Department of Veterinary Medicine, National Chiayi University, Chiayi City, Taiwan.
Theriogenology. 2012 Oct 1;78(6):1329-38. doi: 10.1016/j.theriogenology.2012.05.034. Epub 2012 Aug 13.
Because it is difficult to differentiate male and female Columbidae birds (e.g., Columba livia) on the basis of morphology, detection of DNA fragments associated with Chromobox-Helicase-DNA binding genes or female-specific genes have been widely used. The objective was to establish a loop-mediated isothermal amplification system involving the 18S ribosomal RNA gene and a female-specific gene for sex identification of Columba livia birds. Unlike polymerase chain reaction (PCR), random amplification polymorphic DNA-PCR and amplified fragment length polymorphism-PCR, target DNA was amplified under isothermal conditions (the entire process was completed in <60 min). By modulating various parameters involved in amplification, e.g., concentrations of MgSO(4), betaine, Bst polymerase, and deoxynucleotide triphosphates, as well as the relative ratio of outer/inner primers and temperatures, optimal conditions for both targets were established that had equal detection limits (62.5 ng). To simplify sex determination, direct observations of the presence of white precipitate (derived from magnesium pyrophosphates) were used for positive samples, which was compared with the whitish ring which formed in a negative sample after addition of CuSO(4). This approach was a rapid alternative to electrophoresis or turbidimetry. DNA extracted from the blood and feathers of various birds were tested using loop-mediated isothermal amplification; results were consistent with a standard PCR. Thus, the assay was a simple, accurate, fast, and economical alternative suitable for veterinary practice.
由于雄鸽和雌鸽（例如，鸽子）在形态学上难以区分，因此广泛应用检测与 Chromobox-Helicase-DNA 结合基因或雌性特异性基因相关的 DNA 片段来进行鉴定。本研究旨在建立一种涉及 18S 核糖体 RNA 基因和雌性特异性基因的环介导等温扩增系统，用于鉴定鸽子的性别。与聚合酶链反应（PCR）、随机扩增多态性 DNA-PCR 和扩增片段长度多态性-PCR 不同，目标 DNA 是在等温条件下扩增的（整个过程在<60 分钟内完成）。通过调节扩增过程中的各种参数，例如 MgSO(4)、甜菜碱、Bst 聚合酶和脱氧核苷酸三磷酸的浓度，以及外/内引物的相对比例和温度，可以为两个靶标建立最佳条件，其检测限相等（62.5ng）。为了简化性别鉴定，可以直接观察到白色沉淀（来源于焦磷酸镁）的存在来判断阳性样本，与阴性样本在添加 CuSO(4)后形成的白色环进行比较。与电泳或比浊法相比，这种方法更快捷。使用环介导等温扩增法对来自各种鸟类的血液和羽毛提取的 DNA 进行了测试，结果与标准 PCR 一致。因此，该检测方法是一种简单、准确、快速且经济的替代方法，适用于兽医实践。
