UDG-LAMP with Bst v5.9: Collaboration without Contamination

Problem and Background (200 words max)

Loop-mediated amplification (RT-LAMP) meets many of the criteria required for functional alternatives to RT-PCR for COVID testing. However, it has logistical and scientific limitations. Problem-1: The supply chain is/was controlled by a small number of companies. When the reagents are imported this leads to impractically long lead times and, due to taxes and middle-men, to 50-100% price mark-ups. For a low-middle income country like the Philippines, the higher costs are borne by those with much lower ability to pay -- a ‘double whammy’. There is not yet national infrastructure to create local supply. Problem-2: The sensitivity of LAMP comes at the expense of primer complexity and a preponderance for false positive results. One origin is DNA contamination from neighbouring tests via aerosols and liquid contamination of instruments/pipettes etc. The open-sourced Bst v5.9 enzyme has many of the characteristics to make it a good functional replacement for commercial Bst enzymes -- thereby addressing Problem 1. And yet, it is unknown whether v5.9 is able to acceptably incorporate dUTP, a property essential for its use with the UDG system designed to minimise DNA contamination. This project seeks to answer Problem-2 with v5.9 enzyme and in so doing, address Problem-1.

Solution summary in simple terms

From a healthcare perspective, the Philippines (and other LMICs) needs COVID tests that are 1. affordable in the context of their economy and healthcare system and 2. portable and ‘rugged’ in the context of use in an isolated rural primary care setting. For a technical solution, we need the ability to locally manufacture component enzymes at scale and cheaply and we need these enzymes to be robust to contaminating DNA that can cause false positives when the test is performed in less-than-ideal facilities. For (1), the open-source enzyme “v5.9” will provide a locally-produced, cost-effective alternative to the commercial enzymes, further driving down testing costs and ‘removing the middle-man’. For (2), we will check whether a test kit using v5.9 enzyme will be robust to DNA contamination by finding out if it can work in tandem with another enzyme (UDG) designed to minimize DNA contamination and maximise ‘ruggedness’. 

Solution summary in technical terms

The need for novel, open-access functional alternatives to the proprietary enzymes used in molecular diagnostic assays is well acknowledged. Of the three core enzymes required for UDG-RT-LAMP (RT, Bst, UDG, Hsieh et al., 2014), this project focuses on the Bst strand-displacing polymerase. Specifically, we will build on the work of Milligan et al., (2018) who described a thermostable Bst as a candidate alternative polymerase and on that of Alekseenko et al. (2020) who demonstrated a SARS CoV2 RT-LAMP assay with v5.9. With only two publications on v5.9, our first objective is to replicate this work and, importantly from a manufacturing perspective, determine the simplest (one vs two column) purification procedure. Our subsequent experiments will extend these authors’ work comparing the kinetic and amplification properties of v5.9 with Bst from NEB. Crucially, to minimise potential for DNA carry-over and false positives when used in non-ideal conditions, we will characterise v5.9 for its ability to incorporate dUTP using SYTO9 fluorescence kinetics, melt curve analysis and agarose gel electrophoresis. If there is sufficient incorporation of dUTP into DNA amplicons to be viable in UDG-RT-LAMP then v5.9 will solve our need for a locally sourced UDG-compatible polymerase for RT-LAMP. 

State of advancement of the project (100 words max)

The Bayan Biomedical team at Ateneo de Manila University has hired biochemists and molecular biologists for three related COVID diagnostics projects funded by the Government; it is these projects that will provide the additional resources to enable this project. We have received the v5.9 plasmid and can begin creating DH5 alpha and BL21 clones immediately while the out-standing reagents (ATc, heparin-agarose, UDG) are sourced. Finally, we have a team of 15 undergraduate students, a subset of whom are ready to use the microgrant to support their final year thesis research on Bst v5.9 enzyme.    

Project Timeline

• Month 1: Cloning of DH5 alpha E.coli containing pATetO 6xHis v5.9 plasmid in parallel with ordering of reagents (ATc [Abcam], Heparin-agarose [Merck], UDG [NEB]). 
• Month 2: Phase 1 - Expression and Purification of v5.9 DNA polymerase based on protocols from Milligan et al., 2018 and Alekseenko et al., 2020. Both enzyme preparations will be referenced against Bst 2.0 Warmstart (NEB). 
• Month 3: Phase 2 - Characterization of v5.9 DNA Polymerase: Comparison with commercial enzyme, in particular the ability of v5.9 to incorporate dUTP as part of the UDG system (Hsieh et al., 2014; Kil et al., 2015).