Development of a sample preparation platform for bacterial and viral pathogens

Abstract

Molecular diagnostics plays a crucial role in reducing the impact of infectious diseases on a population. A key stage during the process is sample preparation, viewed as a bottleneck in the field of diagnostics. There are currently no standalone sample preparation systems able to process a range of sample types in a streamlined and reliable manner away from specialised infrastructure. The contribution to knowledge, described in this thesis, demonstrates a proof-of-concept sample preparation platform that was designed for deployment in the developed and developing world. The focus was placed on processing cloacal swab samples collected from chickens, and respiratory swab samples collected from humans, for the amplification and detection of poultry related infections and SARS-CoV-2, respectively, in a separate platform. A lysis protocol (the boiling method) was tested on cloacal samples before conducting both PCR amplification and colorimetric LAMP. 100 mg of a cloacal sample was suspended within 200 μL of a buffer, which was then incubated at 92 °C for 15 minutes. Performing the boiling method in a PBS buffer was found to offer the highest DNA yield (182 ng/μL), while the highest A280/260 ratio was found with the TE/PK buffer (1.06). Both the PCR and LAMP methods showed successful amplification of E. Coli following a 1:10 dilution step. A sample preparation platform was developed to accept cloacal and respiratory (NP and OP) samples on a swab, lyse the cells using thermal lysis, and dispense 1 – 3 μL droplets into PCR tubes. As it was not possible to produce droplets within this range using standard dripping configurations, an EHD module was employed to overcome the effects of surface tension at a metallic capillary. The platform was designed using Solidworks 2020, and optimised using COMSOL simulations. The platform was then fabricated using 3D printing techniques and tested. The platform demonstrated capabilities of performing cell lysis in a single thermal heating step at 92 °C. Droplets with a volume of 1.3 μL were dispensed on-demand, 86% smaller than possible with standard dripping configurations. The multidisciplinary work presented in this thesis reduced the complexity of sample preparation into a ready-to-assemble platform. Although universal sample preparation has yet to be fully achieved, the research conducted in this thesis may offer the first step towards the realisation of this goal.