香港瘰螈 eDNA 引物和 TaqMan 探针的设计与确认

环境 DNA(eDNA)技术是近年来新兴的一种野外水生生物调查方法。本研究旨在设计一组香 港瘰螈(Paramesotriton hongkongensis)特异的引物和 TaqMan 探针并确立此 eDNA 方法在水样中的实 用性，以期进一步调查其野外种群分布。测定 11 种瘰螈物种的线粒体细胞色素 b(Cyt b)基因序列， 然后使用 Primer Express 3.0 软件设计引物和 TaqMan 探针。通过比对 NCBI 基因库及进行退火温度梯度 测试，验证引物和 TaqMan 探针的特异性。通过测试不同浓度的引物和 TaqMan 探针，优化 qPCR 扩增 效率。然后检测养殖不同数量香港瘰螈的水缸水体中 eDNA，以评估已建立的 qPCR 方法灵敏度。同时， 测定香港瘰螈 eDNA 降解速度，以估计其在水中的持续时间。实验结果显示，本研究所设计的引物和 探针只对香港瘰螈呈阳性扩增，而对同属的其他 10 个物种均呈阴性。优化的 qPCR 效率为 93.9%、最 低检测浓度为10 DNA拷贝数。已建立的qPCR方法能灵敏地检测出在养殖1只香港瘰螈24 h实验水 缸水体的 DNA 拷贝数为(13.56 ± 3.35)/ml 水样。另外，降解实验发现，采用 0.45 μm 孔径滤膜已有 效检测香港瘰螈eDNA并能监测15 d。本研究成功设计并建立了一种能在水环境中检测香港瘰螈是否 存在的 eDNA 技术，以期应用于野外考察。

Environmental DNA (eDNA) detection method is considered as a new survey tool for the wildlife aquatic animals in recent years. To further investigate the Hong Kong newt (Paramesotriton hongkongensis) population distribution in the wild, we thus aimed to design a set of specific primers and TaqMan probe and verify the feasibility of this eDNA method for water samples. The mitochondrial cytochrome b genes of 11 Paramesotriton species were sequenced, and then the primers and TaqMan probe were designed using Primer Express Software 3.0. The primers and TaqMan probe specificity were verified by comparing to the NCBI GenBank and testing with the annealing temperature gradient. The qPCR amplification efficiency was optimized by testing different concentrations of primers and TaqMan probe. Then, eDNA was detected in the water samples in aquarium tanks with different numbers of Hong Kong newt to assess the sensitivity of the established qPCR method. Also, the decay rate of Hong Kong newt eDNA was determined to estimate its lasting time in water. The results showed that the designed primers and TaqMan probe only amplified eDNA of Hong Kong newt but not the other 10 closely related newts (Table 3 and 4). The optimized qPCR achieved the efficiency of 93.9 % and the limit of detection down to 10 DNA copies (Fig. 2 and Table 5). The established qPCR method was sensitive enough to detect 13.56 ± 3.35 DNA copies/ml in the aquarium tank with 1 Hong Kong newt after housing for 24 h (Fig. 3). In addition, the decay rate experiment demonstrated that the use of 0.45 μm pore size filter membrane was effective to detect and monitor the Hong Kong newt eDNA in 15 d (Fig. 6). In this study, an eDNA detection method was successfully designed and established to detect the presence or absence of Hong Kong newt in water environments, which can be potentially utilized in the field inspection.