Application of Loop-Mediated Isothermal Amplification (LAMP) in Sex Identification of Parrots Bred in Egypt

Abstract

Over 400 of the 3800 tropical avian species are endangered or threatened. One of many solutions to conserve animal biodiversity is breeding animals in zoos or private animal farms. Animal breeding programs are difficult to implement in species with sexual monomorphism, such as parrots. Molecular biology methods offer a solution to determine the sex of these species. Therefore, in this study, we aimed to test the performance of PCR and LAMP techniques on sex identification for 21 parrot species belonging to three families, i.e., Psittacidae, Cacatuidae, and Psittaculidae. We established a protocol for DNA isolation from feathers in our laboratory and found optimal conditions for PCR and LAMP. We showed that the LAMP method with the use of the PSI-W primers set, developed by Centeno-Cuadros, functions in 17 previously untested species. Moreover, we found that further improvements are required in universal LAMP primers for the detection of parrot DNA, which are necessary for confirmation of the male sex. The LAMP method also proved to be more sensitive for female sex identification in contrast to the reference PCR test. Therefore, we conclude that LAMP is a suitable method for the routine diagnostic sex identification of parrots.

Keywords: LAMP; PCR; loop-mediated isothermal amplification; noninvasive; parrots; sex determination.

Figure 1

Representative results of gel electrophoresis of LAMP products amplified with the use of PSI-W primers (A), UCE primers (B), and CIC-Z primers (C). Amplification products were separated on 2.5% agarose gel. (A) Lane 1—100 bp DNA Ladder (Transgen Biotech, Beijing, China), Lane 2—Amazona aestiva, Lane 3—Amazona farinosa; (B) Lane 1—100 bp DNA Ladder (Transgen Biotech), Lane 2—Amazona amazonica, Lane 3—Psittacula krameri; (C) Lane 1—100 bp DNA Ladder (Transgen Biotech), Lane 2—Poicephalus senegalus, Lane 3—Poicephalus meyeri. Each panel includes NTC lane which is a representative image of No Template Control LAMP reaction with specific primers.

Figure 2

Results for LAMP amplification with parrot DNA for (A) females and (B) males. DNA samples positive in PCR were subjected to LAMP analysis with primer sets PSI-W, UCE, and CIC-Z. Parrot species and samples numbers are shown on the y-axis. Names of method (PCR) or primer set (PSI-W, UCE, CIC-Z) are shown on the x-axis. Each rectangle shows result of amplification, and the colours correspond to the reaction outcome: blue—positive, red—negative, white—reaction was not performed for this sample.

Figure 3

Results for LAMP amplification in samples with negative PCR results. DNA samples negative in PCR were subjected to LAMP analysis with primer sets PSI-W, UCE, and CIC-Z. Parrot species and samples numbers are shown on the y-axis. Names of method (PCR) or primer set (PSI-W, UCE, CIC-Z) are shown on the x-axis. Each rectangle shows results of amplification and the colours correspond to the reaction outcome: blue—positive, red—negative, white—reaction was not performed for this sample.