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Using Recombinase-Aid Amplification Combined with Pyrococcus furiosus Argonaute for Rapid Sex Identification in
Shenluan Tan1, Fanwen Zeng2, Wanhuan Zhong3
1State 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.
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.
