While, according to the World Health Organization, the successful application of antiretroviral therapy reduced global deaths from HIV/AIDS by 24% between 2005 and 2011, a corresponding rise in viral resistance to these drugs has been measured. This rise is of particular concern in low-resource settings that are collectively burdened with over two-thirds of worldwide HIV infections while lacking access to robust diagnostic assays. In response to this need, the Lutz Lab, in collaboration with the Frenkel Lab at Seattle Children’s Research Institute and the Lai Lab at UW BIOE, aims to engineer a low-cost paper-based oligonucleotide ligation assay (OLA) for drug-resistant HIV. In order to lower the chance of user errors and reduce diagnostic turnaround time, it is desirable to be able to detect a set of six point mutations indicative of drug-resistance to first-line antiretroviral treatment in a single device. However, with current techniques we are limited to being able to test for one point mutation per device. To address this challenge, we have investigated a DNA hybridization method of OLA probe capture. Using this method, each point mutation is assigned a specific DNA “barcode” that could be used to generate spatially distinct capture lines in a diagnostic device. We have designed a set of six engineered barcodes that display highly specific and sensitive binding to their engineered complements. Furthermore, the performance of this DNA barcode system has been compared to barcodes constructed from non-natural nucleic acid analogs in order to develop a method that optimizes sensitivity and specificity of probe capture. The completion of this project brings the OLA one step closer to being able to make a previously expensive, technology intensive, and operationally complicated test, accessible to those in low-resource settings.

HIV remains a major global health problem due to high transmission levels and emergence of drug resistant strains. According to WHO, in 2014, sub-Saharan Africa had 25 million people living with HIV and 70% of new HIV infections globally. A high number of circulating drug resistant HIV remains undetected due to lack of affordable HIV genotyping methods. The lab based oligonucleotide ligation assay (OLA) is a highly sensitive method for detecting HIV drug resistance as an alternative to the expensive and complex complete-genome sequencing, but its reliance on moderately expensive lab equipment prevents its universal access.  Our lab, in collaboration with the Frenkel Lab (Seattle Children’s Research Institute) and the Lai Lab (UW BioE), focuses on developing a simplified OLA-based HIV drug resistance test. The ligation step of the OLA relies on the specificity of DNA ligase to target single nucleotide polymorphisms (SNP’s) that can confer HIV drug resistance. However, ligation currently requires a thermal cycler to cycle between 95 °C to de-hybridize target DNA and 37 °C for annealing of oligonucleotide probes to target DNA. Eliminating the temperature cycling component of ligation would be a key advantage because thermal cyclers are not widely available in low resource settings. We measured ligation at intervals from 25 °C to 70 °C and were able to pinpoint an optimal temperature range for isothermal ligation. We plan to further increase ligation yield, and therefore detection signal, by optimizing the concentrations of salts and cofactors, adjusting probe lengths, and modifying probe sequences.  With the development of isothermal ligation, the OLA will be further simplified for potential widespread use in low resource settings. This will allow HIV infected individuals to be accurately diagnosed and receive the proper line of HIV drugs, controlling transmission rates and reducing the evolution of drug resistant HIV within entire populations. 

In developed regions, laboratory-based molecular assays are routinely performed to enable early diagnosis and improve patient outcomes. However, several key technology gaps prevent the widespread use of molecular assays in low-resource areas. For instance, the lack of trained laboratory personnel in resource-poor settings can limit their capacity to perform labor-intensive tests. Additionally, many commonly used enzymes (e.g. polymerase and ligase) require costly cold chain storage. To simplify molecular assays for use in sub-optimal laboratories, we are developing a dried reagent platform for drying, storing, and dispensing test reagents. Our current work is focused on optimizing the platform for PCR and ligation, which are key steps in the Oligonucleotide Ligation Assay (OLA)-based HIV drug-resistance test being developed by the Frenkel lab (Seattle Children’s Research Institute) and the Lai and Lutz labs (UW Bioengineering). Multiple formulas containing preservatives up to 10% w/v were evaluated based on their assay compatibility and enzyme-stabilizing effect during freeze-drying. We found that the presence of trehalose and poly(ethylene glycol) in the dried reagent mixtures best maintained assay performance for both PCR and ligation. Preliminary data also showed that ligation performance of the new formula with the dried reagent platform was comparable to the performance of freshly prepared mixtures and that the dried reagents remained stable after 28-days of ambient storage (<15% relative humidity, 20-25°C). Long-term storage studies with the preserved PCR mixture are still in progress but we have demonstrated that lyophilized mixtures stored overnight produce amplified products in the same range as those from freshly prepared PCR mix. Future work will aim to achieve dried reagent stability up to 3-months and incorporate the platform into the first prototype of our drug-resistance test. Ultimately, the platform will reduce user manipulation and assay preparation time for the OLA and could be expanded to simplify other complex molecular assays.