DEVELOPMENT AND APPLICATION OF REAL-TIME AND LATERAL FLOW DIPSTICK RECOMBINASE POLYMERASE AMPLIFICATION ASSAYS FOR RAPID SPECIES IDENTIFICATION OF PELTEOBAGRUS FULVIDRACO

In order to effectively prevent illegal fishing of wild fishery resources in the Yangtze River, it is crucial to provide technical support for the “ten-year ban on fishing in the Yangtze River” and develop methods that can quickly and accurately identify fish species on site. This study focuses on Pelteobagrus fulvidraco and utilizes isothermal amplification technology, specifically Recombinase Polymerase Amplification (RPA), to develop two rapid on-site detection methods: Real-time fluorescent RPA and lateral flow RPA test strips, which are ideally suited for the swift in-field detection of P. fulvidraco DNA. We developed an RPA assay based on either Real-time fluorescent detection (Real-time RPA assay) or lateral flow dipstick (RPA-LFD assay) for rapid, accurate, and sensitive detection of P. fulvidraco DNA, facilitating effective management of the fishing ban in the Yangtze River basin. Primers and probes for both assays were designed to target conserved regions of the mitochondrial cytochrome c oxidase subunit I (COI) gene in P. fulvidraco. Tests demonstrated that both the Real-time RPA and RPA-LFD assays exhibited good specificity at 37℃. The Real-time RPA assay achieved a detection limit of 10² copies of P. fulvidraco DNA per reaction, with amplification completed within 20min, though it requires a constant temperature amplification instrument. In contrast, the RPA-LFD assay offers a more convenient operation with a shorter amplification time of only 10 minutes for 10² copies of P. fulvidraco DNA. Additionally, both methods were tested on 149samples for known fish species, yielding results consistent with actual species identification. The Real-time fluorescence RPA and lateral flow dipstick RPA methods developed in this study for detecting P. fulvidraco DNA have the advantages of easy operation, intuitive results, high accuracy, and no dependence on laboratory instruments and equipment. They have good prospects for rapid on-site detection applications. In addition, it overcomes the problems of high professional threshold for morphological species identification and difficulty in identifying damaged tissue samples. The detection targets can span the entire life cycle of eggs, fish fry, and adult fish, providing essential technical support for aquatic product supervision and law enforcement.