DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid.
DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid.
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DOI：
10.1128/msystems.00619-21
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发表时间：
2021-06-29
期刊：
影响因子：
6.4
通讯作者：
Wiedmann M
中科院分区：
文献类型：
--
作者：
Ganda E;Beck KL;Haiminen N;Silverman JD;Kawas B;Cronk BD;Anderson RR;Goodman LB;Wiedmann M
食品中存在的核酸的非靶向测序可以为食品安全和原产地的检测以及产品篡改和错误标签问题提供信息。将这些技术应用于食品分析可能会揭示有价值的见解，这些见解是通过有针对性的测试根本无法获得的，从而导致将这些技术应用于食品工业的努力。然而，在应用这些方法之前，必须验证在过程的每一步都使用了最合适的方法：收集原始材料，实验室方法，数据分析和解释。这项研究的重点是收集主要材料，在这种情况下，DNA。我们使用牛乳作为模型，以（i）评价市售试剂盒从接种牛乳中提取核酸的能力，（ii）评价用于牛乳的宿主DNA消除方法，以及（iii）开发和评价用于牛乳中宿主DNA消除的基于选择性裂解-单叠氮丙啶（PMA）的方案。我们的研究结果表明，基于磁性的核酸提取方法是最好的牛奶核酸分离。去除宿主DNA仍然是牛奶非靶向测序的一个挑战，这突出了在食品检测中应始终考虑个体基质特征的发现。一些报告的方法引入了对特定类型微生物的偏见，这在食品安全中可能特别成问题，因为检测革兰氏阴性病原体和卫生指标至关重要。需要不断努力开发和验证新的方法，用于在具有来自单个宿主的大量DNA的样品中进行非靶向宏基因组学。重要性跟踪我们食品中存在的细菌群落有可能为食品安全和产品来源提供信息。为此，使用化学方法或市售试剂盒提取样品中存在的全部遗传物质，并使用下一代平台进行测序，以提供微生物组成的快照。因为食物中存在高等生物的遗传物质（例如，牛奶或牛肉中的牛，面粉中的小麦）的细菌含量大约是细菌含量的1 000倍，因此在收集感兴趣的信息方面存在挑战。此外，特定的细菌特征可以使它们更容易或更难检测，从而为这个问题增加了另一层复杂性。在这项研究中，我们展示了使用不同方法检测特定细菌的能力的影响，并强调了确保对每个特定样本使用最合适方法的必要性。
Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis may reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering of primary material, laboratory methods, data analysis, and interpretation. The focus of this study is on gathering the primary material, in this case, DNA. We used bovine milk as a model to (i) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk, (ii) evaluate host DNA depletion methods for use with milk, and (iii) develop and evaluate a selective lysis-propidium monoazide (PMA)-based protocol for host DNA depletion in milk. Our results suggest that magnetically based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting the finding that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety, where the detection of Gram-negative pathogens and hygiene indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host. IMPORTANCE Tracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of the microbial composition. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around 1,000 times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods for the ability to detect specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.