Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification

Abstract

Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.

FIG 1

(A) Limit of detection. pol and LTR primer/probe sets were tested on samples of 100, 40, 20, 10, 5, 3, 1, and 0 copies of an exact sequence-matched HIV-1 DNA template. NTC, no-template control. (B) Fluorescent detection of low-copy-number DNA with the pol primer/probe set. Fluorescent detection of 10 copies of exact sequence-matched DNA. Reactions were scored positive when the change in fluorescence exceeded 200 units. No false positives were detected with HIV-negative genomic DNA alone (NTC controls).

FIG 2

Assessment of the real-time HIV-1 RPA pol assay to amplify and detect diverse viral variants. The pol RPA primer and probe sequences, shown at the bottom, are aligned with the 56 pol sequences tested to highlight the exact location and number of polymorphisms relative to the primer and probe sequences.