Poster Presentation 14th Lorne Infection and Immunity 2024

CRISPR diagnostics: The versatile, rapid & accurate detection of pathogens (#154)

Lewis Williams 1
  1. WEHI, Parkville, VICTORIA, Australia

PCR has been long been considered the diagnostic gold-standard. However, its technical requirements restrict PCR’s utility to a laboratory setting and with high costs for setup and operation. Whilst inconvenient in metropolitan areas, it becomes critically limiting for remote communities of Australia and smaller health clinics. CRISPR-based diagnostics have become increasingly promising as a screening technique or even replacement for PCR. We have established a k-mer and pan-genome bioinformatic pipeline to generate novel primer and CRISPR guide combinations for subsequent use in the RPA isothermal amplification. Enhancements to the RPA assay has yielded the ability to amplify single copy numbers whilst being able to transfer this reaction directly into the CRISPR assay without further purification. Finally, subsequent CRISPR detection has also been substantially improved, with a 10-fold improvement on the sensitivity published in the literature through extensive optimisation of assay conditions. In combination, we have developed an assay can that operate continuously at a single temperature, is at least 30 minutes faster than PCR, can detect as few as 5 copies of DNA all whilst retaining complete specificity. We have adapted our platform for the detection of Human T-cell Lymphotropic Virus-1c (HTLV-1c) but the detection of many other pathogens is possible. We aim to transform our research into a diagnostic platform that is more appealing to communities and hospitals, encourages rates of testing and community awareness of HTLV-1.