Abstract
Many techniques are currently in use to study microbes. These can be aimed at detecting, identifying, and characterizing bacterial, fungal, and viral species. One technique that is suitable for high-throughput analysis is flow cytometry-based fluorescence in situ hybridization, or Flow-FISH. This technique employs (fluorescently labeled) probes directed against DNA or (m)RNA, for instance targeting a gene or microorganism of interest and provides information on a single-cell level. Furthermore, by combining Flow-FISH with antibody-based protein detection, proteins of interest can be measured simultaneously with genetic material. Additionally, depending on the type of Flow-FISH assay, Flow-FISH can also be multiplexed, allowing for the simultaneous measurement of multiple gene targets and/or microorganisms. Together, this allows for, e.g., single-cell gene expression analysis or identification of (sub)strains in mixed cultures. Flow-FISH has been used in mammalian cells but has also been extensively employed to study diverse microbial species. Here, the use of Flow-FISH for studying microorganisms is reviewed. Specifically, the detection of (intracellular) pathogens, studying microorganism biology and disease pathogenesis, and identification of bacterial, fungal, and viral strains in mixed cultures is discussed, with a particular focus on the viruses EBV, HIV-1, and SARS-CoV-2.
Highlights
A recent, unreviewed preprint employed telomere Flow-FISH to study SARS-CoV-2, and showed that telomeres are of comparable length in both COVID-19 patients and age-matched controls, indicating that no increased cellular attrition occurs in COVID-19 patients [94]
Due to its single-cell approach, Flow-FISH assays provide more information compared to conventional diagnostic tests
Flow-FISH has even been suggested as a tool for clinical and/or diagnostic applications in human immunodeficiency virus 1 (HIV-1) therapy [20]
Summary
A myriad of techniques is available for the detection, identification, and characterization of bacterial, fungal, and viral species. One technique that is suitable for this purpose in a high-throughput fashion is flow cytometry-based fluorescence in situ hybridization (Flow-FISH) [2,3,4] This technique employs highly specific probes directed against DNA or (m)RNA specific to the transcript or (microorganism) species of interest. By making use of (online) tools (e.g., the Stellaris Probe Designer by Biosearch Technologies), probe set design is straightforward Due to this relatively easy design process, Flow-FISH can be a valuable tool in research settings where no good (fluorescently labeled) antibodies are available for the target of interest (i.e., difficult to stain cytokines such as IL-21 [2]), when no protein product is formed (i.e., noncoding RNAs such as microRNAs [9,10]), or when studying (model) organisms for which the antibody toolbox has not yet been perfected or developed (i.e., fruit-eating bats [11]).
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