Sex Identification of a Multispecies Carinatae Birds by Chicken EE0.6 Gene Using Real-Time Recombinase-Aid Amplification Assay.

The difficulty in bird sex identification has made molecular sexing an important way to solve this problem. The conventional polymerase chain reaction (PCR) methods are time-consuming and dependent on laboratory equipment. Recombinase-aided amplification (RAA) is a rapid, specific, sensitive, and cost-effective isothermal nucleic acid amplification technique. Hence, a rapid birds sexing method based on real-time RAA targeting the unique conserved sequence 0.6-kb EcoRI fragment (EE0.6) gene of Carinatae birds has been established and showed good specificity at 39°C for 20 min. The limit of detection for the real-time RAA assay was determined to be 10 pg., which is 10 times more sensitive than the conventional PCR assay. For real clinical samples, the real-time RAA assay was successfully determined sex in a subset of nine bird species and was 100% consistent with the conventional PCR assay. Consequently, the present real-time RAA assay proves to be a powerful on-site detection tool that can be used for an efficient and reliable birds sexing for further studies on sex ratio and captive management.