Codeposition of dNTPs detection for rapid LAMP-based sexing of bovine embryos

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method with high specificity and rapidity under isothermal conditions. According to the LAMP method, a rapid and simple detection system was established for bovine embryo sexing. Two sets of primers were designed by targeting the bovine male-specific sequence and bovine common sequence respectively. The reaction condition of the detection system was optimized within 60 min under isothermal conditions of 65 degrees C by detection of the reaction mixture on agarose gel. Especially, the primers F2 and B2 could replace the F3 and B3 as outer primers, making the primer design simpler and the amplification efficiency higher. Additionally, codeposition of dNTPs was firstly performed to detect the reaction products by addition of 1 microl 0.1 mM CuSO(4), the visible ring-shaped deposit was found in the middle of the reaction tube with negative mixture. It could be employed as an alternative method in the detection of the reaction products in place of the time-consuming electrophoresis or the turbidity meter. Furthermore, the embryo sexing system was carried out in the embryo transfer and achieved 98% of efficiency and 99.5% of accuracy.