Cross-subtype detection of HIV-1 using reverse transcription and recombinase polymerase amplification

Abstract

A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10-30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7%) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV.

Keywords: Diagnostic; Human immunodeficiency virus; Infant HIV; Point of care; Reverse transcription recombinase polymerase amplification.

Figure 1. Comparison of HIV-1 RPA assays using real time fluorescence detection

Real time fluorescence amplification curves using the previously published pol HIV-1 RPA assay (dashed lines) (Boyle et al., 2013) and new Twist Alpha HIV-1 RPA assay (solid lines) to amplify 100 copies of sequenced matched HIV-1 DNA (black) or negative controls (grey). Data is shown for triplicate amplifications under each reaction condition. The gaps at 5 minutes reflects the mix step where reagent tubes were removed from the device, mixed and reinserted to complete amplification.