A universal and accurate real-time PCR assay for psittacine sex determination

The parrot's sex misrepresentation has detrimental consequences for consumers, parrots, and the economy. It hinders breeding efforts, harms parrots' well-being, fuels illegal trade, and pushes them towards extinction. Traditional methods for sexing parrots are often inaccurate, cumbersome, time-consuming, limited to specific species or lack sufficient validation studies. To address the limitations of traditional sexing methods, we developed and fully validated a universal real-time PCR assay with TaqMan probes for accurate sex determination of parrot species commonly subjected to sexing fraud. The assay was highly accurate, specific, and robust, with 100% concordance with the conventional PCR method. It demonstrated universal applicability for all 38 parrot species from 34 genera (n = 1087), achieving 100% accuracy with sensitivity down to 0.031 ng of DNA input. Single-blind testing showed the assay's capability to work with casework and fieldwork situations. The utilization of the CHD gene specific to psittacines in combination with real-time PCR enables simultaneous determination of species and sex, enhancing its utility in safeguarding parrots from fraud and supporting forensic inquiries.