Simple Inexpensive Assay for Five Common HIV Resistance Mutations



 DESCRIPTION (provided by applicant): Only a few inexpensive drugs can be used in the developing world for the treatment of AIDS. Several of these target the HIV reverse transcriptase (RT). Accordingly, patient therapy with these drugs often fails when the gene encoding RT undergoes mutation. Thus, the WHO recently reported that after 12 months of treatment with anti-RT drugs, patients most often relapsed when the following mutations arose in RT: (a) for nevirapine, K103N and Y181C; (b) for tenofovir and d4T, K65R; (c) for 3TC and FTC, M184V; and (d) for thymidine analogs, D67N. Therefore, both for surveillance and for immediate patient care, the NIAID and the CDC issued an SBIR solicitation for commercial technology transfer to develop an assay that could quickly and inexpensively detect these five mutations in patient samples in a resource-limited environment. The solicitation sought, as a benchmark specification, a level of detection (LOD) of 10,000 molecules/mL. The work proposed here will deliver this assay with a much better LOD (10-100 molecules/mL), exploiting technology developed under an NIAID R01 to the FfAME that ends in 2014. Thus, an STTR grant format has been chosen to deliver an assay with the following features that make it easy and inexpensive: 1. The assay will do multiplexed amplification for regions of the RT gene that contain resistance-conferring mutations in one assay, avoiding the cost of five separate assays for each of the alleles. This performance specification is possible because of FfAME-Firebird self-avoiding molecular recognition systems (SAMRS). 2. The assay will exploit the isothermal helicase-dependent amplification (HDA), not standard PCR. This avoids both the cost of a PCR instrument and the power demands of thermal cycling. Multiplexed HDA relies on technology recently developed in the Benner laboratory under the NIAID R01 grant, including SAMRS-reverse transcriptase HDA, which has a level of detection (LOD) of 10 ~ 100 molecules. 3. To ensure high coverage, multiple primers covering ~90% of the sequence diversity surrounding the target site will be used. SAMRS, by preventing primer-primer interactions, makes this multiplicity of primers possible, and allows them to be expanded, without needing to redesign the multiplex. 3. The assay will amplify target xNA before SNP detection, exploiting the very low noise of nested PCR using the FfAME-Firebird technology known as "artificially expanded genetic information systems" (AEGIS). 4. The assay will detect amplicons using "orthogonal beacons", also developed here. These also rely on AEGIS technology to suppress background noise, allowing detection by eye of as few as 1010 amplicons. 5. Readout will use immobilized beacons, with fluorescence generated by a hand-held battery-operated LED, with diagnosis made on the spot or, if captured by a cell phone camera, at a remote evaluation center. 6. Should cross-reactivity be observed, in Phase 2, it will be reduced using aminoxy reversible terminators with engineered polymerases, another innovation coming from the Benner laboratory.