INIAV - National Institute for Agrarian and Veterinarian Research, Rua dos Lagidos, Lugar da Madalena, Vairão, Vila do Conde, Portugal.
LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal.
Methods Mol Biol. 2021;2246:1-15. doi: 10.1007/978-1-0716-1115-9_1.
Fluorescence in situ hybridization (FISH) is a molecular biology technique that enables the localization, quantification, and identification of microorganisms in a sample. This technique has found applications in several areas, most notably the environmental, for quantification and diversity assessment of microorganisms and, the clinical, for the rapid diagnostics of infectious agents. The FISH method is based on the hybridization of a fluorescently labeled nucleic acid probe with a complementary sequence that is present inside the microbial cell, typically in the form of ribosomal RNA (rRNA). In fact, an hybridized cell is typically only detectable because a large number of multiple fluorescent particles (as many as the number of target sequences available) are present inside the cell. Here, we will review the major steps involved in a standard FISH protocol, namely, fixation/permeabilization, hybridization, washing, and visualization/detection. For each step, the major variables/parameters are identified and, subsequently, their impact on the overall hybridization performance is assessed in detail.
荧光原位杂交（FISH）是一种分子生物学技术，可实现样品中微生物的定位、定量和鉴定。该技术已在多个领域得到应用，尤其是在环境领域，用于定量和评估微生物的多样性，以及在临床领域，用于快速诊断传染病原体。FISH 方法基于荧光标记的核酸探针与微生物细胞内存在的互补序列（通常以核糖体 RNA（rRNA）的形式存在）的杂交。实际上，杂交细胞通常仅可检测到，因为在细胞内存在大量多个荧光颗粒（与可用的靶序列数量一样多）。在这里，我们将回顾标准 FISH 方案涉及的主要步骤，即固定/渗透、杂交、洗涤和可视化/检测。对于每个步骤，确定主要变量/参数，然后详细评估它们对整体杂交性能的影响。
