Fluorescence In Situ Hybridization

Within the last 10 years, fluorescence in situ hybridization has become established in microbiological diagnostics, using labeled DNA probes directed against the bacterial 16S-rRNA [25, 26]. The ongoing focus of microbiological diagnostics on molecular genetics and pathogenomics has helped to introduce FISH technology to both clinical laboratories and research facilities. FISH provides information about the presence, number, morphology, spatial distribution, and species of microorganisms. To date, FISH has yielded important insights into the structure of complex microbiological communities in humans, animals, and the environment. Furthermore, rRNA-based antibiotic resistance mechanisms are detectable by means of specific FISH probes [27]. FISH has been successfully used to identify pathogens in smears or tissue preparations from humans and animals; however, the number of target organisms has to be above the detection level, for microscopic techniques [28, 29]. FISH using peptide nucleic acid probes (PNA-FISH) is a novel diagnostic technique combining FISH with the unique performance of PNA probes to provide rapid and accurate diagnosis of infectious diseases. Both FISH and PNA-FISH are well suited to routine application and enable clinical microbiology laboratories to report information important for patient treatment within a time frame that is unreachable using classic biochemical methods [30]. However, the FISH technique still remains quite laborious, it is restricted to the sensitivity of light microscopy, and is only to a limited extent suitable for laboratory automation. A further drawback of the FISH method is the limited number of probes which can be applied in one hybridization experiment. This limitation becomes a distinct bottleneck when this technique is used in clinical diagnostics. In addition, there is a need for multiple probes to check for false-positive and false-negative results caused by individual probes used for identification of the selected target organism. Thus, FISH is yet not appropriate for high-throughput applications or the simultaneous detection of a multitude of bacteria.

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