Probes

The oligonucleotide probes used to identify bacteria are usually short DNA molecules, between 15 to 25 nucleotides long. The shorter the probe, the lower the probe can tolerate mismatches. The probes can be labeled with a variety of compounds (chemiluminescence, fluorescence dye, peroxidase, lectin, etc.) and be used in combination with corresponding detection methods. The most common probe labeling involves enzymatically linked reporter molecules like digoxigenin, alkaline phosphatase, or horseradish peroxidase. These probes need an additional step after the hybridization procedure with fluorescent anti-DIG or use tyramid signal amplification (TSA) detection kit. The TSA kit consists of a fluorescent tyramide, which would be radicalized by horseradish peroxidase and then bind intracellularly to arominatic amino acids (tyrosine, phenylalanine, and tryptophan). The signal intensity may be increased 10- to 20-fold by using the TSA kit (Schonhuber, 1997; Juretschko, 1999). For laboratory equipped with fluorescent microscope, the use of fluorescent labeled probe is ideal; it usually produces strong signal and less background. Probes can be designed on different phylogenetic levels, specific for domain, phylum, family, genus, or species.

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