Ribotyping and automated ribotyping of listeria monocytogenes

Abstract

Ribotyping is a molecular technique that allows identification and typing of bacteria to the strain level. It is based on restriction endonuclease cleavage of total genomic DNA followed by electrophoretic separation, Southern blot transfer, and hybridization of transferred DNA fragments with a radiolabeled ribosomal operon probe. Following autoradiography, only those bands containing a portion of the ribosomal operon are visualized. The number of fragments generated by ribotyping is a reflection of the multiplicity of rRNA operons present in a bacterial species. Automated Ribotyping—AR (RiboPrinter) is a commercially available instrument with a high level of reproducibility and standardization. The RiboPrinter automates all of the steps in the process from cell lysis to data capture and database comparisons. Further, reagent cassettes including the enzymes, enzyme conjugated-hybridization probe, electrophoretic gel, and membrane have been developed to deliver consistent performance. Data capture is accomplished via a CCD camera and the gel patterns obtained stored in a digitized format, making it easier to compare results among laboratories and to exchange data electronically.