Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA
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 t...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wolters Kluwer
2024-03-01
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| Series: | Chinese Medical Journal |
| Online Access: | http://journals.lww.com/10.1097/CM9.0000000000003081 |
| _version_ | 1797257331523715072 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Lin Yuan |
| collection | DOAJ |
| description | 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 <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4+ T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8+ T-cell counts.
Conclusions:. This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials. |
| first_indexed | 2024-04-24T22:35:57Z |
| format | Article |
| id | doaj.art-959c6dce537e4cafa2d960d5af435dbe |
| institution | Directory Open Access Journal |
| issn | 0366-6999 2542-5641 |
| language | English |
| last_indexed | 2024-04-24T22:35:57Z |
| publishDate | 2024-03-01 |
| publisher | Wolters Kluwer |
| record_format | Article |
| series | Chinese Medical Journal |
| spelling | doaj.art-959c6dce537e4cafa2d960d5af435dbe2024-03-19T10:28:51ZengWolters KluwerChinese Medical Journal0366-69992542-56412024-03-01137672973610.1097/CM9.0000000000003081202403200-00014Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNALin Yuan0Zhiying Liu1Xin Zhang2Feili Wei3Shan Guo4Na Guo5Lifeng Liu6Zhenglai Ma7Yunxia Ji8Rui Wang9Xiaofan Lu10Zhen Li11Wei Xia12Hao Wu13Tong Zhang14Bin Su15Yanjie Yin1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China2 Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China2 Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China1 Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, ChinaAbstract. 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 <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4+ T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8+ T-cell counts. Conclusions:. This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.http://journals.lww.com/10.1097/CM9.0000000000003081 |
| spellingShingle | 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 Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA Chinese Medical Journal |
| title | Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA |
| title_full | Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA |
| title_fullStr | Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA |
| title_full_unstemmed | Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA |
| title_short | Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA |
| title_sort | development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated hiv 1 dna |
| url | http://journals.lww.com/10.1097/CM9.0000000000003081 |
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