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Chapter 1 : Applications of Fluorescence In Situ Hybridization in Diagnostic Microbiology
Category: Clinical Microbiology; Bacterial Pathogenesis
The presence of a sufficient number of target molecules is critical in detecting conferred fluorescence of probe-target hybrids and permits direct visualization of intact organisms by epifluorescence microscopy. This direct approach to visualization of organisms using fluorescence in situ hybridization (FISH) simultaneously provides information on phylogenetic relationships of organisms, their spatial distribution in the sample matrix, their relative abundance, and their relative physiologic activity. An incubator, a water bath, and an epifluorescence microscope are essential, with the microscope being most critical for successful FISH performance. The main goal in designing specific probes is to find a suitable and unique region within the 16S or 23S rRNA that permits discrimination of target from nontarget organisms. Once a probe has been designed and confirmed to be specific for the motif desired by using the appropriate databases, it must be evaluated against a series of reference organisms. In case of a low number of ribosomes, the simultaneous application of two fluorescence-monolabeled probes targeting two different regions of rRNA with the same specificity would theoretically result in a twofold amplification of signal intensity when applied to the test samples. The chapter talks about expanded techniques combined with FISH. FISH, based upon either DNA or PNA oligonucleotide probes, is a rapid diagnostic method capable of challenging traditional culture techniques for the direct and accurate identification not only of nonfastidious pathogens but also of fastidious, slow-growing, and difficult-to-cultivate organisms.
Examples of the application of FISH in clinical microbiology
Commonly used fluorescent dyes and their characteristics
Sequences and characteristics of useful probes for diagnostic microbiology
Selected examples of PNA-FISH application in clinical microbiology