Development and Evaluation of a Rapid CRISPR-based Pathogen Detection Platform

Thesis Abstract

The rapid and accurate detection of pathogens is a critical bottleneck in clinical diagnostics and public health surveillance, particularly in settings with limited laboratory infrastructure where turnaround time directly impacts patient outcomes and outbreak containment. This study aims to develop and evaluate a rapid CRISPR-based pathogen detection platform that integrates amplification-free CRISPR-Cas sensors with portable readout to deliver actionable results within 30 minutes at the point of care. Specific objectives are (i) to design a multiplex CRISPR-Cas12a/Cas13a assay targeting a panel of clinically relevant bacterial and viral pathogens (including Escherichia coli O157H7, Staphylococcus aureus, Salmonella enterica, SARS-CoV-2, and Influenza A) and to optimize reaction conditions for minimal cross-reactivity; (ii) to engineer a microfluidic-compatible cartridge and a smartphone-enabled readout for field deployment; (iii) to establish analytical performance metrics, including limit of detection (LOD), linear dynamic range, specificity, and time-to-result, under varying matrix conditions (blood, saliva, and environmental swabs); (iv) to evaluate diagnostic performance in a prospective clinical study and in simulated field scenarios, and (v) to perform a cost-effectiveness analysis and deployment feasibility assessment in resource-constrained settings. The methodological framework adopts a design-based research approach that iterates from bench-scale assay development to field-ready deployment. The population comprises clinical specimens (n=600 across four pathogen panels) and field samples (n=200 environmental and point-of-care specimens). A formative pilot (n=120) informs the optimization of guide RNA design, reaction chemistry, and cartridge architecture. Data collection instruments include standardized laboratory protocols, a structured field-use questionnaire, and digital readouts from the smartphone-based analyzer. For analytical rigor, the study employs quantitative methods such as receiver operating characteristic (ROC) analysis to determine diagnostic accuracy, with sensitivity, specificity, positive predictive value, and negative predictive value calculated against gold-standard qPCR/viral culture reference methods. Regression analyses evaluate the relationship between sample matrix complexity and LOD, while ANOVA tests compare performance across pathogen panels and matrices. The analytical framework incorporates time-to-result as a continuous outcome, analyzed via Cox proportional hazards modeling to identify predictors of rapid detection. To elucidate user experience and implementation barriers, thematic analysis of qualitative feedback (n=40 field operators) is performed following the Braun and Clarke approach, aided by NVivo software. A feasibility model assesses deployment readiness using a modified Technology Acceptance Model (TAM) and a cost-effectiveness model from the health system perspective. Expected findings include (i) a multiplex CRISPR-based assay achieving LODs in the low single-copy to high-copy range depending on the pathogen, with overall diagnostic sensitivity and specificity exceeding 92% and 98% respectively across tested matrices; (ii) a cartridge-based microfluidic workflow reducing manual handling and achieving a total time-to-result under 30 minutes; (iii) robust performance under clinically relevant inhibitors and varying sample viscosities, with minimal cross-reactivity due to carefully designed guide sequences and reaction conditions; (iv) favorable user acceptance and operational feasibility in field settings, supported by qualitative insights on workflow integration; and (v) a favorable cost-effectiveness profile compared with standard laboratory-based molecular diagnostics in low-resource contexts. The study contributes to knowledge by delivering a validated, rapid, portable CRISPR-based diagnostic platform with demonstrated usability in non-laboratory environments, expanding the applicability of CRISPR diagnostics to point-of-care public health and outbreak response. It advances the theoretical understanding of integrating CRISPR biology with microfluidics and mobile technology to achieve field-deployable diagnostics, and it provides a pragmatic framework for evaluating deployment viability in diverse health systems. The primary conclusion anticipates that the integrated platform can provide timely, accurate pathogen detection with substantial reductions in time-to-result and per-test cost, thereby enhancing clinical decision-making and outbreak control. Policy and practice recommendations emphasize standardized training for field operators, integration with electronic health records, supply-chain considerations for reagent stability, and staged scale-up strategies in alignment with national diagnostic guidelines.