Novel molecular marker for RAA-LFD visual detection of Cynoglossus semilaevis sex

Due to the significant sex dimorphism, Cynoglossus semilaevis has long been a species of research interest in the field of artificial sex manipulation. The existence of pseudo-males both in the natural habitat and aquaculture enterprises also is indicative of the importance for identification of the genetic sex in this species. In the present study, there was elucidation of a novel molecular marker for utilizing the recombinase aided amplification-lateral flow dipstick (RAA-LFD) visual system to identify the genetic sex of C. semilaevis. This 533 bp novel marker is a differential single copy fragment between the Z and W chromosome of C. semilaevis and exists only in the W chromosome. After primer designing and probe labeling, this marker has been utilized in a RAA isothermal amplification system. There were 49 C. semilaevis specimens evaluated for genetic sex identification using both PCR-agarose gel electrophoresis based InDel marker detection and the novel RAA-LFD system. The results from conducting evaluations with the two methods were consistent in all samples. Also, results from sensitivity analysis with use of the RAA-LFD system indicated the detection system was effective and reliable from 10⁸ copy number to 10¹. With use of the RAA reaction, there was only need to utilize a constant temperature of 37 ℃ for specific DNA amplification within 30 min. The combination use of RAA with LFD resulted in more efficient and convenient sex determination with there being a lesser technical threshold.