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

ABSTRACT 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.