摘要
【目的】建立LAMP法快速鉴定水牛早期胚胎性别体系,为预知性别水牛胚胎移植奠定基础。【方法】分别采用化学裂解法和非化学裂解法提取水牛早期胚胎DNA,对比不同方法对G3PDH基因扩增效果的影响;基于水牛SRY基因和12S r RNA基因分别设计雄性特异性引物及雌、雄性共用引物用于LAMP法胚胎性别鉴定,同时以PCR对LAMP法鉴定结果进行验证。【结果】化学裂解法对水牛早期胚胎DNA具有良好的裂解效果,其有效检出率为94.1%。优化的LAMP法对水牛早期胚胎性别鉴定的准确率与检出率均为100.0%,检测灵敏度为2×10-5 ng。LAMP扩增产物中加入1%SYBR green I,于紫外光下观察,阳性呈绿色,阴性呈淡黄色;自然光下观察,阳性呈黄色,阴性呈橙色。以LAMP法对37枚水牛IVF早期胚胎性别进行鉴定,其中雄性为28枚,雌性为9枚。PCR验证结果与LAMP法鉴定结果一致。【结论】用化学裂解法提取水牛早期胚胎DNA,再根据水牛SRY基因及12S r RNA基因设计引物进行LAMP法水牛胚胎性别鉴定切实可行。
【Objective】The present experiment was conducted to establish LAMP method for sexing buffalo embryos to provide some references for embryo transfer. 【Method】DNA was extracted from early embryos of buffalo by using chemical lysis and non-thermal lysis methods respectively to compare efficiency of different methods for amplifying G3 PDH gene.Then based on SRY gene and 12 S r RNA gene, the male-specific primer and common primer of male and female were designed to identify embryos sex by using LAMP method. Meanwhile the results of embryo sexing by using LAMP method were verified by using PCR method. 【Result 】The results showed that chemical lysis was the best method for cracking embryonic DNA, and its effective detectable rate was 94.1%. The optimized LAMP method＇s accuracy and detectable rate were all 100.0% in sex identification, and its sensltivity was 2 ×10-5ng. In addition, after adding 1% SYBR green I to amplification products of LAMP, under ultraviolet light, the positive samples were found to be green, and the negative samples were found to be faint yellow; under natural light, the positive samples were found to be yellow, and the negative samples were found to be orange. The sexes of 37 IVF early embryoes were identified, the identification results showed that there were 28 males and 9 females. Furthermore, the identification result of PCR method was consistent with that of LAMP method. 【Conclusion】The DNA of buffalo embryos is extracted using chemical lyses method, and the sex of embryo can be identified effectively by using LAMP method, after designing primers based on SRY gene and 12 S r RNA gene,therefore, which is a workable method for sex identification.
出处
《南方农业学报》
CAS
CSCD
北大核心
2015年第6期1106-1110,共5页
Journal of Southern Agriculture
基金
广西科学研究与技术开发计划项目(桂科攻1123005-3
桂科重14121003-3-2)
