We describe the development and testing of qPCR assays to detect four species of amphibians and reptiles of conservation concern in the South Central United States through environmental DNA (eDNA) samples. The target species include the Ringed Salamander (Ambystoma annulatum), Three-toed Amphiuma (Amphiuma tridactylum), Crawfish Frog (Rana areolata), and Chicken Turtle (Deirochelys reticularia). A set of primers and probes amplifying a 64–72 bp target regions were designed for each species from DNA sequence data for either the mitochondrial Cytochrome Oxidase I or Cytochrome B gene. All assays were assessed for target specificity, with no evidence of amplification in closely related or sympatrically-distributed non-target species. In vitro tests indicate that all assays consistently detect focal species down to concentrations of 2 × 10^− 9 pg/µL. We evaluated the utility of qPCR assays on the eDNA samples collected during field surveys across Eastern Oklahoma, focusing on counties with vouchered historical records for the target species. Although detection rates were low for field applications of the assays, positive detection of Ambystoma annulatum, Rana areolata, and Deirochelys reticularia, but not Amphiuma tridactylum, were recorded. These assays can provide a practical tool for a non-invasive, genetic monitoring program that allows for both rapid detection and tracking of native aquatic and semi-aquatic amphibian and reptile species of conservation concern.

我们描述了通过环境DNA（eDNA）样本检测和检测qPCR分析方法的开发和测试，以检测美国中南部地区四种具有保护意义的两栖动物和爬行动物。目标物种包括环状Sal（Ambystoma annulatum），三趾Am（Amphiama tridactylum），小龙虾蛙（Rana areolata）和乌龟鸡（Deirochelys reticularia））。从线粒体细胞色素氧化酶I或细胞色素B基因的DNA序列数据中，为每个物种设计了一组扩增64-72 bp目标区域的引物和探针。评估了所有测定法的靶标特异性，没有在紧密相关或同伴分布的非靶标物种中扩增的证据。体外测试表明，所有测定法都能始终检测到浓度低至2×10 ^{− 9} pg / µL的病原菌。我们评估了在整个俄克拉荷马州野外调查期间收集的eDNA样品上qPCR测定的效用，重点是具有目标物种历史记录的县。尽管在现场检测中检出率很低，但阳性检出环孢菌，记录了林蛙和网纹藻（Deirochelys reticularia），但未记录两栖两栖动物。这些测定法可为非侵入性遗传监测程序提供实用工具，该程序允许快速检测和追踪自然保护方面的本地水生和半水生两栖动物和爬行动物。