Recombinase Polymerase Amplification Combined With the Programmable Nuclease Pf Ago for Rapid Detection of Perkinsus olseni in Shellfish

Perkinsus olseni is a major pathogenic parasite that threatens shellfish aquaculture and can trigger mass mortality and considerable economic losses. Conventional culture-based assays and PCR-based detection protocols for P. olseni recommended by the World Organization for Animal Health (OIE) involve long assay durations and heavy dependence on sophisticated instruments, making them unsuitable for on-site rapid screening. To establish a simple, rapid and highly sensitive detection method for early warning of P. olseni at aquaculture sites, this study integrated isothermal recombinase polymerase amplification (RPA) with the specific-recognition capacity of PfAgo nuclease to construct an RPA-PfAgo detection assay. The assay achieved favourable specificity with a limit of detection as low as 30 copies. Its practical applicability was validated using 55 real-world shellfish samples. Performing at constant temperatures, the RPA-PfAgo detection assay features a simple procedure and short turnaround time, effectively addressing the drawbacks of conventional detection approaches. This assay is suitable for shellfish seed quarantine and routine pathogen surveillance in shellfish farms, and provides solid support for the early prevention and control of perkinsosis caused by P. olseni.