Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification
Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification
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DOI：
10.1016/j.theriogenology.2003.12.007
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发表时间：
2004-09-01
期刊：
影响因子：
2.8
通讯作者：
Minamihashi, A
中科院分区：
文献类型：
--
作者：
Hirayama, H;Kageyama, S;Minamihashi, A
环介导等温扩增（LAMP）是一种新型的DNA扩增方法，其在等温条件下特异性地扩增靶序列。通过反应混合物的浊度检测LAMP的产物，无需电泳。本研究的目的是建立一种基于LAMP技术的牛早期胚胎性别鉴定方法。实验一优化了LAMP技术用于胚胎性别鉴定的DNA提取方法。采用热处理、NaOH处理和蛋白酶K-Tween 20（PK-TW）处理三种方法提取单个卵裂球DNA。提取DNA后，用两种LAMP反应（雄性特异反应和雌雄共同反应）进行性别鉴定。性别鉴定正确率为88.9- 94.4%，不同方法间无差异。在接下来的实验中评估基于LAMP的胚胎性别鉴定的敏感性和准确性。对于1至5个活组织检查细胞，正确确定性别的样本比例为75-100%。最后，检查体内衍生的胚胎以验证基于LAMP的胚胎性别鉴定的有用性，并将其中一些新鲜的性别鉴定的胚胎转移到受体动物中。性别鉴定所需时间<1h。妊娠率为57.4%，出生的所有小牛均为预测性别（12只雄性和21只雌性）。因此，基于LAMP的胚胎性别鉴定方法能够准确地确定性别，适合于田间应用。(c)2004爱思唯尔公司All rights reserved.
Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was < 1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application. (c) 2004 Elsevier Inc. All rights reserved.