Lim Shaun W, Lance Shea T, Stedman Kenneth M, Abate Adam R
Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA, 94158, USA.
Biology Department and Center for Life in Extreme Environments, Biology Department, Portland State University, Portland, OR, USA.
J Virol Methods. 2017 Apr;242:14-21. doi: 10.1016/j.jviromet.2016.12.009. Epub 2016 Dec 29.
Characterizing virus-host relationships is critical for understanding the impact of a virus on an ecosystem, but is challenging with existing techniques, particularly for uncultivable species. We present a general, cultivation-free approach for identifying phage-associated bacterial cells. Using PCR-activated cell sorting, we interrogate millions of individual bacteria for the presence of specific phage nucleic acids. If the nucleic acids are present, the bacteria are recovered via sorting and their genomes analyzed. This allows targeted recovery of all possible host species in a diverse population associated with a specific phage, and can be easily targeted to identify the hosts of different phages by modifying the PCR primers used for detection. Moreover, this technique allows quantification of free phage particles, as benchmarked against the "gold standard" of virus enumeration, the plaque assay.
表征病毒与宿主的关系对于理解病毒对生态系统的影响至关重要，但现有技术面临挑战，尤其是对于不可培养的物种。我们提出了一种通用的、无需培养的方法来鉴定与噬菌体相关的细菌细胞。利用PCR激活细胞分选技术，我们对数百万个单个细菌进行检测，以确定特定噬菌体核酸的存在。如果存在核酸，则通过分选回收细菌并分析其基因组。这使得能够有针对性地从与特定噬菌体相关的多样化群体中回收所有可能的宿主物种，并且通过修改用于检测的PCR引物，可以轻松地针对性识别不同噬菌体的宿主。此外，该技术能够对游离噬菌体颗粒进行定量，以病毒计数的“金标准”噬菌斑测定法为基准。
