Development and Application of an eDNA-Based MIRA-LFD Assay for the Detection of the Chinhai Spiny Newt

The Chinhai spiny newt (Echinotriton chinhaiensis) is a rare amphibian species endemic to China. Its specialized habitat and small size make conventional field surveys and population monitoring not only time-consuming and labor-intensive, but also challenging. To address this challenge, we developed a visual detection method for habitat monitoring by integrating environmental DNA (eDNA) analysis with multi-enzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) technologies. A specific mitochondrial gene fragment was selected as a target. MIRA primers and probes were designed and optimized through comparative sequence analysis with sympatric species, including crucian carp, bullfrogs, and polar fire shrimp. The assay conditions were subsequently refined by optimizing reaction temperature and test strip development time. Results indicate that using aquatic eDNA as a template, the optimized MIRA system incubated at 37 °C for 30 min, followed by a 20 min LFD development, produces clear and interpretable results. The assay demonstrated a DNA detection limit of 5.1 × 10−4 ng/μL, corresponding to a sensitivity higher than that of conventional PCR. The complete MIRA-LFD procedure can be performed within 50 min, and its identification results are consistent with those from high-throughput sequencing. This established method enables specific, efficient, and rapid detection of the Chinhai spiny newt in aquatic environments. It provides a practical technical tool for habitat surveys of this species, improving efficiency while reducing labor and resource demands. Furthermore, this approach offers a potential reference for habitat studies of other rare animal species.