Ribotyping

Ribotyping is a molecular technique for bacterial identification and characterization that uses information from rRNA-based phylogenetic analyses.^[1] It is a rapid and specific method widely used in clinical diagnostics and analysis of microbial communities in food, water, and beverages.^[1]

All bacteria have ribosomal genes, but the exact sequence is unique to each species, serving as a genetic fingerprint. Therefore, sequencing the particular 16S gene and comparing it to a database would yield identification of the particular species.^[2]

Technique

Ribotyping involves the digestion of bacterial genomic DNA with specific restriction enzymes. Each restriction enzyme cuts DNA at a specific nucleotide sequence, resulting in fragments of different lengths.^[3]

Those fragments are then run on a Gel electrophoresis, where they are separated according to size: the application of electrical field to the gel in which they are suspended causes the movement of DNA fragments (all negatively charged due to the presence of phosphate groups) through a matrix towards the positively charged end of the field. Small fragments move more easily and rapidly through the matrix, reaching a bigger distance from the starting position than larger fragments.

Following the separation in the gel matrix, the DNA fragments are moved onto nylon membranes and hybridized with a labelled 16S or 23S rRNA probe. This way only the fragments coding for such rRNA are visualised and can be analyzed.^[4] The pattern is then digitized and used to identify the origin of the DNA by a comparison with reference organisms in a computer database.^[1]

Conceptually, ribotyping is similar to probing restriction fragments of chromosomal DNA with cloned probes (randomly cloned probes or probes derived from a specific coding sequence such as that of a virulence factor).^[5]

See also

• Genotyping

References