King Saud University, College of Pharmacy, Pharmacology Department, Riyadh, Saudia Arabia.
Theriogenology. 2011 Jul 1;76(1):83-7. doi: 10.1016/j.theriogenology.2011.01.020. Epub 2011 Mar 11.
The objective of the experiment of this paper is to develop and improve in the sexing method for preimplantation embryos of water buffalo (Bubalus bubalis) using loop-mediated isothermal amplification (LAMP) reaction. Embryo sexing has been recognized to control effectively the sex of offspring in the embryo transfer industry. A rapid and simple detection system was established by adding ethidium bromide (EB) or 5 μl of CuSO4 (3M) to the product of LAMP reaction. The result of these additions after 2 min was a color change and a precipitate. 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. The in vitro produced buffalo embryos were divided into one to eight pieces using a microblade attached to a micromanipulator. The cell number in each piece was counted before sexing. Sexing of DNA samples extracted from one to five biopsies cells was performed by LAMP. After biopsy, the remaining part of the embryos was used to confirm the sex by polymerase chain reaction (PCR). Fifty buffalo embryos were used and the accuracy of sex prediction was 100% when the blastomeres dissociated from a morula exceeds three. In conclusion, the present procedure without turbidity meter and electrophoresis was reliable and applicable for sexing the water buffalo embryos.
本论文实验的目的是开发和改进利用环介导等温扩增（LAMP）反应对水牛（Bubalus bubalis）胚胎进行性别鉴定的方法。胚胎性别鉴定已被认为可有效控制胚胎移植产业中后代的性别。通过在 LAMP 反应产物中添加溴化乙锭（EB）或 5 μl 3M 的 CuSO4，可以建立一种快速简单的检测系统。添加后 2 分钟，结果会发生颜色变化和沉淀。它可以替代耗时的电泳或浊度计，作为检测反应产物的替代方法。使用微刀片和微操作器将体外生产的水牛胚胎分成一到八块。在进行性别鉴定之前，对每一块的细胞数量进行计数。通过 LAMP 对从一到五个活检细胞中提取的 DNA 样本进行性别鉴定。活检后，使用聚合酶链反应（PCR）对剩余的胚胎部分进行性别确认。使用了 50 个水牛胚胎，当从桑葚胚分离的分裂球超过三个时，预测性别准确率为 100%。总之，本程序无需浊度计和电泳，是一种可靠且适用于水牛胚胎性别鉴定的方法。
