B-227 Effects of Buffer Optimization and the Use of Engineered Reverse Transcriptase and Taq DNA Polymerase Variants for Improved Activity and Inhibitor Tolerance in POC MDx Applications

Background Point of care (POC) molecular diagnostics (MDx) enable same-day diagnosis, revolutionizing patient care and disease surveillance. The requirement for DNA purification is a limitation for POC devices because it increases both the cost and time to get from sample to answer. In order to enable the use of crude lysates or skip extraction altogether, we engineered new reverse transcriptase (RT) and Taq DNA polymerase (Taq DNAP) enzymes with higher inhibitor tolerance and polymerization speed, and evaluated them with custom inhibitor-tolerant buffers. Methods M-MLV RT and Taq DNAP were engineered by directed evolution, rational design and in-silico modeling to achieve increased activity and inhibitor tolerance. These engineered variants were characterized by RT-qPCR and qPCR assays using a respiratory panel targeting SARS-CoV-2, influenza A and influenza B. The highest performing RT and Taq DNAP variants were then used in buffer optimization experiments designed for direct PCR, in which reactions were challenged with increasing concentrations of inhibitors such as saliva, nasopharyngeal swabs, urine, blood and representative inhibitor components. Performance was measured by limit of detection (LOD) sensitivity and quality of amplification curves. Results The engineered RT and Taq DNAP variants showed increased activity and inhibitor tolerance above their wild-type counterparts. This increase in activity also led to a decrease in the required cycling time, enabling faster PCR. The importance of buffer attributes such as pH control and salt content were clearly demonstrated, and PCR additives were shown to be crucial in maintaining tolerance to specific sample types. Conclusion The combination of both advanced buffer design and engineered RT and Taq DNAP variants enables direct PCR, which will alleviate the limitation of DNA purification on POC devices. Increased activity and inhibitor tolerance were shown to enable shorter RT-PCR/PCR cycling times, which further decreases assay times needed for POC devices.