Development of Paper-Based Point-of-Care Diagnostics for Detection of Human Respiratory Pathogens Using Loop-Mediated Isothermal Amplification (LAMP)

Paper-based microfluidic analytical devices (µPADs) are ideal for pathogen detection at the point-of-care (POC) due to their ease of production, user-friendliness, and scalability. This project focuses on developing a spatially multiplexed µPAD for detecting respiratory pathogens using Loop-mediated Isothermal Amplification (LAMP). This is a robust method suited for clinical settings with the goal to enhance patient treatment and enable the detection of emerging pathogens, particularly during public health emergencies. It will provide a diagnosis that is typically only available through costly laboratory-based testing.

LAMP amplifies nucleic acids in the presence of a specific pathogen, with six primers, binding to eight distinct regions of the target genome to drive the specificity of the reaction. Nucleotide incorporation during phosphodiester bond polymerization releases protons, causing a red-to-yellow colour change with the pH indicator phenol red. The Verma lab previously designed a µPAD which incorporates pre-dried reagents on chromatography paper reaction pads. These 5 x 6 mm pads are arranged in a rectangular array, annealed to a Melinex backing with a neutral adhesive, and separated by polystyrene spacers to prevent crosstalk. Nasal swabs or saliva samples from patients were diluted in water and added to rehydrate reagents in the pads. The device is then sealed and heated at 65°C for 60 minutes in a water bath, and the results are visually read by the user.

The device was previously tested using saliva samples, and the Verma Lab found the limit of detection (LOD) was 200 copies per µL with 98% accuracy using digital image analysis (97% analytical sensitivity, 100% specificity) and 91% accounting for colour perception using user surveys (71% analytical sensitivity, 100% specificity; n=4 users). The devices included a no-primer control to ensure accuracy and prevent false positives.

We are taking this design and furthering its diagnostic capabilities to target Influenza A/B, Respiratory Syncytial Virus (RSV), and further validating its use for merging strains such as SARS-CoV-2. We intend to perform this validation by spiking viruses cultured in vitro and determine the diagnostic performance of the device with primer sets targeting the HA gene of Influenza A/B, the junction region between the G and F genes of RSV, and the continued targeting of the junction region between ORF-7A and ORF-7B regions of SARS-CoV-2 previously investigated by the Verma lab.

By changing a single component on our diagnostic platform, the µPADs can be adapted to detect emerging pathogens, making them a valuable tool for sentinel surveillance and public health monitoring. The µPADs are expected to perform similarly in other respiratory pathogens of public health interests, including Influenza A/B and RSV in clinical and contrived samples, as demonstrated using the SARS-CoV-2 virus. These devices offer an inexpensive and accessible option for diagnosing acute respiratory illnesses at the POC. This targeted approach allows for pathogen-specific treatment, improving patient outcomes compared to the current symptom-based management. Furthermore, these µPADs offer an invaluable tool for sentinel surveillance which can serve as an early warning system for pathogen outbreaks and support efforts to track the evolution of respiratory diseases.