Freen-van Heeren Julian J
Independent Researcher, Amsterdam, The Netherlands.
BioTech (Basel). 2021 Oct 11;10(4):21. doi: 10.3390/biotech10040021.
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.
目前有许多技术用于研究微生物。这些技术可用于检测、鉴定和表征细菌、真菌和病毒物种。一种适用于高通量分析的技术是基于流式细胞术的荧光原位杂交技术，即流式荧光原位杂交（Flow-FISH）。该技术使用针对DNA或（m）RNA的（荧光标记）探针，例如靶向感兴趣的基因或微生物，并在单细胞水平上提供信息。此外，通过将流式荧光原位杂交与基于抗体的蛋白质检测相结合，可以同时测量感兴趣的蛋白质和遗传物质。另外，根据流式荧光原位杂交检测的类型，还可以进行多重检测，从而同时测量多个基因靶点和/或微生物。这些共同使得例如单细胞基因表达分析或混合培养物中（亚）菌株的鉴定成为可能。流式荧光原位杂交已应用于哺乳动物细胞，但也广泛用于研究各种微生物物种。在此，对使用流式荧光原位杂交技术研究微生物进行综述。具体讨论了（细胞内）病原体的检测、微生物生物学和疾病发病机制的研究以及混合培养物中细菌、真菌和病毒菌株的鉴定，特别关注了爱泼斯坦-巴尔病毒（EBV）、人类免疫缺陷病毒1型（HIV-1）和严重急性呼吸综合征冠状病毒2（SARS-CoV-2）。
