Peering below the diffraction limit: robust and specific sorting of viruses with flow cytometry
Year: 2016
Type: article
Abstract: BACKGROUND: Viruses are incredibly diverse organisms and impact all forms of life on Earth; however, individual virions are challenging to study due to their small size and mass, precluding almost all direct imaging or molecular analysis. Moreover, like microbes, the overwhelming majority of viruses cannot be cultured, impeding isolation, replication, and study of interesting new species. Here, we introduce PCR-activated virus sorting, a method to isolate specific viruses from a heterogeneous population. Specific sorting opens new avenues in the study of uncultivable viruses, including recovering the full genomes of viruses based on genetic fragments in metagenomes, or identifying the hosts of viruses. METHODS: PAVS enables specific sorting of viruses with flow cytometry. A sample containing a virus population is processed through a microfluidic device to encapsulate it into droplets, such that the droplets contain different viruses from the sample. TaqMan PCR reagents are also included targeting specific virus species such that, upon thermal cycling, droplets containing the species become fluorescent. The target viruses are then recovered via droplet sorting. The recovered virus genomes can then be analyzed with qPCR and next generation sequencing. RESULTS AND CONCLUSIONS: We describe the PAVS workflow and demonstrate its specificity for identifying target viruses in a heterogeneous population. In addition, we demonstrate recovery of the target viruses via droplet sorting and analysis of their nucleic acids with qPCR.
Source: Virology Journal
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $3,290
Funders: National Science Foundation, Portland State University, National Institutes of Health, Defense Advanced Research Projects Agency, Advanced Research Projects Agency, National Human Genome Research Institute, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of Biomedical Imaging and Bioengineering
Awards: G2021490073, G2993362680, Gigapixel digital PCR in Giant Unilamellar Vesicles, G3637141366, G4208297642, CAREER: Massively-parallel single-cell genomics and transcriptomics with droplet microfluidics, G549111027, Microfluidic immunoprofiling for biomarker discovery in rheumatoid arthritis, G5931632516, G6671297155, Extreme Virus Morphology: The Structure and Assembly of SSV1, the Prototypical Fusellovirus, G7152168685, Accurate, long-read sequencing with droplet-microfluidic barcoding, G9000068819, G9000236248
OpenAlex ID: W2557260101