Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

PLoS One. 2020 May 14;15(5):e0233085. doi: 10.1371/journal.pone.0233085. eCollection 2020.

Abstract

Highly sensitive detection of HIV-1 nucleic acids is of critical importance for evaluating treatment interventions superimposed on combination antiretroviral therapy (cART) in HIV-1 infected individuals. SIV infection of rhesus macaques models many key aspects of human HIV-1 infection and plays a key role in evaluation of approaches for prevention, treatment and attempted eradication of HIV infection. Here we describe two droplet digital PCR (ddPCR) assays, a ddPCR DNA assay and an RT-ddPCR RNA assay for detecting simian immunodeficiency virus (SIV) on the RainDance platform. We demonstrate that RainDance ddPCR can tolerate significantly higher cell DNA input without inhibition on a per reaction basis (compared to both qPCR and Bio-Rad ddPCR), thus allowing a large quantity of sample to be analyzed in each reaction. In addition, the combination of a high processivity RT enzyme and RainDance ddPCR could overcome inhibition in severely inhibited (99.99% inhibition in qPCR quantification) viral RNA samples. These assays offer valuable tools for assessing low level viral production/replication and strategies for targeting residual virus in the setting of cART suppression of viral replication. The methodologies presented here can be adapted for a broad range of applications where highly sensitive nucleic acid detection is required.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Animals
  • DNA, Viral / analysis
  • DNA, Viral / genetics
  • HIV Infections / drug therapy
  • HIV Infections / virology
  • HIV-1 / genetics
  • Humans
  • Macaca mulatta
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / statistics & numerical data
  • RNA, Viral / analysis
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Simian Acquired Immunodeficiency Syndrome / virology
  • Simian Immunodeficiency Virus / genetics*
  • Simian Immunodeficiency Virus / isolation & purification*
  • Viral Load

Substances

  • DNA, Viral
  • RNA, Viral