Periodic Reporting for period 2 - FreeATPOC (Towards an instrument-free future of molecular diagnostics at the point-of-care)
Periodo di rendicontazione: 2022-01-01 al 2023-12-31
All assays were developed upon extensive optimization of relevant steps, especially the sample pre-treatment for lysis at room temperature and under conditions compatible with the subsequent isothermal amplification. Regarding the HIV test, we demonstrated the detection of as little as 10 viral copies per reaction (or <1000 copies per ml of plasma) after isothermal (RPA) amplification at 37oC using our newly developed molecular lateral flow strip (LFS)-based technology. Such a high performance assay, leading to semi-quantitative HIV detection in patients’ plasma at the point of care is unique and has never been reported before. The same protocol and methodology were also applied to influenza detection, reaching a detection limit of 50 copies per reaction. Moreover, the HIV and influenza assays were validated by end users in a hospital environment, both in Europe and S. Africa, demonstrating the feasibility of the method. Finally, the universal and generic nature of the LFS molecular assay has been demonstrated during Xylella pathogens detection in the field using crude plant tissue. It is noted that the two assays developed for HIV and influenza detection are already commercially available and can be obtained for research purposes in the form of two kits.
Our newly developed and patented methodology, based on a molecular Lateral Flow Strip assay for amplicons’ detection and a 3D-printed LFS reader and relevant software, is an attractive solution for diagnostics at the POC using minimal instrumentation. The low cost of the reader (<70 euros), together with the simplicity in operation and rapid and semi-quantitative nature of the detection make the above innovation an attractive solution for global diagnostics. The method, currently at TRL=4-5, will form the main result for future exploitation and is the basis of FREE@POC business plan. Further innovations also produced within FREE@POC include a portable instrumentation which relies on the combination of paper microfluidics with an acoustic (SAW) biochip. This method also forms a generic platform suitable for nucleic acids detection but also antibodies and antigens. Such a flexible system has not been reported so far and a new patent application has been filed for this purpose. Overall, the project has resulted in several new approaches and methods, leading to two patent applications, two small pilot productions of HIV and influenza kits and a robust and attractive methodology for Xylella detection in the field using crude plant tissue.
All the above are expected to contribute significantly in advancing of the state of the art in the field of diagnostics and produce useful tools for application in the field. The fact that we are targeting both human and plant-borne pathogens is enhancing the impact of the work; potential applications could be found in the healthcare, but also agro/food safety, and could be implemented in both the developing and developed countries.