Lim Shaun W, Tran Tuan M, Abate Adam R
UC Berkeley-UCSF Graduate Program in Bioengineering, University of California San Francisco, San Francisco, California, United States of America.
Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, California, United States of America.
PLoS One. 2015 Jan 28;10(1):e0113549. doi: 10.1371/journal.pone.0113549. eCollection 2015.
Microbial systems often exhibit staggering diversity, making the study of rare, interesting species challenging. For example, metagenomic analyses of mixed-cell populations are often dominated by the sequences of the most abundant organisms, while those of rare microbes are detected only at low levels, if at all. To overcome this, selective cultivation or fluorescence-activated cell sorting (FACS) can be used to enrich for the target species prior to sequence analysis; however, since most microbes cannot be grown in the lab, cultivation strategies often fail, while cell sorting requires techniques to uniquely label the cell type of interest, which is often not possible with uncultivable microbes. Here, we introduce a culture-independent strategy for sorting microbial cells based on genomic content, which we term PCR-activated cell sorting (PACS). This technology, which utilizes the power of droplet-based microfluidics, is similar to FACS in that it uses a fluorescent signal to uniquely identify and sort target species. However, PACS differs importantly from FACS in that the signal is generated by performing PCR assays on the cells in microfluidic droplets, allowing target cells to be identified with high specificity with suitable design of PCR primers and TaqMan probes. The PACS assay is general, requires minimal optimization and, unlike antibody methods, can be developed without access to microbial antigens. Compared to non-specific methods in which cells are sorted based on size, granularity, or the ability to take up dye, PACS enables genetic sequence-specific sorting and recovery of the cell genomes. In addition to sorting microbes, PACS can be applied to eukaryotic cells, viruses, and naked nucleic acids.
微生物系统常常呈现出惊人的多样性，这使得对稀有且有趣的物种进行研究具有挑战性。例如，对混合细胞群体的宏基因组分析通常由最丰富的生物体的序列主导，而稀有微生物的序列即使能被检测到，也只是低水平的。为了克服这一问题，在进行序列分析之前，可以使用选择性培养或荧光激活细胞分选（FACS）来富集目标物种；然而，由于大多数微生物无法在实验室中培养，培养策略往往会失败，而细胞分选需要能够唯一标记感兴趣细胞类型的技术，对于不可培养的微生物来说这通常是不可能的。在此，我们引入一种基于基因组内容对微生物细胞进行分选的非培养策略，我们称之为PCR激活细胞分选（PACS）。这项技术利用基于微滴的微流控技术，与FACS类似，它利用荧光信号来唯一识别和分选目标物种。然而，PACS与FACS的重要区别在于，该信号是通过对微流控液滴中的细胞进行PCR检测产生的，通过合理设计PCR引物和TaqMan探针，能够以高特异性识别目标细胞。PACS检测具有通用性，所需优化极少，而且与抗体方法不同，无需获取微生物抗原即可开发。与基于大小、粒度或摄取染料能力对细胞进行分选的非特异性方法相比，PACS能够实现基因序列特异性的细胞分选和细胞基因组的回收。除了分选微生物外，PACS还可应用于真核细胞、病毒和裸核酸。
