Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA
| Autor: | Lin Yuan, Zhiying Liu, Xin Zhang, Feili Wei, Shan Guo, Na Guo, Lifeng Liu, Zhenglai Ma, Yunxia Ji, Rui Wang, Xiaofan Lu, Zhen Li, Wei Xia, Hao Wu, Tong Zhang, Bin Su, Yanjie Yin |
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| Jazyk: | angličtina |
| Rok vydání: | 2024 |
| Předmět: | |
| Zdroj: | Chinese Medical Journal, Vol 137, Iss 6, Pp 729-736 (2024) |
| Druh dokumentu: | article |
| ISSN: | 0366-6999 2542-5641 00000000 |
| DOI: | 10.1097/CM9.0000000000003081 |
| Popis: | Abstract. Background:. Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA. Methods:. The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4+) T-cell counts, CD8+ T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. Results:. The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6–6.5 copies/reaction) with linearity over a 5-log10-unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8–16.6 copies/reaction) with linearity over a 3-log10-unit range. Total HIV DNA in CD4+ T cells was positively associated with integrated HIV DNA (r = 0.76, P |
| Databáze: | Directory of Open Access Journals |
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