The Development of a Rapid Assay to Diagnose Infant HIV Using Recombinase Polymerase Amplification
David Boyle*1, D Lehman2, M Singhal1, O Piepenburg3, M Parker3, P Munday3, N Armes3, and J Overbaugh2
1PATH, Seattle, WA, US; 2Fred Hutchinson Cancer Res Ctr, Seattle, WA, US; and 3TwistDx Ltd, Cambridge, UK
Background: Early diagnosis and treatment of HIV in infants greatly reduces mortality rates. However, current infant HIV diagnostics cannot reliably be performed in low resource settings, and shipping samples to appropriate test laboratories can significantly delay treatment. Recombinase polymerase amplification (RPA) is a novel technology that is an ideal HIV diagnostic because it amplifies DNA at a constant temperature in the range of 25°C to 42°C, thus reducing the need for complex thermocycling equipment. Results can be assessed via different detection methods, including in real time using a low-cost fluorescence reader or by end-point detection using immunochromatographic strips (ICS). Amplification and detection are rapid, generating results in only 15 minutes.
Methods: We screened RPA primer/probe sets targeting conserved regions identified in pol, gag, and LTR for primer/probes that consistently amplified an exact sequence-matched template at <50 copies. Candidate assays were further screened for sensitivity using dilutions of pre-quantified HIV DNA from 40 to 0.5 copies per test. The candidate primers/probe sets were then tested against a panel of 15 viruses including subtypes A, D, and C to test whether these RPA assays tolerate expected HIV-1 genetic diversity.
Results: We screened 3 to 6 forward and reverse RPA primers and an internal probe for each region in pol, gag, and LTR (resulting in 63 primer/probe combinations) for the ability to amplify an exact matched DNA sequence. We identified 2 primer/probe sets in pol that both amplified with 100% specificity. One primer set could detect as few as 1 to 3 copies of sequence-matched HIV DNA, however detection <40 copies was not consistent. The second primer/probe set amplified DNA at 2 copies in 100% of tests and at 1 copy in 28% of tests. The first primer/probe set amplified 7 of 15 diverse HIV variants, and the second primer/probe set amplified 10 of 15 variants. Of 15 virus variants, 13 were amplified by either one or the other assay, suggesting multiplexing could be effective.
Conclusions: RPA permits the detection of proviral DNA in <15 minutes with a current limit of detection of 1 to 3 copies per test. Cross-subtype testing suggests that RPA can amplify highly diverse target sequences, an essential feature for a DNA-based HIV diagnostic assay. The use of degenerate primers or multiplexing may allow increased sensitivities across subtypes.