Development of a Rapid and Cost-Effective Multiplex PCR Assay for the Simultaneous Identification of Three Commercially Important Sea Squirt Species (Halocynthia spp.)

We developed and validated a rapid, cost-effective multiplex PCR assay targeting mitochondrial cytochrome c oxidase subunit I (COX1) to discriminate three commercially important sea-squirt species, <i>Halocynthia roretzi</i>, <i>H. aurantium</i> and <i>H. hilgendorfi ritteri</i>. Species-specific forward primers were designed from interspecific single-nucleotide polymorphisms within the barcode region and combined with a common reverse primer in a single reaction. Specificity was confirmed in all tested individuals (<i>n</i> = 7 per species) without cross-amplification. Sensitivity tests demonstrated consistent amplification down to 0.1 ng of template DNA, matching or surpassing detection limits reported for other food-authentication markers. Because the entire reaction including DNA extraction can be completed within three hours and requires only basic laboratory equipment, the method is well suited for quality control laboratories, border inspections and routine monitoring of processed products. The COX1 multiplex PCR set proposed here provides a reliable tool to enhance traceability, protect consumer choice, and support regulatory enforcement in the sea-squirt supply chain.