Development of loop-mediated isothermal amplification for rapid diagnostics

Thesis

The urgent need for rapid, reliable diagnostic technologies has been underscored by global health challenges like the COVID-19 pandemic. Conventional diagnostic methods, such as Polymerase Chain Reaction (PCR), are effective but rely on complex infrastructure, making them less accessible for decentralized or resource-limited settings. Loop-Mediated Isothermal Amplification (LAMP) technology offers a faster and simpler alternative, but traditional LAMP assays face limitations, including the inability to quantify pathogen load, cold-chain dependency, limited multiplexing, and interference from sample pH.

This thesis addresses these gaps through multiple innovations, resulting in the development of OxLAMP, a third-generation LAMP assay designed for versatile point-of-care diagnostics. Key advancements include the advancement of quantitative LAMP (qLAMP ) for pathogen quantification, akin to PCR’s CT values. qLAMP was verified by 64 clinical samples (25 positives and 39 negatives) and LAMP threshold time (TT) values showed linear relationships to pathogen load. Novel lyophilisation and Matrix-Assisted Room Temperature (MART) drying techniques for reagent stabilization were developed, demonstrating capabilities of preserving reagents for six months at 45°C, eliminating the need for cold-chain storage. Additionally, the OxLAMP platform was further developed by incorporating pH-independent colorimetric diagnostic capabilities, improving robustness and ensuring consistent diagnostic accuracy across pH ranges of 2.0 to 9.0. Custom primer sets were designed and validated for pathogens, including six variants of SARS-CoV-2, seven different bacterial bloodstream pathogens, and Herpes Simplex Virus (HSV), enabling multiplex detection of pathogens within a single platform. False positives, a common challenge in LAMP, were eliminated through stringent primer design and experimental validation, with a specificity of 100% in clinical testing.

These innovations culminated in the integration of the OxLAMP assay into a multiplex diagnostic device, the Sinopharm Colorimetric Photometer, which facilitates high-throughput and quantitative pathogen detection from clinical samples. Validation using 20 clinical specimens (14 positives and 6 negatives) demonstrated the platform’s reliability and diagnostic accuracy across diverse healthcare settings.

By addressing key limitations in traditional diagnostic tools, OxLAMP bridges the gap between laboratory innovation and real-world application. This work not only enhances the scalability and accessibility of rapid diagnostic technologies but also provides a robust framework for managing current and emerging infectious diseases in both clinical and resource-limited environments.

Authors: Zhou, JX

• DOI: 10.5287/ora-e91vpgja6
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: point-of-care diagnostics; lyophilisation of molecular assays; loop-mediated isothermal amplification; quantitative LAMP; clinical sample validation
• Subjects: Herpes simplex virus; Point-of-care testing; COVID-19 (Disease); Biomedical engineering; Pathogens