Laboratório de Embriologia Molecular e Transgênese, Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil.
Theriogenology. 2012 Feb;77(3):694-8. doi: 10.1016/j.theriogenology.2011.09.005. Epub 2011 Oct 13.
In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.
在这项研究中，我们使用聚合酶链反应（PCR）对第一 PCR 产物（二次 PCR）进行重扩增，并使用 qPCR 检测方法，从怀孕母马血浆中的循环游离胎儿 DNA（ccffDNA）中检测性别决定区 Y（SRY）基因，以确定胎儿性别。从 20 匹 5-13 岁的纯种母马妊娠的最后 3 个月的血浆中分离出 ccffDNA（在产驹后对胎儿性别进行了验证）。作为对照，我们使用了 2 匹非怀孕母马和 2 匹处女母马的血浆，以及非模板对照。通过 DNA 测序证实了与 SRY-PCR 产物相对应的 182 个核苷酸序列。基于 SRY/PCR，11 只雄性胎儿中的 8 只和 9 只雌性胎儿中的 9 只被正确识别，因此敏感性为 72.7%（对于雄性胎儿），整体准确性为 85%。此外，使用 SRY/2nd-PCR 和 qPCR 技术，敏感性和准确性分别为 90.9%和 95%。总之，本研究显然是首次报道使用 ccffDNA 对母马进行胎儿性别鉴定。
