# Visualization of Sex Identification in Red‐Crowned Crane ( Grus japonensis ) via Recombinase‐Aided Amplification Combined With Pyrococcus furiosus Argonaute Assay

## TL;DR

This paper introduces a new method to identify the sex of red-crowned cranes using a combination of recombinase-aided amplification and a biosensor, allowing for quick and accurate results.

## Contribution

The study introduces a novel RAA-PfAgo system for rapid and visual sex identification in red-crowned cranes.

## Key findings

- The RAA-PfAgo system achieved detection limits between 0.35 ng/μL and 0.035 ng/μL.
- The system showed strong green fluorescence in females and no fluorescence in males under blue light.
- Results matched those of conventional PCR methods.

## Abstract

The red‐crowned crane (
Grus japonensis
), 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
Pyrococcus furiosus
Argonaute (PfAgo) biosensor have facilitated clinical detection. In this study, an innovative sex identification system was developed by integrating RAA and PfAgo in red‐crowned cranes. The RAA‐PfAgo system identified both females and males with remarkable accuracy. Via proper design of primers set, gDNA and probe, the RAA‐PfAgo 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‐PfAgo 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.

This study presents an innovative approach for sex identification in the red‐crowned crane (
Grus japonensis
) by combining recombinase‐aided amplification (RAA) with the
Pyrococcus furiosus
Argonaute (PfAgo) biosensor. The RAA‐PfAgo system enables rapid, accurate, and highly sensitive molecular sexing of these sex‐monomorphic birds, with a key advantage being the visual detection of fluorescence. The system achieves a detection limit as low as 0.35 ng/μL and demonstrates consistent performance with traditional PCR methods.