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.

Keywords: EE0.6; birds; real‐time recombinase‐aid amplification; sex identification.

FIGURE 1

Primer sets screening for the basic recombinase‐aided amplification (RAA) assay. The RAA amplifies products using four primer sets were subjected to electrophoresis on a 2% agarose gel, respectively. M: DL 2000 DNA Marker.

FIGURE 2

Specificity test results of the real‐time recombinase‐aided amplification (RAA) assay. The RAA reactions were incubated at 39°C for 20 min. Males: Negative control.

FIGURE 3

Sensitivity evaluation of recombinase‐aided amplification (RAA) and PCR assays. (A) Limit of detection of the conventional PCR assay run with USP1/SUP3 and SINT‐F/SINT‐R primer sets. (B) The results of real‐time RAA with different concentrations (100 ng⁻¹pg) of female Gallus gallus domesticus DNA. The RAA reactions were incubated at 39°C for 20 min. (C) Probit regression analysis of the data collected from the eight real‐time RAA repeats using GraphPad Prism 8.0 software. The limit of detection at 95% probability (10 pg/reaction) is depicted by a red rhomboid. (D) Semi‐logarithmic regression of the data collected from the eight real‐time RAA repeats using GraphPad Prism 8.0 software. The data were represented as the mean.