Hale Vanessa L, Tan Chia L, Knight Rob, Amato Katherine R
Department of Biological Sciences, Purdue University, West Lafayette, IN 47906, USA.
San Diego Zoo Institute for Conservation Research, Escondido, CA 92027, USA.
J Microbiol Methods. 2015 Jun;113:16-26. doi: 10.1016/j.mimet.2015.03.021. Epub 2015 Mar 27.
Studies of the gut microbiome have become increasingly common with recent technological advances. Gut microbes play an important role in human and animal health, and gut microbiome analysis holds great potential for evaluating health in wildlife, as microbiota can be assessed from non-invasively collected fecal samples. However, many common fecal preservation protocols (e.g. freezing at -80 °C) are not suitable for field conditions, or have not been tested for long-term (greater than 2 weeks) storage. In this study, we collected fresh fecal samples from captive spider monkeys (Ateles geoffroyi) at the Columbian Park Zoo (Lafayette, IN, USA). The samples were pooled, homogenized, and preserved for up to 8 weeks prior to DNA extraction and sequencing. Preservation methods included: freezing at -20 °C, freezing at -80 °C, immersion in 100% ethanol, application to FTA cards, and immersion in RNAlater. At 0 (fresh), 1, 2, 4, and 8 weeks from fecal collection, DNA was extracted and microbial DNA was amplified and sequenced. DNA concentration, purity, microbial diversity, and microbial composition were compared across all methods and time points. DNA concentration and purity did not correlate with microbial diversity or composition. Microbial composition of frozen and ethanol samples were most similar to fresh samples. FTA card and RNAlater-preserved samples had the least similar microbial composition and abundance compared to fresh samples. Microbial composition and diversity were relatively stable over time within each preservation method. Based on these results, if freezers are not available, we recommend preserving fecal samples in ethanol (for up to 8weeks) prior to microbial extraction and analysis.
随着近期技术的进步，对肠道微生物群的研究变得越来越普遍。肠道微生物在人类和动物健康中发挥着重要作用，并且肠道微生物群分析在评估野生动物健康方面具有巨大潜力，因为可以从非侵入性收集的粪便样本中评估微生物群。然而，许多常见的粪便保存方案（例如在-80°C下冷冻）不适用于野外条件，或者尚未经过长期（超过2周）储存的测试。在本研究中，我们从美国印第安纳州拉斐特市哥伦比亚公园动物园圈养的蜘蛛猴（Ateles geoffroyi）中收集了新鲜粪便样本。在进行DNA提取和测序之前，将样本合并、匀浆并保存长达8周。保存方法包括：在-20°C下冷冻、在-80°C下冷冻、浸入100%乙醇中、应用于FTA卡以及浸入RNAlater中。在粪便收集后的0（新鲜）、1、2、4和8周，提取DNA并对微生物DNA进行扩增和测序。比较了所有方法和时间点的DNA浓度、纯度、微生物多样性和微生物组成。DNA浓度和纯度与微生物多样性或组成无关。冷冻和乙醇样本的微生物组成与新鲜样本最相似。与新鲜样本相比，FTA卡和RNAlater保存的样本的微生物组成和丰度最不相似。在每种保存方法中，微生物组成和多样性随时间相对稳定。基于这些结果，如果没有冷冻设备，我们建议在进行微生物提取和分析之前，将粪便样本保存在乙醇中（长达8周）。
