Using Recombinase-Aid Amplification Combined with Pyrococcus furiosus Argonaute for Rapid Sex Identification in Flamingo (Phoenicopteridae)
by
and
Shenluan Tan
1,
Fanwen Zeng
2,
Wanhuan Zhong
3,
Tanzipeng Chen
2,
Xuanjiao Chen
2,
Li Li
1,
Hengxi Wei
1,* and
Shouquan Zhang
1,* 1
State Key Laboratory of Swine and Poultry Breeding Industry, National Engineering Research Center for Breeding Swine Industry, Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, China
2
Guangzhou Zoo & Guangzhou Wildlife Research Center, Guangzhou 510075, China
3
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650201, China
*
Authors to whom correspondence should be addressed.
Animals 2025, 15(1), 7; https://doi.org/10.3390/ani15010007
Submission received: 31 October 2024
/
Revised: 13 December 2024
/
Accepted: 20 December 2024
/
Published: 24 December 2024
(This article belongs to the Section Birds)
Simple Summary
Flamingos (Phoenicopteridae), known for their red feathers, are challenging to sex due to their similar appearance in males and females. While PCR is a reliable method for sex identification, it requires lab equipment and is time-consuming. To overcome these limitations, researchers developed a rapid, accurate system for greater flamingos (Phoenicopterus roseus) using the RAA-PfAgo technique. This method determines sex in less than an hour using fluorescence or blue light detection and can identify 0.6 ng of DNA. Field tests confirmed 100% accuracy compared to PCR. RAA-PfAgo provides a specific, sensitive, and accurate reference method for greater flamingo sexing.
Abstract
Flamingos (Phoenicopteridae) are among the oldest birds worldwide and are loved by people for their bright red feathers. In addition, flamingos are sexually monomorphic birds, and distinguishing between males and females is difficult. The polymerase chain reaction (PCR) is widely used for sex identification. However, the PCR method requires a precise thermal cycler in the laboratory and is time-consuming. Therefore, developing a rapid, sensitive, and accurate method to identify the sex of flamingos is crucial. In this study, we established a sex identification system using a recombinase-aided amplification-Pyrococcus furiosus Argonaute (RAA-PfAgo) technique for greater flamingo (Phoenicopterus roseus). The greater flamingo-RAA-PfAgo system can identify unknown-sex greater flamingos in less than 1 h and can be visualized using a fluorescent detector or blue light. The results showed that optimal RAA-PfAgo conditions could detect 0.6 ng of genomic DNA and effectively differentiate between males and females. Random sample evaluations revealed that the system had a 100% coincidence rate compared with conventional PCR. In conclusion, this study provides a sensitive, specific, and accurate reference method for greater flamingo sexing.
