UBC Theses and Dissertations
Detection of campylobacter jejuni using a hybrid paper/polymer-based microfluidic device based on the recombinase polymerase amplification and lateral flow assay Chen, Yunxuan
Campylobacter jejuni is recognized as the most common species in the genus Campylobacter to cause foodborne diseases globally. Main reservoirs harboring C. jejuni are poultry products that are associated with the majority of illnesses, making a demand for effective detection methods for point-of-care diagnostics. The gold standard method involving tedious microbiological culture isolation and temperature-varied polymerase chain reaction (PCR) detection cannot achieve the use either on site or in resource-reduced settings. We aimed to integrate paper-based DNA extraction, isothermal nucleic acid amplification and lateral flow detection on a hybrid paper/polymer-based microfluidic device that it is portable and easy to use outside laboratory. Recombinase polymerase amplification (RPA) was conducted isothermally and benefited from short incubation time (10-20 min). Hybrid paper/polymer-based microfluidic device was fabricated by wax printing on the chromatography paper containing a cellulose paper dipstick for DNA extraction to avoid pipetting, RPA reaction zones and attached with a lateral flow dipstick for endpoint diagnostics. Overall, RPA reaction was completed in 20 min and demonstrated 100% specificity to C. jejuni against 9 other Campylobacter subspecies strains and 11 non-Campylobacter strains. The limit of detection was 46 CFU/mL with DNA extracted on the cellulose paper, 100-fold more sensitive than PCR. The sensitivity was reduced on the integrated hybrid paper/polymer-based microfluidic device, showing the limit of detection of 460 CFU/mL. In addition, the microfluidic device was capable of detecting C. jejuni spiked at concentrations ranging from 10 to 10² CFU/mL on chicken breast meat after enrichment for 5-10 h. For spiking C. jejuni >10³ CFU/mL, the microfluidic device managed to confirm positive results immediately without bacterial enrichment. RPA reagents and primers remained stable on paper at room temperature for 12 h and could be lyophilized on paper for 3 days with consistent sensitivity. For storage time within 25 days, the sensitivity of the microfluidic device was reduced to 10³ CFU/mL. The use of the hybrid paper/polymer-based microfluidic device enabled detection of Campylobacter contamination in food samples with high specificity and sensitivity. It showed the potential as a reliable point-of-care diagnostic platform for in-field conditions owning to its low-cost, portability, and simplicity.
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Attribution-NonCommercial-NoDerivatives 4.0 International