Isothermal amplification techniques offer an alternative to “classical” PCR and are more suitable to point-of-use technology. For a general overview, see FAN’s analytical method explainers. Recent advances have enabled the development of microfluidic chip platforms, which integrate micro-scale channels, pumps, chambers, valves, and sensors onto a single substrate for fluidic control. This integration enables simultaneous sample pretreatment, component separation, detection, and biochemical analysis on a single platform.
Recombinase polymerase amplification (RPA) is an isothermal method that has gained attention due to its low instrument dependency, high sensitivity, and rapidity. RPA reactions can be conducted at near-ambient temperatures (37–42 °C) within 20 minutes, and results can be interpreted via fluorescence signals or lateral flow dipstick by incorporating sequence-species probes. The exo probe, typically 46–52 nucleotides in length, is widely used in real-time RPA detection. The design of primers and exo probes in RPA assays offers potential for seamless integration with microfluidic chip platforms.
In this study (purchase required) the researchers integrated RPA into a microfluidic chip to develop an assay for identifying commonly marketed codfish species prone to adulteration: Atlantic cod (G. morhua), sablefish (A. fimbria) and toothfish (D. eleginoides and D. mawsoni).
They reported that the assay demonstrated high specificity and sensitivity, with detection limits of 10 copies/μL recombinant plasmid or 103 fg/μL genomic DNA. Application to 141 commercial seafood products resulted in 100 % identification accuracy for the three target species, and revealed a 32 % inconsistency between product labels and genetic identities, involving substitutions with Pacific cod, pollock, and other species.
They conclude that on-chip RPA assay offers a rapid, high-throughput, and reliable tool for seafood authentication and potential mislabeling surveillance.
Photo by Patrick Boucher on Unsplash
