRNA观察到偏差为0.94±0.36 log₁₀。无模板对照在半巢式qPCR中始终为阴性，但在一些孔中ddPCR产生阳性信号。因此，假阳性信号可能影响了本研究中ddPCR的检测能力。数字PCR在HIV核酸定量方面很有前景，但假阳性信号需要进一步关注。CA HIV RNA的定量检测有潜力改善对抗逆转录病毒治疗患者的监测，并用于旨在根除HIV的临床研究，但在广泛使用之前应由多个实验室进行交叉验证。
Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.
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