M)RNA, as an example focusing on a gene or microorganism of interest and offers facts on the single-cell level. Additionally, by combining Flow-FISH with antibody-based protein detection, proteins of interest is often measured simultaneously with genetic materials. Additionally, dependant upon the sort of Flow-FISH assay, Flow-FISH also can be multiplexed, making it possible for to the simultaneous measurement of multiple gene targets and/or microorganisms. Collectively, this permits 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 examine varied microbial species. Here, the use of Flow-FISH for studying microorganisms is reviewed. Especially, the detection of (intracellular) pathogens, learning microorganism biology and disease pathogenesis, and identification of bacterial, fungal, and viral strains in mixed cultures is discussed, that has a individual focus on the viruses EBV, HIV-1, and SARS-CoV-2. Keywords and phrases: bacteria; DNA; Flow-FISH; fungi; protein; RNA; viruses; flow cytometryCitation: Freen-van Heeren, J.J. Flow-FISH being a Device for Learning Bacteria, Fungi and Viruses. BioTech 2021, ten, 21. https://doi.org/ ten.3390/biotech10040021 Academic Editor: Paolo Iadarola Obtained: 13 August 2021 Accepted: eight October 2021 FeTPPS custom synthesis Published: eleven October1. Introduction A myriad of techniques is accessible to the detection, identification, and characterization of bacterial, fungal, and viral species. Most usually, these approaches probe the genome of microorganisms, i.e., sequencing of 16S ribosomal RNA, to recognize bacterial species [1]. Even so, these techniques are frequently unable to offer information and facts with regards to the relative abundance, or, in the situation of intracellular microorganisms, the percentage of contaminated cells. Ideally, a single-cell technique is utilized for that detection and characterization of (intracellular) microorganisms. One particular Sulprostone GPCR/G Protein strategy that is certainly suitable for this objective within a high-throughput fashion is flow cytometry-based fluorescence in situ hybridization (Flow-FISH) [2]. This technique employs hugely unique probes directed against DNA or (m)RNA unique to your transcript or (microorganism) species of curiosity. These probes can be right labeled using a fluorophore but are sometimes also visualized via sequential binding measures with, e.g., biotin and streptavidin. Flow-FISH may also be multiplexed, enabling for measuring quite a few RNA species [5]. On top of that, Flow-FISH assays may also be combined with fluorescently labeled antibodies [6]. When focusing on mRNA, this permits to the concomitant measurement of mRNA and protein from the identical gene [3,5,6]. Of note, Flow-FISH has lately even been employed to the cell sorting of dwell bacteria [7,8]. By producing use of (online) tools (e.g., the Stellaris Probe Designer by Biosearch Technologies), probe set design is straightforward. As a consequence of this somewhat quick design and style system, Flow-FISH can be quite a valuable tool in research settings wherever no excellent (fluorescently labeled) antibodies are available to the target of interest (i.e., difficult to stain cytokines this kind of as IL-21 [2]), when no protein solution is formed (i.e., noncoding RNAs such as microRNAs [9,10]), or when studying (model) organisms for which the antibody toolbox has not but been perfected or developed (i.e., fruit-eating bats [11]). Furthermore, Flow-FISH assaysPublisher’s Note: MDPI stays neutral with regard to jurisdictional claims i.
