ABSTRACT: Accurate detection of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) is critical for wildlife disease research; however, false negatives in detection do occur. Here we compared different DNA extraction methods to determine the threshold for Bd detection and identify an optimal extraction method to improve detection and quantification of the pathogen. We extracted both lab-created cell suspension standards using PrepMan Ultra, Chelex resin, and 3 spin column DNA extraction kits (Qiagen DNeasy Blood and Tissue, Zymo Quick DNA miniprep, and IBI gMAX mini kit), and further compared extraction methods using field-collected samples. We found that when extracting Bd DNA from cells in lab-created culture, the spin column extraction methods and PrepMan Ultra were equivalent, while the resin method detected higher Bd DNA quantities, especially at higher loads. However, when swabs from live animals were analyzed, low Bd quantities were more than twice as likely to be detected using a spin column extraction than with the PrepMan Ultra extraction method. All tested spin column extraction methods performed similarly across both field and lab samples. Samples containing low Bd quantities yielded inconsistent detection and quantification of Bd DNA copies regardless of extraction method. To manage imperfect detection of Bd, we suggest that presence/absence analyses are more informative than attempting to quantify Bd DNA when quantities are low. Overall, we recommend that a cost-benefit analysis of target species susceptibility and epidemiology be taken into consideration when designing an experiment to determine the most appropriate DNA extraction method to be used, because sometimes detecting low Bd quantities is imperative to the study, whereas in other situations, detecting low DNA quantities is less important.

中文翻译：

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优化的拭子样品的巴氏梭菌树状DNA提取会导致检测不完善，尤其是在感染强度较低时。

摘要：准确检测两栖类真菌病原菌Badchochochytrium dendrobatidis（Bd）对于野生动物疾病研究至关重要。但是，在检测中确实会出现假阴性。在这里，我们比较了不同的DNA提取方法，以确定Bd检测的阈值，并确定了一种最佳提取方法，以改善对病原体的检测和定量。我们使用PrepMan Ultra，Chelex树脂和3个旋转柱DNA提取试剂盒（Qiagen DNeasy血液和组织，Zymo Quick DNA miniprep和IBI gMAX mini试剂盒）提取了实验室创建的细胞悬浮液标准品，并进一步比较了使用现场收集样品。我们发现提取Bd时实验室创建的培养物中的细胞DNA，旋转柱提取方法和PrepMan Ultra相当，而树脂法检测到的Bd DNA量更高，尤其是在较高的负载下。但是，对活体拭子进行分析时，使用PrepMan Ultra提取方法检测到的低Bd量是使用旋转柱提取法检测到的可能性的两倍以上。所有测试过的离心柱萃取方法在现场和实验室样品中的表现相似。无论提取方法如何，含有低Bd量的样品都无法对Bd DNA拷贝进行检测和定量。管理不完善的Bd检测，我们建议当数量较少时，存在/不存在分析比尝试量化Bd DNA更具信息性。总体而言，我们建议在设计实验以确定要使用的最合适的DNA提取方法时，应考虑对目标物种的易感性和流行病学进行成本效益分析，因为有时检测低Bd量对研究至关重要。在其他情况下，检测低DNA量的重要性降低。