Zhang Z-P, Zhang Y, Liu J-P, Zhang J-T, An Z-X, Quan F-S, Zhang L, Cai X, Pu S W J
Institution of Bio-Engineering, Northwest Sci-Tech University of Agriculture and Forestry, Yang ling, China.
Reprod Domest Anim. 2009 Feb;44(1):116-21. doi: 10.1111/j.1439-0531.2007.01006.x. Epub 2008 Nov 18.
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.
环介导等温扩增技术（LAMP）是一种新型的核酸扩增方法，在等温条件下具有高特异性和快速性。根据LAMP方法，建立了一种用于牛胚胎性别鉴定的快速简便检测系统。分别针对牛雄性特异性序列和牛共同序列设计了两组引物。通过在65℃等温条件下于60分钟内对反应混合物进行琼脂糖凝胶检测，优化了检测系统的反应条件。特别地，引物F2和B2可以替代F3和B3作为外部引物，使引物设计更简单且扩增效率更高。此外，首次通过添加1微升0.1 mM CuSO₄进行dNTPs共沉积以检测反应产物，在阴性混合物的反应管中间发现了可见的环形沉积物。它可以作为一种替代方法来检测反应产物，以取代耗时的电泳或浊度计。此外，胚胎性别鉴定系统在胚胎移植中进行，效率达到98%，准确率达到99.5%。
