Section of Food Microbiology and Fermentation, Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Rederiksberg, Denmark.
Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark.
Viruses. 2019 Jul 20;11(7):667. doi: 10.3390/v11070667.
The human gut microbiome (GM) plays an important role in human health and diseases. However, while substantial progress has been made in understanding the role of bacterial inhabitants of the gut, much less is known regarding the viral component of the GM. Bacteriophages (phages) are viruses attacking specific host bacteria and likely play important roles in shaping the GM. Although metagenomic approaches have led to the discoveries of many new viruses, they remain largely uncultured as their hosts have not been identified, which hampers our understanding of their biological roles. Existing protocols for isolation of viromes generally require relatively high input volumes and are generally more focused on extracting nucleic acids of good quality and purity for down-stream analysis, and less on purifying viruses with infective capacity. In this study, we report the development of an efficient protocol requiring low sample input yielding purified viromes containing phages that are still infective, which also are of sufficient purity for genome sequencing. We validated the method through spiking known phages followed by plaque assays, qPCR, and metagenomic sequencing. The protocol should facilitate the process of culturing novel viruses from the gut as well as large scale studies on gut viromes.
人类肠道微生物组（GM）在人类健康和疾病中发挥着重要作用。然而，尽管在理解肠道细菌居民的作用方面已经取得了相当大的进展，但对于 GM 的病毒成分知之甚少。噬菌体（phages）是攻击特定宿主细菌的病毒，可能在塑造 GM 方面发挥重要作用。尽管宏基因组学方法已经发现了许多新的病毒，但由于它们的宿主尚未确定，这些病毒仍然在很大程度上未被培养，这阻碍了我们对其生物学作用的理解。现有的病毒组分离方法通常需要相对较高的输入量，并且通常更侧重于提取高质量和高纯度的核酸进行下游分析，而较少关注具有感染能力的病毒的纯化。在这项研究中，我们报告了一种高效的方法的开发，该方法需要的样本输入量较低，可产生含有仍具有感染性的噬菌体的纯化病毒组，并且其纯度足以进行基因组测序。我们通过噬菌斑测定、qPCR 和宏基因组测序对已知噬菌体进行了验证。该方法应该有助于从肠道中培养新型病毒以及对肠道病毒组进行大规模研究。
