Isothermal Nucleic Acid Assays Based on Nucleic Acid Sequence Based Amplification (NASBA) and Recombinase Polymerase Amplification (RPA) for HIV-1 Diagnosis and Management in Low Resource Settings
Rohrman, Brittany Ann
Richards-Kortum, Rebecca Rae
Doctor of Philosophy
Over two-thirds of the estimated 35 million people worldwide infected with HIV live in the developing world. Nucleic acid tests (NATs) are necessary for early infant diagnosis and for monitoring patients receiving therapy. However, NATs cost $50-100 USD per test and require expensive thermal cycling equipment that may be unavailable in the developing world. This thesis presents two low-cost NATs for HIV-1 diagnosis and management that are based on isothermal amplification, which eliminates the need for expensive thermal cycling equipment. In one assay, HIV-1 viral RNA is detected using nucleic acid sequence based amplification (NASBA) and a custom lateral flow test. This assay costs about \$16 USD and only requires a heat block. When coupled with NASBA, the lateral flow test detected concentrations of synthetic RNA spanning the entire clinical range. When the assay was evaluated using pediatric plasma samples, the sensitivity (61%) and limit-of-detection (10,000 HIV-1 copies/mL plasma) were lower because of the genetic diversity of the samples, and the specificity was lower (88%) due to amplicon contamination. In the other assay, HIV-1 proviral DNA is amplified using recombinase polymerase amplification (RPA). This assay, which costs about \$5 per test, was integrated into a paper and plastic microfluidic device. The device was capable of amplifying 10 copies of plasmid HIV-1 DNA to detectable levels in 15 minutes. The assay was then adapted for real-time quantification. On average, the assay predicted sample concentrations within one order of magnitude of the correct concentration. In addition, a method for incubating RPA reactions without external equipment was developed. Using human body heat for incubation, all RPA reactions with 10 copies of plasmid HIV-1 DNA and 95% of reactions with 100 copies of plasmid HIV-1 DNA tested positive. Finally, concentrations of background DNA found in whole blood were shown to prevent the amplification of target DNA by RPA. To address this problem, three sequence-specific capture methods were developed to enrich target DNA concentration relative to background DNA concentration. These methods may be enable detection of high proviral loads in 0.1 mL infant blood samples but require improvement to detect lower proviral loads.