Tan Shenluan, Zhan Tongtong, Zeng Fanwen, Chen Xuanjiao, Chen Tanzipeng, Li Li, Wei Hengxi, Zhang Shouquan, Zuo Kejing
State Key Laboratory of Swine and Poultry Breeding Industry, National Engineering Research Center for Breeding Swine Industry, Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science South China Agricultural University Guangzhou China.
Beijing Zoo, Beijing Key Laboratory of Captive Wildlife Technologies Beijing China.
Ecol Evol. 2025 Jul 21;15(7):e71780. doi: 10.1002/ece3.71780. eCollection 2025 Jul.
The red-crowned crane (), a Class I protected animal in China, inhabits Northeast Asia, including China, Russia, and Japan. As sex-monomorphic birds, red-crowned cranes cannot be directly distinguished between females and males through observation. Molecular methods are accurate and stable for sex identification in birds and are widely used in zoos and farms. With the development of isothermal techniques, recombinase-aided amplification (RAA) has provided novel insights into bird sexing owing to its low equipment dependence and rapid amplification. Advancements in the Argonaute (Ago) biosensor have facilitated clinical detection. In this study, an innovative sex identification system was developed by integrating RAA and Ago in red-crowned cranes. The RAA-Ago system identified both females and males with remarkable accuracy. Via proper design of primers set, gDNA and probe, the RAA-Ago system can complete visual detection, with detection limits between 0.35 ng/μL and 0.035 ng/μL under optimal conditions. The test samples exhibited strong green fluorescence in females, whereas no fluorescence was observed in males under blue light. The results of RAA-Ago in the field were consistent with those obtained using conventional PCR. This study provides a high degree of rapidity, accuracy, and sensitivity for the sex identification of red-crowned cranes.
丹顶鹤是中国一级保护动物，栖息于东北亚地区，包括中国、俄罗斯和日本。作为性别单态性鸟类，无法通过观察直接区分丹顶鹤的雌雄。分子方法在鸟类性别鉴定中准确且稳定，在动物园和养殖场中广泛应用。随着等温技术的发展，重组酶辅助扩增（RAA）因其对设备依赖性低和扩增快速，为鸟类性别鉴定提供了新的思路。Argonaute（Ago）生物传感器的进步促进了临床检测。在本研究中，通过将RAA和Ago整合，开发了一种用于丹顶鹤的创新性别鉴定系统。该RAA-Ago系统对雌雄的鉴定具有显著的准确性。通过合理设计引物组、基因组DNA（gDNA）和探针，RAA-Ago系统能够完成可视化检测，在最佳条件下检测限介于0.35 ng/μL至0.035 ng/μL之间。测试样本在雌性中呈现强烈的绿色荧光，而在蓝光下雄性未观察到荧光。RAA-Ago在野外的检测结果与传统聚合酶链反应（PCR）的结果一致。本研究为丹顶鹤的性别鉴定提供了高度的快速性、准确性和灵敏度。
