2012 Global WACh & W.H. Coulter Foundation Pilot Grant Awardee:
The long-term goal of the 2012 seed project is to develop a point-of-care assay for HIV drug resistance by reducing a complex laboratory process into a rapid and easy-to-use format. The existing laboratory-based oligonucleotide ligation assay (OLA) developed by the Frenkel lab allows highly-specific identification of HIV drug resistance, but the complexity of the process (sample preparation, nucleic acid amplification, ELISA- based detection) has restricted adoption in some low resource labs of the developing world.
As a first goal, the team will simplify each step of the assay – specimen preparation, PCR (nucleic acid amplification and ligation), and target detection – to provide a less complex test for low resource laboratories; these simplifications will be the first steps to developing a point-of-care assay appropriate for use outside the laboratory.
The role of the Lai lab is to develop specimen preparation technologies that can enable nucleic acid target enrichment from a larger whole blood volume in two simple steps, mixing and separation. This approach will provide a great number of viral templates to be submitted to PCR to allow simplifications in the amplification and ligation steps (described below) without losing sensitivity. To date we have designed and synthesized stimuli-responsive polymers that form complexes with oligonucleotides. Next the team will utilize these polymer reagents for isolating DNA from the specimens.
The role of the Frenkel lab in the Coulter/GlobalWACh seed project is to adapt the OLA from two nested rounds of PCR to a single round of PCR and modify the ligase reaction so that it can be performed in the same “tube” (well). The team plans to pursue these advancements using DNA for a test that would be performed prior to ART to detect transmitted resistance, and in a slightly different assay using plasma RNA that would be used in individuals taking ART to detect “virologic failure” and proceed directly to testing for drug resistance. (This latter assay was added after the award of the Coulter WaCH grant.) To date the team has simplified the nested to a single round of PCR using plasma. Next the team will work on proceeding directly to the ligase reaction, and using on applying the single-round PCR to blood DNA isolated by Dr. Lai.
The role of the Lutz lab is to reduce the multi-step laboratory detection process into a rapid and simple strip format that can be read by eye. In the existing assay, detection is based on the enzyme-linked immunosorbent assay (ELISA) with additional stringency wash steps to remove non-target (non-ligated) oligonucleotides, and it requires comparison of two or more target signals detected by an instrument. The team is currently adapting the chemistry and assay steps into a paper strip format that will report colored lines for resistant and non-resistant targets. With the Frenkel lab, the team has produced PCR-amplified and ligated products to be used for testing, and has prepared detection strips that show faint signals for the ligated targets.