Microbial Characterization at Accugenix using the RiboPrinter®
Automated ribotyping uses restriction enzymes, gel electrophoresis and chemiluminescent probes to generate a DNA fingerprint for each sample. Accugenix uses both EcoRI and PvuII enzymes as the standard to obtain greater discrimination than utilizing only one enzyme. The resulting DNA fingerprints from both restriction enzymes are compared to others in a database and designated to a RiboGroup®. Accugenix will perform custom comparisons for any samples or sets of samples upon request, all at no additional charge to you.
Tracking sources of contamination
If a sterility failure occurs, customers can send samples to Accugenix, and we can assist you in determining the root cause. We will first identify the isolates by 16S rDNA sequencing. If the 16S sequence is different, you can be sure that the isolates are different strains. Further characterization is necessary for those isolates that have identical 16S sequences and can be done using the automated RiboPrinter®.
Another useful application of automated ribotyping is the creation of a map of your microbial environment. Here’s how it works: a client-specific database is created to archive fingerprints of isolates from production facilities. When a contaminant is encountered, you can quickly pinpoint where you have seen that particular strain before. This greatly reduces the time and cost associated with identifying the source of contamination. Likewise for the nutraceutical and probiotics industries, Accugenix will customize a library of your strains of interest to enable you to verify their identity with certainty.
Method of Analysis for Automated Ribotyping-BacRib
| Pure, Isolated Colony | Samples must be 72 hours old or less and contain well isolated pure colonies. Any sample received that is more than 72 hours old will be subcultured upon arrival for fresh growth. Turnaround time for the sample will begin upon successful subculture. |
| Heat Treatment | Samples are harvested placed in buffer and vortexed before being transferred to the Sample Carrier and heat treated. Each sample is heated to inactivate the enzymes that degrade DNA. |
| Cell Lysis and Digestion | The cells are lysed to release the DNA and other cell contents. The DNA is then cut into fragments with restriction enzymes. Our standard characterization test involves cutting each sample with 2 separate enzymes (EcoRI and PvuII) in 2 separate digests. Analysis with 2 enzymes results in greater discriminatory power than analysis with a single enzyme. |
| Electrophoresis | The DNA fragments are separated by size using gel electrophoresis. The fragments are then transferred to a membrane. |
| DNA Probe Hybridization | The DNA probe for the ribosomal genes is hybridized to the fragments and a chemiluminescent agent is applied to cause the fragments to “light up” and show a banding pattern. |
| Report Generation | A report is generated that displays the 2 banding patterns obtained for the sample after cutting the sample with the 2 restriction enzymes, EcoRI and PvuII. |
Result Analysis
The Accugenix Microbial Characterization Report include your sample code, the two banding patterns resulting from digestions of the isolate with EcoRI and PvuII, as well as the RiboGroup® to which the isolate is assigned.
The greatest value of Microbial Characterization lies in the ability to retrospectively compare similar organisms of interest to one another, whether you’re concerned with tracking the source of contamination or verifying a known organism. At no additional cost, Accugenix will perform historical comparisons on your previous samples to provide you with the information you need in a Comparison Report. Simply call our Technical Support group to discuss your needs.
The Microbial Characterization Comparison Report displays the sample information and Riboprint® patterns for each sample requested. The sample at the top of the list is selected as the pattern to which subsequent patterns are compared. A pattern similarity value is calculated for each sample displayed in the Comparison Report. The patterns of both enzyme cuts are included in one Comparison Report. An Accugenix Data Analyst will provide a summary interpretation of the samples’ similarity.
