Real-Time Monitoring on the Chinese Giant Salamander Using RPA-LFD.

The Chinese giant salamander (), listed as an endangered species under "secondary protection" in China, faces significant threats due to ecological deterioration and the expansion of human activity. Extensive field investigations are crucial to ascertain the current status in the wild and to implement effective habitat protection measures to safeguard this species and support its population development. Traditional survey methods often fall short due to the elusive nature of the , presenting challenges that are time-consuming and generally ineffective. To overcome these obstacles, this study developed a real-time monitoring method that uses environmental DNA (eDNA) coupled with recombinase polymerase amplification and lateral flow strip (RPA-LFD). We designed five sets of species-specific primers and probes based on mitochondrial genome sequence alignments of and its close relatives. Our results indicated that four of these primer/probe sets accurately identified distinguishing it from other tested caudata species using both extracted DNA samples and water samples from a tank housing an individual. This method enables the specific detection of genomic DNA at concentrations as low as 0.1 ng/mL within 50 min, without requiring extensive laboratory equipment. Applied in a field survey across four sites in Huangshan City, Anhui Province, where is known to be distributed, the method successfully detected the species at three of the four sites. The development of these primer/probe sets offers a practical tool for field surveying and monitoring, facilitating efforts in population recovery and resource conservation for .