Fu Q, Zhang M, Qin W S, Lu Y Q, Zheng H Y, Meng B, Lu S S, Lu K H
Animal Reproduction Institute, Guangxi Key Laboratory for Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530005, China.
Theriogenology. 2007 Dec;68(9):1211-8. doi: 10.1016/j.theriogenology.2007.07.007. Epub 2007 Oct 24.
The polymerase chain reaction (PCR) is an efficient method for sexing embryos. The objective of this study was to develop an accurate and reliable method for sexing swamp buffalo (Bubalus bubalis) embryos. The SRY gene from swamp buffalo genomic DNA was amplified by PCR, using primers based on the sequence of the Holstein SRY gene. This fragment was sequenced based on a BLAST search; the SRY gene was highly conserved. Using a Southern blot, there was a strong signal in genomic DNA only from male swamp buffalo. Two pairs of nested primers, targeted to amplify the swamp buffalo SRY conserved region, were designed for sex identification. Simultaneously, the G3PDH gene was co-amplified to serve as an internal control. A multiplex-nested PCR system was optimized by varying the following individually: concentrations of Mg(2+) and dNTPs, ratio of concentrations of primers and numbers of cycles. Biopsies of 27 IVF-derived embryos and 24 embryos fertilized with Y-chromosome-bearing sperm were examined. Using optimized procedures, clear signals following PCR amplification were obtained from all embryo samples; PCR amplification accuracy was further verified by comparing PCR and dot blots. We concluded that this PCR technique was highly reliable for sexing swamp buffalo embryos.
聚合酶链反应（PCR）是一种鉴定胚胎性别的有效方法。本研究的目的是开发一种准确可靠的沼泽水牛（Bubalus bubalis）胚胎性别鉴定方法。基于荷斯坦牛SRY基因序列设计引物，通过PCR扩增沼泽水牛基因组DNA中的SRY基因。根据BLAST搜索对该片段进行测序；SRY基因高度保守。通过Southern印迹法，仅在雄性沼泽水牛的基因组DNA中检测到强信号。设计了两对靶向扩增沼泽水牛SRY保守区的巢式引物用于性别鉴定。同时，共同扩增G3PDH基因作为内对照。通过分别改变Mg(2+)和dNTPs的浓度、引物浓度比和循环次数，优化了多重巢式PCR系统。对27个体外受精胚胎和24个用携带Y染色体精子受精的胚胎进行活检。采用优化程序，所有胚胎样本经PCR扩增后均获得清晰信号；通过比较PCR和斑点印迹进一步验证了PCR扩增的准确性。我们得出结论，这种PCR技术在鉴定沼泽水牛胚胎性别方面高度可靠。
