Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo, Hokkaido, 060-8589, Japan.
Graduate School of Human Development and Environment, Kobe University, 3-11, Tsurukabuto, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
Anal Sci. 2023 May;39(5):721-728. doi: 10.1007/s44211-023-00289-6. Epub 2023 Mar 1.
Loss of biodiversity is a serious concern, and amphibians are particularly threatened. Most small salamanders in Japan are endangered. Distributional information is fundamental to the conservation of these rare species; however, small salamanders are generally difficult to locate or catch. Environmental DNA analysis is an effective survey method for monitoring such rare species. The conventional polymerase chain reaction (PCR) method, which combines PCR amplification with subsequent electrophoresis, and the real-time PCR method, which uses fluorescent material, are commonly used for this purpose. In this study, a comparison of these two detection methods was conducted using a rare salamander species, Hynobius boulengeri, as a model case. We compared three points: (i) detection sensitivity, (ii) influence of environmental factors related to detection, and (iii) time and financial costs of the two methods. To perform this comparison, we developed a real-time PCR detection assay, conducted field surveys, and compared the time and financial costs of conventional and real-time PCR methods. The comparison showed no statistical difference in the detection sensitivity from field samples, and the effects of environmental factors tended to be similar. In addition, the financial cost was lower for the conventional PCR method while the time cost was lower for the real-time PCR method. Therefore, selecting eDNA detection methods based on objectives, time, and financial costs will promote efficient monitoring and contribute to the conservation of rare species.
生物多样性的丧失是一个严重的问题，而两栖动物尤其受到威胁。日本的大多数小型蝾螈都处于濒危状态。分布信息对于保护这些稀有物种至关重要；然而，小型蝾螈通常很难定位或捕捉。环境 DNA 分析是监测这些稀有物种的有效调查方法。传统的聚合酶链反应 (PCR) 方法，将 PCR 扩增与随后的电泳相结合，以及使用荧光物质的实时 PCR 方法，通常用于此目的。在这项研究中，以稀有的日本山溪鲵 (Hynobius boulengeri) 为模型案例，对这两种检测方法进行了比较。我们比较了三个方面：（i）检测灵敏度，（ii）与检测相关的环境因素的影响，以及（iii）两种方法的时间和财务成本。为了进行这种比较，我们开发了一种实时 PCR 检测方法，进行了实地调查，并比较了传统和实时 PCR 方法的时间和财务成本。比较结果表明，从实地样本中检测到的灵敏度没有统计学上的差异，并且环境因素的影响趋于相似。此外，常规 PCR 方法的财务成本较低，而实时 PCR 方法的时间成本较低。因此，根据目标、时间和财务成本选择 eDNA 检测方法将促进有效的监测，并有助于稀有物种的保护。
