多重PCR鉴定水牛胚胎性别的研究

Sex identification of buffalo embryo using multiple-PCR

【目的】建立多重PCR鉴定水牛胚胎性别的技术体系,为下一步优化多重巢式PCR方法奠定基础。【方法】采用热裂解法、蛋白酶K+二硫苏糖醇(DTT)法和吐温-20+DTT法分别提取水牛卵裂球DNA,比较3种方法对G3PDH基因的扩增效果;同时设计1对检测引物和1对内标引物用于胚胎性别鉴定,提取胚胎DNA后进行多重PCR检测。【结果】吐温-20+DTT法具有良好的裂解效果,其有效检出率为97.1%;而经吐温-20+DTT法抽提的DNA用于多重PCR检测,其胚胎鉴定率达到100.0%。【结论】采用吐温-20+DTT法处理水牛胚胎样品后再进行多重PCR性别鉴定可以简化操作步骤、提高鉴定效率,但样品数量不宜太少。

[Objective]The present experiment was conducted to develop a fast and accurate PCR based method for sex identification of buffalo embryos by PCR for providing the basis of optimizing multiple nested PCR method. [Method]Three methods to extract DNA from buffalo blastomere, including thermal lyses, proteinase K＋DTT and DTT＋Tween-20, were used and the isolated DNAs were comparatively analyzed for their amplification efficiency using primers based on G3PDH gene of buffalo. The embryonic DNA was amplified by using pairs of detection primer and an internal primer in multiple-PCR amplification. [Result]The results indicated that DTT＋Tween-20 methods were the best for cracking embryonic DNAs and gave 97.1% detectable rate, and 100% embryo identification rate in sex identification using multiple-PCR amplification. [Conclusion]The embryo sample must be initially treated with DTT＋Tween-20 for the lyses of cells so as to completely extract the DNA followed by amplification using multiple-PCR method. The present method has simplified operating steps and therefore, many samples can be processed in less time compared to the other sex identification methods.