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Lyophilized open source RT-LAMP preprint
We’re excited to finally present our latest work detailing a fully open source RT-LAMP reactions assembled from three public domain enzymes including lyophilization protocols! The corresponding preprint is available on medRxiv.
A collaborative effort
The work expands on our previous RT-LAMP work done in the 2020/2021 pandemic and we finally release it to the world and hope to keep detailed protocols easy to access through this website. It’s a collaborative piece of research performed jointly by the RT-LAMP team at the Vienna BioCenter and experienced LAMP researchers at the West African Centre for Cell Biology of Infectious Pathogens (WACCBIP) in Accra, Ghana.
An open source UDG
In the preprint, you’ll read about how we made a third LAMP enzyme compatible with our open source ethos. The enzyme in question is uracil-DNA-glycosylase (UDG), a DNA repair enzyme that has found great use and utility in amplification techniques like PCR and LAMP, where it prevents carryover contamination and thus helps keep false positives to a minimum. LAMP, because of the nature of its “explosive” amplification, is very prone to false positives. We’ve seen first hand how a small mishandling of LAMP reactions can lead to false positives through carryover contamination. Something as simple as taking out finished reactions from an incubator and handling them in gloves can cause contamination. But with the dUTP/UDG cross-contamination prevention system, the danger is greatly reduced. We show cross-contamination prevention activity of an open source UDG enzyme during reaction setup and its thermolability. The UDG we work with has been both published and patented, but the patent has expired, making it free for anyone to use. You can already find the His-tagged thermolabile UDG we’ve used for our study on Addgene under the Plasmid #172197.
Freeze-drying for storage and shipment
The next step in our puzzle was to figure out how the RT-LAMP enzymes and reactions can be freeze-dried for storage and shipment. Stability at room temperature enabled the reaction to be shipped without costly dry ice or heavy ice packs. It also enables LAMP testing in places where stable electrical power for -20°C freezer storage isn’t guaranteed. To achieve this, we present two protocols to lyophilize either just the enzymes or a reagent mix containing enzymes with dNTPs and primers. We show that a quick amplification and a clear color change we’ve come to know and love with RT-LAMP can be still achieved after lyophilizing and reconstituting the mixtures. What’s more, enzymes stored at 37°C for weeks can perform on par with those stored in the freezer! While lyophilization for RT-LAMP has been described in the literature, this is the first report of colorimetry-compatible enzyme-primer-dNTP mix.
Two validation studies
The last crucial task was to perform clinical validation of this assay. We chose to do a side-by-side comparison of specificity and sensitivity on clinical samples in Austria, where the enzymes were made and optimized, and in the labs of WACCBIP in Ghana. We sent the lyophilized RT-LAMP reagent mix from Europe to Africa without any cooling system, at ambient temperature. Felix Ansah Ph.D, a co-author of this preprint, then reconstituted the enzymes and used them to test 192 patient samples, comparing the LAMP results to RT-qPCR values. The results show a robust detection of samples with higher viral load than 1000 viral RNA copies per microliter, a threshold of infective sample.
In the coming days, we hope to update this webpage with lyophilization protocols contained in the manuscript and to spread the message further. To our understanding, this is the first case of fully open source RT-LAMP containing all three enzymes necessary for sensitive and specific detection. We also hope that clear lyophilization protocols are a resource for the community and can help researchers design RT-LAMP assays for various applications, be it in epidemic response or pathogen surveillance.