Paired Capture and FISH Detection of Individual Virions Enable Cell-Free Determination of Infectious Titers

Abstract

Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are “infectious,” thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).

Single-Cell Genotyping of Single-Nucleotide Mutations Using In Situ Allele-Specific Loop-Mediated Isothermal Amplification

“…Fluorescence in situ hybridization (FISH) is an underlying technology to provide genomic and transcriptomic information in the spatial cellular context . Particularly, the capacity of synthesizing a set of fluorophore-labeled nucleic acid probes fuels FISH to visualize RNAs with single-molecular resolution, thus enabling the precise quantification of gene expression in single cells. , However, the FISH principle can hardly resolve minor changes, such as SNMs in target sequences, due to the limited specificity based on hybridization. The coupling with nucleic acid amplification with FISH has dramatically improved the gain for cellular imaging.…”