Detection of microorganisms by fluorescence in situ hybridization using peptide nucleic acid

Fluorescence in situ hybridization (FISH) is a 30-year-old technology that has evolved continuously and is now one of the most well-established molecular biology techniques. Traditionally, DNA probes are used for in situ hybridization. However, synthetic molecules are emerging as very promising alternatives, providing better hybridization performance and making FISH procedures easier and more efficient. In this chapter, we describe a universal FISH protocol, using nucleic acid probes, for the detection of bacteria. This protocol should be easily applied to different microorganisms as a way of identifying in situ relevant microorganisms (including pathogens) and their distribution patterns in different types of samples.This work was financially supported by: the project UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE funded by national funds through FCT/MCTES (PIDDAC); the project POCI-01-0145-FEDER-030431, the project PTDC/DTP-PIC/4562/2014 - POCI-01-0145-FEDER-016678, the project POCI-01-0145-FEDER-029961, and the project POCI-01-0145-FEDER031011 funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; and the project “LEPABE-2-ECO-INNOVATION”—NORTE-01-0145- FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors also thank FCT for the PhD Fellowship SFRH/BD/138883/2018.