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    Global Health Technologies

    Development of a reverse transcription recombinase polymerase amplification assay targeting long terminal repeat regions of the HIV-1 genome to enable HIV-1 RNA viral quantification at point-of-care

    (J-363) Development of a reverse transcription recombinase polymerase amplification assay targeting long terminal repeat regions of the HIV-1 genome to enable HIV-1 RNA viral quantification at point-of-care

    Friday, October 13, 2023
    3:30 PM – 4:30 PM PDT
    Location: Exhibit Hall - Row J - Poster # 363

      Presenting Author(s)

    • SN

      Sami Nguyen

      Undergraduate Student Researcher
      Department of Bioengineering, University of Washington, United States

      • Co-Author(s)

    • EK

      Enos Kline

      Research Scientist
      University of Washington, United States

      • Primary Investigator(s)

    • BL

      Barry R. Lutz, PhD (he/him/his)

      Associate Professor
      University of Washington, United States

      • Last Author(s)

    • Nuttada Panpradist, PhD (she/her/hers)

      Postdoctoral Fellow
      Department of Global Health, University of Washington
      Seattle, Washington, United States

    Introduction::

    People living with HIV (PLH) have the potential to live nearly normal lives with proper anti-retroviral therapy. Monitoring HIV RNA levels is crucial in determining viral load suppression and ensuring effective treatment. When the RNA level in PLH is below 1000 copies/mL, the risk of transmission is considered negligible. Therefore, there is a pressing need for low-cost and efficient treatment monitoring for HIV to be incorporated into routine healthcare practices, especially in low- and medium-income countries with high HIV burdens. Previously, our research group introduced the use of target mimicking internal control in conjunction with an isothermal reverse transcription recombinase polymerase to enable semi-quantitation of HIV-RNA [1]. Although this assay demonstrated rapidity, its sensitivity needed enhancement. This study aimed to investigate a novel primer and probe design, focusing on the long terminal repeat regions of HIV-1. The performance of this new design was demonstrated in the context of point-of-care testing of our portable Harmony heater and reader device [2], depicted in Fig 1A.



    Materials and Methods:: Primers and exonuclease probes were specifically designed to target regions within the long terminal repeats (LTR) of the HIV-1 genome, similar to those utilized in a sensitive, clinically-validated RT-PCR assay [3]. To determine the characteristics of the primer and probe, such as their lengths and compositions, we followed the manufacturer’s recommendation [4]. A total of 38µL of exo-RPA reactions (TwistDx) was prepared, incorporating 420 nM forward primer (IDT), 420 nM of reverse primer (IDT), 240 nM of exo probe (IDT), and 4 units of Omniscript RT (Invitrogen). Subsequently, 2µL of HIV RNA template was added to achieve the final concentrations of 0, 1, 10, 100, or 1000 copies/µL, with four technical replicates for each condition. The prepared mixture was then analyzed using the Harmony device [2]. Raw signals were exported from the Harmony mobile application, and the time to detection for each HIV RNA concentration was calculated and reported as means ± standard errors.

    Results, Conclusions, and Discussions::

    Our newly developed real-time RT-RPA LTR exhibited a high level of analytical sensitivity, detecting as low as 1 copy/µL of HIV RNA, which is comparable to our reference RT-PCR assay. Despite this high sensitivity, our RT-PCR LTR assay has several advantages over traditional RT-PCR methods. One of the key advantages lies in its rapidity, with a remarkably short time to detection of < 15 minutes (Figs. 1B and 1C). In contrast, the clinical RT-PCR assay typically demands approximately 1.5 hours for operation, depending on the heating and cooling speed of the thermal cycler employed. This substantial time difference is critical in facilitating prompt diagnosis. Moreover, our RT-RPA LTR assay is designed to be cost-effective and only relies on an in-house heater and reader device at a cost of < $250. This affordability offers an attractive option for operating in resource-limited regions where access to expensive equipment may be challenging. Our next step includes incorporating target mimicking internal control in our assay. This addition will enable the semi-quantitation of HIV RNA, providing insights into viral load levels and treatment response in PLH.



    Acknowledgements (Optional): :

    Acknowledgments: We thank other Lutz lab members: Robert Atkinson, Michael Roller, Daniel Leon, Dr. Amy Oreskovic, Qin Wang, Dr. Ian Hull, and Jack Henry Kotnik for their previous contributions to the development and evaluation of the Harmony device for SARS-CoV-2 RNA detection. This work was funded by NIH R01AI145486. The funder has no role in study design, experimental results, and interpretation.



    References (Optional): :

    [1] Hull IT, Kline EC, Gulati GK, et al. Isothermal Amplification with a Target-Mimicking Internal Control and Quantitative Lateral Flow Readout for Rapid HIV Viral Load Testing in Low-Resource Settings. Anal Chem. 2022 Jan 18;94(2):1011-1021. doi: 10.1021/acs.analchem.1c03960. Epub 2021 Dec 17. Erratum in: Anal Chem. 2022 Apr 19;94(15):6071. PMID: 34920665; PMCID: PMC9219584

    [2] Panpradist N, Kline EC, Atkinson RG, et al. Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection. Sci Adv. 2021;7(51):eabj1281. doi:10.1126/sciadv.abj1281

    [3] Arvold ND, Ngo-Giang-Huong N, McIntosh K, et al. Maternal HIV-1 DNA load and mother-to-child transmission. AIDS Patient Care STDS. 2007 Sep;21(9):638-43. doi: 10.1089/apc.2006.0169. PMID: 17919090.

    [4] https://www.twistdx.co.uk/product/twistamp-exo/