Development of a Rapid Sex Identification Method for Newborn Pigeons Using Recombinase Polymerase Amplification and a Lateral-Flow Dipstick on Farm

SIMPLE SUMMARY: The sex of a bird is important for aviculture, scientific research, and conservation. Sex identification is usually not easy, even if the bird’s appearances and sex organs are examined more closely. In monomorphic birds—or most birds during young, molecular sexing—there is a requirement for a fast and accurate identification method. We have designed a pair of DNA primers that is unique to the W chromosome of pigeon, which was unique to the female; further, RPA and LFD are combined for the purposes of a portable field detection for a sex identification method for birds (i.e., pigeons). The minimal-equipped on-farm approach was tested on pigeon sexing and the results have been 100% correct, so far. The concept of this study could spread to any kind of bird to meet the needs and achieve the goals of bird studies and businesses. ABSTRACT: According to pigeon racing rules in Taiwan, the pigeon raiser must decide which juveniles will be chosen as soon as possible. Differentiating the sex of young pigeons based on appearances, and other traditional methods, can be time-consuming and require several pieces of equipment. Recombinase polymerase amplification (RPA) combined with a lateral-flow dipstick (LFD) could further simplify the presentation of amplification results. A designed reverse primer and probe were labeled with biotin and FAM (fluorescein), respectively, to serve as ligands in the LFD. With the addition of a designed forward primer, the RPA-LFD can be used to perform sex identification of pigeon DNA. The optimal conditions were determined to require at least 6.3 pg of the DNA template, a temperature of 37 °C, and a reaction time of at least 20 min. Under these conditions, the test band area on the strip appeared as a dark color if the sample contained female template DNA, whereas the male DNA samples did not produce any test signal in any of the conditions. The results of random samples using RPA-LFD under the optimal conditions agreed with the results of the same samples determined by PCR-agarose gel electrophoresis. The approach in this study represents a rapid and accurate method for pigeon sexing.