Performing ecological surveys for secretive, fossorial snakes is challenging. Traditional survey methods involve visual observation under artificial cover objects (ACOs); this is labor-intensive and requires multiple consistent surveys of suitable habitats. Detection of snake DNA deposited under ACOs represents an innovative method for species detection. However, for terrestrial species, common issues with soil-based methods include the challenges of adequately removing enzyme inhibitors that reduce environmental DNA (eDNA) detection and potential photodegradation of DNA taken from surface samples. These issues may be circumvented by obtaining swabs and soil samples directly from the underside of ACOs for eDNA analysis. We demonstrate the application of this method in surveys of sharp-tailed snake (Contia tenuis), an endangered species under the Canadian Species at Risk Act. We describe the design and validation of a new quantitative real-time polymerase chain reaction (qPCR)-based eDNA eCOTE3 assay with high specificity and sensitivity for sharp-tailed snake. We developed a practical and robust protocol for obtaining eDNA samples by swabbing the underside of ACOs and collecting soil samples under ACOs. Traditional surveys were conducted over two successive years (2018–19) on 220 paired ACOs at 110 sites monitored between 12 and 30 times each. Of the 6,060 ACO visits, only 24 resulted in sharp-tailed snake observations (0.4% success rate) illustrating the considerable difficulty in detecting these snakes. During this same time, 109 swabs were taken directly from the undersides of ACOs and 78 soil samples were collected from a subset of these ACOs. Of the 24 occurrences where sharp-tailed snakes were visually observed, 13 of 23 ACO swabs (57%) and nine of 20 soil samples (45%) tested positive for DNA. eDNA deposition is likely low because of the small size and behavior of this cryptic species, yet DNA was detected from soil exposed to captured snakes for only 10 min. Nevertheless, sharp-tailed snake eDNA was detected at eight sites (9%) from ACO swabs (n = 86) and seven sites (13%) from soil samples (n = 56) where snakes were not observed. This is an overall detection rate of 25% (14/56) for swab and soil samples testing positive in sites where both were tested, representing a substantial reduction in the effort required for detection of this species. Given the time-consuming nature of traditional surveys, eDNA holds great promise as a complementary survey tool for this terrestrial species. While further work is needed to delineate DNA deposition rates, this work represents a significant advance in monitoring a challenging species.

对隐秘的化石蛇进行生态调查具有挑战性。传统的调查方法涉及在人工覆盖物体（ACO）下进行目视观察；这是劳动密集型的，需要对合适的栖息地进行多次一致的调查。检测 ACO 下沉积的蛇 DNA 代表了一种创新的物种检测方法。然而，对于陆生物种，基于土壤的方法的常见问题包括充分去除酶抑制剂的挑战，这些酶抑制剂会减少环境 DNA (eDNA) 检测和从表面样本中提取的 DNA 的潜在光降解。通过直接从 ACO 底部获取拭子和土壤样本进行 eDNA 分析，可以避免这些问题。我们展示了这种方法在尖尾蛇（Contia tenuis）调查中的应用)，属于《加拿大濒危物种法》规定的濒危物种。我们描述了一种新的基于定量实时聚合酶链反应 (qPCR) 的 eDNA eCOTE3 检测的设计和验证，该检测对尖尾蛇具有高特异性和敏感性。我们开发了一种实用且强大的协议，用于通过擦拭 ACO 的底部并在 ACO 下收集土壤样本来获取 eDNA 样本。传统调查连续两年（2018-19 年）对 110 个站点的 220 个配对 ACO 进行了 12 到 30 次监测。在 6,060 次 ACO 访问中，只有 24 次观察到尖尾蛇（成功率为 0.4%），说明检测这些蛇相当困难。在同一时间，直接从 ACO 的底部采集了 109 个拭子，并从这些 ACO 的一个子集中收集了 78 个土壤样本。在 24 次目视观察到尖尾蛇的事件中，23 个 ACO 拭子中有 13 个 (57%) 和 20 个土壤样本中有 9 个 (45%) 的 DNA 检测呈阳性。由于这种隐匿物种的体型小和行为，eDNA 沉积可能很低，但从暴露于捕获的蛇的土壤中仅检测了 10 分钟的 DNA。尽管如此，在 ACO 拭子 (n = 86) 的八个地点 (9%) 和未观察到蛇的土壤样本 (n = 56) 的七个地点 (13%) 检测到尖尾蛇 eDNA。对于拭子和土壤样本在测试地点均呈阳性的总体检测率为 25% (14/56)，这表明检测该物种所需的工作量大幅减少。鉴于传统调查的耗时性质，eDNA 有望作为这种陆生物种的补充调查工具。虽然需要进一步的工作来描述 DNA 沉积率，