Microbial Characterization on the RiboPrinter® System

A Laboratory Service offered at Accugenix.

 

 

 

Technical Overview of the RiboPrinter® Method

The RiboPrinter® system is an automated system used at Accugenix. for characterizing bacterial samples. It generates a DNA fingerprint of regions of the ribosomal RNA genes (5S, 16S, 23S and the spacer region including Glu-tRNA) that is unique to the organism at the strain level. This DNA fingerprint is known as a RiboPrint® pattern. The RiboPrinter® system groups together (characterizes) samples whose RiboPrint® patterns fall within a fixed degree of similarity. A customer’s database of patterns is dynamic, as it changes and expands with the addition of each new sample that is processed.

RiboPrint® pattern example:

We will build and maintain a custom library for each customer who submits samples for ribotyping. Each time the customer submits a new sample it will be compared to all of their other previous samples in their custom library and assigned to a RiboGroup.

The RiboPrinter® software will assign a sample to an existing RiboGroup if it determines that the sample has a similarity index of 0.90 or greater to that group. If the sample represents a new strain in the custom library, it will be assigned to its own RiboGroup. The characterization report the customer will receive will include the sample code, the RiboPattern® for the sample and the RiboGroup to which it has been assigned. Upon customer request, Accugenix will perform custom comparisons to a specified subset of samples in their custom library or to all of the samples in their custom library.

The BacRib service includes two different restriction enzyme digests – EcoRI and PvuII – both of which are validated methods. The results will include the patterns from both tests. If two isolates tested with one enzyme result in different RiboPrint® patterns, the isolates can be considered different strains. If two isolates appear to have the same RiboPrint® pattern after testing with one enzyme, it is possible that the two isolates are the same strain. The Accugenix service provides patterns from both restriction enzymes, and comparisons may indicate that they are identical or different.

What are the basic steps involved for each sample submitted for testing on the RiboPrinter®?

  1. Subculture the isolate. A fresh isolate (18-24 hours growth, or for slow growers as soon as there is growth in the 3rd quadrant) is required for the test.
  2. Gram stain the isolate. The Gram stain result (positive or negative) is used to determine the amount of cell material that will be used for the test. Two picks (similar to a toothpick) of the isolate are required for gram positive samples and one pick is required for gram negative samples.
  3. Harvest the cells. A pure colony is necessary.
  4. Heat treatment step. The sample is heat treated to inactivate viable cells.
  5. Lysis and deproteinization step. A lysis agent is added to break open the cells and the cells and then deproteinization cleans up the debris.
  6. Digestion. A restriction enzyme (either EcoRI or PvuII) cuts the DNA into fragments.
  7. Electrophoresis. The fragments are separated by size using gel electrophoresis. These fragments are transferred and immobilized onto a nylon membrane.
  8. DNA Probe hybridization. The membrane is exposed to a series of chemical/enzymatic treatments where the DNA probe for the ribosomal genes is hybridized to the target DNA fragments on the membrane. A chemiluminescent agent is applied to the membrane to cause these fragments to give off light.
  9. RiboPrint® Pattern. The band pattern image is captured by a camera and a RiboPrint® pattern is created and stored by the software.

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