Campylobacter jejuni is recognized as the most common species in the genus Campylobacter that causes foodborne diseases globally. Main reservoirs harboring C. jejuni are poultry productions that are associated with the majority of illnesses, making a demand for prompting effective detection methods to understand the transmission route within agri-food systems. The gold standard diagnostic method involves tedious microbiological culture isolation and nucleic acid amplification, specifically polymerase chain reaction (PCR). This analytical workflow is only applicable in laboratory with expensive equipment and experienced technicians, limiting its use in resource-reduced or on-site settings. Therefore, we aimed to integrate paper-based DNA extraction, isothermal amplification and lateral flow detection on a paper-based microfluidic chip that it is portable and easy to use outside laboratory. Recombinase polymerase amplification (RPA) is conducted isothermally and becomes a prevailing alternative to the traditional PCR due to its simplicity in temperature setting and relatively short incubation time (10-20 mins). The endpoint diagnosis is visualized on a lateral flow strip as part of the paper microfluidics. Paper microfluidics was fabricated by wax printing where reaction zones preloaded with amplification reagents were confined within the hydrophilic area. Lysed nucleic acid was extracted by low cost cellulose paper dipstick, and transported onto microfluidic reaction zone without any pipetting. The integrated paper microfluidics enabled performing sample amplification to end-point-detection within 25 mins. Overall, the complete chip demonstrated 100% specificity to C. jejuni including 6 wild strains isolated from our laboratory among 8 other Campylobacter subspecies strains and 11 non-Campylobacter strains. High sensitivity was achieved with the help of lateral flow strip to reduce the detection limit to 100 CFU/mL in Campylobacter culture, 10-fold improvement than visualizing on agarose gel electrophoresis. In addition, the device was capable of detecting C. jejuni spiked at a concentration of 25 CFU/mL on chicken breast meat after enrichment for 12 hrs. Our results suggested the utility of paper-based microfluidic chip on food sample diagnosis 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.

Detection of Campylobacter jejuni in agri-food products using a paper-based microfluidic device based on recombinase polymerase amplification and lateral flow immunoassay

ACS Spring 2022

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