3 years ago

The Use of Droplet Digital PCR to Quantify HIV-1 Viral Replication in the Colorectal Explant Model.

Ashley Myerski, Aaron Siegel, Jarret Engstrom, Ian McGowan, Rhonda M Brand
Ex vivo explant models are used to characterize in vitro efficacy of pre-exposure prophylaxis (PrEP) agents. Tissue is challenged with virus in culture and HIV-1 p24 levels are quantified with enzyme-linked immunosorbent assay (ELISA) on supernatants collected throughout a 14-21-day incubation. Due to the narrow dynamic range of HIV-1 p24 kits, we evaluated whether droplet digital PCR (ddPCR) provides an alternative method to quantify HIV-1 replication in supernatant samples. We used samples from the MWRI-01 study, which evaluated the pharmacokinetic/pharmacodynamic profile of long-acting rilpivirine using the explant model (McGowan I et al. Lancet HIV 2016). HIV-1 pol RNA was measured with ddPCR, either directly with a one-step method or reversed transcribed to cDNA before ddPCR (two-step method) on supernatants from the MWRI-01 study. Previously analyzed HIV-1 p24 antigen levels (Alliance, Perkin-Elmer) were available for comparison purposes. Both ddPCR methods strongly correlated with HIV-1 p24 and displayed similar patterns of HIV-1 suppression before and after rilpivirine. Compared to the p24 ELISA, two-step and one-step ddPCR reduced the amount of hands-on time by approximately one-half and two-thirds respectively. ddPCR also required less sample and based on p24 vs ddPCR correlation, could potentially reduce the explant culture time from 14 to 10 days (r2=0.78, p<0.001) due to the increased sensitivity of ddPCR. We demonstrate that ddPCR is a suitable alternative to HIV-1 p24 ELISA to quantify HIV-1 infection in the explant model and has the potential to decrease explant culture time.

Publisher URL: http://doi.org/10.1089/AID.2018.0227

DOI: 10.1089/AID.2018.0227

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.