Recombinase polymerase amplification combined with lateral flow dipstick assay for rapid visual detection of A.simplex (s. s.) and A.pegreffii in sea foods

Anisakiasis is a food -borne parasitic disease mainly caused by the third stage of Anisakis simplex (s. s.) and Anisakis pegreffii. Traditional methods for detecting of Anisakis involve morphology identification such as visual inspection, enzyme digestion, and molecular methods based on PCR, but they have certain limitations. In this study, the internal transcribed spacer 1 (ITS 1) regions of Anisakis were targeted to develop a visual screening method for detecting A. simplex (s. s.) and A. pegreffii in fish meat based on recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD). Specific primers and probes were designed and optimized for temperature, reaction time, and detection threshold. LFD produced clear visual results that were easily identifiable after a consistent incubation of 10 -20 min at 37 degrees C. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. In addition, the detection limit is up to 9.5 x 10 -4 ng/ mu L, and the detection of the artificially contaminated samples showed that the developed assay can effectively and specifically detect A. simplex (s. s.) and A. pegreffii , which fully meet the market 's requirements for fish food safety supervision.