Data from: DNA extraction method affects the detection of a fungal pathogen in formalin-fixed specimens using qPCR

Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis.博物馆馆藏是获取野生动物种群历史疫病分布信息的不可或缺的资源库。致病性两栖类蛙壶菌（Batrachochytrium dendrobatidis, Bd）是引发全球两栖动物种群衰退的关键病原，对馆藏标本开展Bd检测有助于深化我们对该病原体出现与传播路径的认知。定量聚合酶链式反应（Quantitative PCR, qPCR）可在最小程度干扰两栖类皮肤组织的前提下实现Bd检测，且其检测灵敏度显著高于组织学方法；因此，开发适用于福尔马林固定标本的Bd DNA qPCR检测方法，可为历史时期Bd的出现与流行率研究提供高效可靠的技术路径。当前针对博物馆标本的Bd检测技术，尚未在模拟馆藏典型标本（包括低病原负荷样本）环境的对照样本中开展有效性评估。本研究以感染状态已知的美洲牛蛙（Lithobates catesbeianus）为实验材料，评估了经3年乙醇保存后福尔马林固定标本的qPCR Bd检测效果。本研究的目标包括：对比当前Bd检测最常用的DNA提取方法PrepMan（PM）与Macherey-Nagel FFPE DNA提取试剂盒（MN）的效果；优化基于PM的Bd检测流程；并提出最大化Bd检出率的实操建议。研究结果显示，当福尔马林固定前的Bd负荷较高时，无论采用何种提取方法，检测成功率均可维持在80%~90%的较高水平；但当感染水平较低时，检测成功率会显著下降。在中等感染水平下，MN DNA提取方法可将Bd检出率提升最高达50%。本研究结果表明，针对馆藏中占多数的低病原负荷两栖类标本，现有检测方法可能会低估Bd感染个体的实际比例。从馆藏博物馆标本中提取DNA的研究人员，应确保所采用的技术能够获得满足后续分析需求的高质量、高通量DNA样本。