Poulos Bonnie T, John Seth G, Sullivan Matthew B
Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, 85721, USA.
Department of Earth Sciences, University of Southern California, Los Angeles, CA, 90089, USA.
Methods Mol Biol. 2018;1681:49-57. doi: 10.1007/978-1-4939-7343-9_4.
Viruses influence ecosystem dynamics by modulating microbial host population dynamics, evolutionary trajectories and metabolic outputs. While they are ecologically important across diverse ecosystems, viruses are challenging to study due to minimal biomass often obtained when sampling natural communities. Here we describe a technique using chemical flocculation, filtration and resuspension to recover bacteriophages from seawater and other natural waters. The method uses iron to precipitate viruses which are recovered by filtration onto large-pore size membranes and then resuspended using a buffer containing magnesium and a reductant (ascorbic acid or oxalic acid) at slightly acid pH (6-6.5). The recovery of bacteriophages using iron flocculation is efficient (>90%), inexpensive and reliable, resulting in preparations that are amenable to downstream analysis by next generation DNA sequencing, proteomics and, in some cases, can be used to study virus-host interactions.
病毒通过调节微生物宿主种群动态、进化轨迹和代谢输出,影响生态系统动力学。虽然病毒在各种生态系统中具有重要的生态意义,但由于在对自然群落进行采样时通常获得的生物量极少,病毒的研究颇具挑战性。在此,我们描述一种利用化学絮凝、过滤和重悬来从海水及其他天然水体中回收噬菌体的技术。该方法利用铁沉淀病毒,通过过滤将其收集到大孔径膜上,然后在微酸性pH值(6 - 6.5)下,使用含有镁和还原剂(抗坏血酸或草酸)的缓冲液进行重悬。利用铁絮凝回收噬菌体高效(>90%)、成本低廉且可靠,所得到的制剂适合通过下一代DNA测序、蛋白质组学进行下游分析,在某些情况下,还可用于研究病毒 - 宿主相互作用。