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Peering below the diffraction limit: robust and specific sorting of viruses with flow cytometry
Shea T Lance1,2,3, David J Sukovich1,2, Kenneth M Stedman4
1Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA.
Virology Journal
|December 2, 2016
Summary
Researchers developed PCR-activated virus sorting (PAVS) to isolate specific viruses from complex mixtures. This method allows for the study of uncultivable viruses and recovery of their genetic material.
Area of Science:
- Virology
- Molecular Biology
- Biotechnology
Background:
- Viruses are diverse and crucial to all life, but their small size and inability to be cultured hinder study.
- Studying individual virions is difficult due to limitations in direct imaging and molecular analysis.
- The majority of viruses cannot be cultured, preventing isolation and detailed investigation of novel species.
Purpose of the Study:
- To introduce a novel method for isolating specific viruses from heterogeneous populations.
- To enable the study of previously uncultivable viruses.
- To facilitate the recovery of viral genomes and identification of virus hosts.
Main Methods:
- Developed PCR-activated virus sorting (PAVS) utilizing microfluidics and flow cytometry.
- Encapsulated virus populations into droplets with TaqMan PCR reagents for specific viral detection.
- Utilized thermal cycling to induce fluorescence in droplets containing target viruses, enabling sorting.
Main Results:
- Demonstrated the specificity of the PAVS workflow in identifying target viruses within mixed populations.
- Successfully recovered target viruses through droplet sorting.
- Analyzed the nucleic acids of recovered viruses using quantitative PCR (qPCR).
Conclusions:
- PAVS provides a powerful tool for isolating specific viruses, including uncultivable ones.
- The method allows for the recovery of viral genomes from complex samples.
- PAVS opens new possibilities for viral discovery and host identification.
