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 months of treatment with anti RT drugs patients most often relapsed when the following mutations arose in RT a for nevirapine K N and Y C b for tenofovir and d T K R c for TC and FTC M V and d for thymidine analogs D N 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 molecules mL The work proposed here will deliver this assay with a much better LOD molecules mL exploiting technology developed under an NIAID R to the FfAME that ends in Thus an STTR grant format has been chosen to deliver an assay with the following features that make it easy and inexpensive 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 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 R grant including SAMRS reverse transcriptase HDA which has a level of detection LOD of molecules To ensure high coverage multiple primers covering 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 The assay will amplify target xNA before SNP detection exploiting the very low noise of nested PCR using the FfAME Firebird technology known as andquot artificially expanded genetic information systemsandquot AEGIS The assay will detect amplicons using andquot orthogonal beaconsandquot also developed here These also rely on AEGIS technology to suppress background noise allowing detection by eye of as few as amplicons 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 Should cross reactivity be observed in Phase it will be reduced using aminoxy reversible terminators with engineered polymerases another innovation coming from the Benner laboratory PUBLIC HEALTH RELEVANCE Five mutations in the gene encoding HIV reverse transcriptase RT are commonly found when therapy with inexpensive anti RT drugs fails Recently the NIAID issued a call for innovative technologies that could inexpensively detect these mutations near points of care as assays based on these technologies could help optimize second line therapeutic regimens in the developing world as well as assist in surveillance around the world The work proposed here will deliver these assays by combining five separate chemical innovations exclusive to the applicant organizations FfAME and Firebird