Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA

被引:123
作者
Kiselinova, Maja [1 ,2 ]
Pasternak, Alexander O. [3 ]
De Spiegelaere, Ward [1 ,2 ]
Vogelaers, Dirk [1 ,2 ]
Berkhout, Ben [3 ]
Vandekerckhove, Linos [1 ,2 ]
机构
[1] Univ Ghent, Dept Internal Med, HIV Translat Res Unit HTRU, B-9000 Ghent, Belgium
[2] Ghent Univ Hosp, Ghent, Belgium
[3] Univ Amsterdam, Acad Med Ctr, Ctr Infect & Immun Amsterdam CINIMA, Dept Med Microbiol,Lab Expt Virol, NL-1105 AZ Amsterdam, Netherlands
来源
PLOS ONE | 2014年 / 9卷 / 01期
基金
比利时弗兰德研究基金会;
关键词
GENE-EXPRESSION ANALYSIS; LOW-LEVEL VIREMIA; DNA COPY NUMBER; ANTIRETROVIRAL THERAPY; ABSOLUTE QUANTITATION; PLASMA VIREMIA; VIRUS; REPLICATION; LOAD;
D O I
10.1371/journal.pone.0085999
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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: R-2 = 0.97, msRNA: R-2 = 0.92) and patient-derived samples (usRNA: R-2 = 0.51, msRNA: R-2 = 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 log(10)). However, a bias of 0.94 +/- 0.36 log(10) 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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页数:8
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