Species Identification of Totoaba (Totoaba macdonaldi) From Swim Bladders by Real‐Time Fluorescence‐Based Recombinase‐Aided Amplification

The increased illegal fishing to supply swim bladders is the current threat to the Totoaba macdonaldi, due to the high commercial value of swim bladders. Species identification is the foundation of the endangered animal protection. Limited by the sophisticated thermal cycling, specialized machine, and sequencing costs, DNA barcoding might not better satisfy the needs of on‐site inspection to the efficiency, convenience, and low cost, although it has reliable results and has been widely used. It is necessary to establish a convenient, rapid, sensitive, and accurate method for customs to inspect the swim bladders in legal or illegal trade on site. In this study, we introduce real‐time fluorescence‐based recombinase‐aided amplification (RF‐RAA) into species identification of swim bladders. Specific primers and probes targeted to Totoaba macdonaldi were designed and the reaction temperature and time were screened. It was applied to the forensic identification of swim bladders confiscated by custom after its sensitivity and specificity were verified. The results showed that (1) the primers of TMcytB F1 and TMcytB R3 targeted to the Cytb had a good amplification at 39°C, 30 cycles (15 min) with good repeatability and stability; (2) the method had good specificity and no cross‐reaction with other 22 swim bladder originated species; (3) the lowest detectable limit (LDL) of this method was 3.7 × 101 copies/μL; (4) about 1.836 kg of all the swim bladders confiscated by Shenzhen customs on 2022 were identified to be Totoaba macdonaldi by using this method. This study provides an effective species identification alternative for competent authorities to tackle the illegal import and market regulation of totoaba swim bladders.