Fluorescence in situ hybridization (FISH) is a well-established technique that allows the detection of microorganisms in diverse types of samples (e.g., clinical, food, environmental samples, and biofilm communities). The FISH probe design is an essential step in this technique. For this, two strategies can be used, the manual form based on multiple sequence alignment to identify conserved regions and programs/software specifically developed for the selection of the sequence of the probe. Additionally, databases/software for the theoretical evaluation of the probes in terms of specificity, sensitivity, and thermodynamic parameters (melting temperature and Gibbs free energy change) are used. The purpose of this chapter is to describe the essential steps and guidelines for the design of FISH probes (e.g., DNA and Nucleic Acid Mimic (NAM) probes), and its theoretical evaluation through the application of diverse bioinformatic tools.
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