Many human diseases are caused by viruses, including acute diseases like Ebola and influenza, and chronic diseases caused by Epstein-Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and Zika virus. Viruses impact every form of life on earth, from applying evolutionary stresses to enhancing the transfer of genes between organisms. In addition, we demonstrate recovery of the target viruses via droplet sorting and analysis of their nucleic acids with qPCR. We describe the PAVS workflow and demonstrate its specificity for identifying target viruses in a heterogeneous population. The recovered virus genomes can then be analyzed with qPCR and next generation sequencing. The target viruses are then recovered via droplet sorting. TaqMan PCR reagents are also included targeting specific virus species such that, upon thermal cycling, droplets containing the species become fluorescent. 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.
PAVS enables specific sorting of viruses with flow cytometry.
Emulsion pcr and uc berkeley full#
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. Here, we introduce PCR-activated virus sorting, a method to isolate specific viruses from a heterogeneous population.
Moreover, like microbes, the overwhelming majority of viruses cannot be cultured, impeding isolation, replication, and study of interesting new species. 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.
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