New Bacterial Library (released June 25, 2007)
New Fungal Library (to be released April 28, 2008)
Bacterial Characterization on the RiboPrinter® System
Reports
Samples & Shipping
ID Methods
How do the new Bacterial and Fungal Libraries benefit me?
With each library update, we add many new species to our libraries in order to give more species level identifications for the organisms you send to us. We also update the names of organisms in the database to reflect current taxonomy.
Will the test services you offer or the price for those services change with the release of the new libraries?
No, the tests that we perform will not change and the prices for these tests will not change.
When can I expect to see identifications produced with the latest Bacterial library?
The new Bacterial library will be in use as of June 25, 2007, so any identification result produced on or after this date will be generated using the new library. The date and time (EST) of report generation is displayed beneath the sample name on each report.
When can I expect to see identifications produced with the latest Fungal library?
The new Fungal library will be in use as of April 28, 2008. Any identification result produced on or after this date will be generated using the new library. The date and time (EST) of report generation is displayed beneath the sample name on each report.
How many species have you added to your Bacterial library and why have you added these?
We have added over 600 species relevant to the markets we serve in order
to improve the coverage of the library. Some of these represent isolates
that would have resulted in a "No Match" or "Genus" level identification
when searched against our previous library. Other organisms added are
species that have been named and published since our last Bacterial
Library release in 2006.
Because of our experience in sequencing hundreds of thousands of
isolates, we were able to choose organisms to add that are important
to industry in identifying unknown environmental isolates.
How many species have you added to your Fungal library and why have you added these?
With the latest update, to be released April 28, 2008, we added 200 species relevant to the markets we serve in order to improve the coverage of the library. These represent some of the isolates that would have resulted in a "No Match" or "Genus" level identification when searched against our previous library.
How will the new organisms you have added to the libraries affect my identification results?
In the past, if you had sent us one of these organisms for identification, you would have received either a "Genus level" or a "No Match" identification result. With the addition of these new organisms, there is a greater probability that your identification will be to the "Species" level.
How can I find out if an organism I am interested is included either in your Bacterial library or in your Fungal libary?
Contact your Account Manager or our Technical Support Department and that information will be provided to you.
What do I do if an organism I need to identify is not represented in your libraries?
The sequence data can still be very useful information for you. Please contact our Technical Support Department to explore other ways we can help you gain valuable information about your sample.
I have often sent in a particular isolate that you have given me a Species level result for, and now I have received a different Species level result for the same isolate. How can this happen?
There are a few reasons why this can happen. We have updated the names
many organisms in our library to reflect current taxonomy. Organism names
can change when it is shown by phylogenetic analysis that an organism has
been previously classified incorrectly, probably based on phenotypic
characteristics. The organism is then reclassified with the appropriate
name based on phylogenetic taxonomy, and the name change is published.
We have updated the names of organisms in our library to reflect these
published changes.
You could also receive a different result for an isolate if the isolate
is a newly identified species, but was previously thought to be part of
another existing species.
For any questions, or for assistance in resolving technical issues
resulting from changes in our library, please contact our Technical
Support Department.
When should I use Bacterial Characterization on the RiboPrinter® system?
We suggest submitting samples for ribotyping on the RiboPrinter® system when you have isolates that have been identified as the same species with the same 16S sequence to determine if they are the same strain or not. For example, if you are investigating the source of contamination found in a sterility failure, it could be necessary to determine the strain-relatedness of your isolates.
How does the RiboPrinter® process work?
The RiboPrinter® system is an automated ribotyping system that generates a DNA fingerprint of the Ribosomal RNA genes. This is accomplished by first cutting the bacterial genome with a restriction enzyme and separating the resulting fragments by gel electrophoresis. Then a DNA probe, chemiluminescent agent, and camera are used to visualize the DNA fragments of interest. The resulting RiboPrint® pattern is then compared against the RiboPrint® patterns of other isolates to determine their similarity.
What does the cost of one Bacterial Characterization test include?
The standard Bacterial Characterization test (BacRib test codes) includes subculture of the isolate (if necessary), gram stain (if necessary), and analysis of the isolate with two restriction enzymes (EcoRI and PVUII). The characterization report will include your sample code, the resulting RiboPrint® patterns for the isolate, and the RiboGroup that the isolate is assigned to. Upon request, Accugenix will perform custom comparisons at no additional cost.
Do you need a live organism to perform the Bacterial Characterization test?
Yes, a live organism less than 72 hours old is required for ribotyping.
This is in contrast to identification by 16S sequencing which does not
require a live organism.
Turnaround time for your sample will begin on the day of receipt if the
sample arrives at Accugenix less than 72 hours old. All samples that are
more than 72 hours old upon receipt will require subculture before
testing can begin. Turnaround time for these samples will begin upon
successful subculture.
How is the due date for an ID report
calculated?
Due dates are calculated from the date the culture is received. Only
business days count for determining the due date.
We provide preliminary reports via email or fax. Please indicate your preference on the identification request form. Hard copies of the report are sent via a traceable courier upon completion of the job. You may request overnight delivery of your reports for an additional nominal fee.
If two organisms have the same 16S sequence, are they the same strain?
They could be the same strain, but 16S rRNA gene sequences are highly conserved at the species level and are generally not useful for detecting strain level differentiation. However, if two isolates have different 16S rRna gene sequences, they can be considered to be different strains. When it is important to determine if two isolates with the same 16S sequence are the same strain or not, we recommend submitting the isolates for testing on the RiboPrinter® system.
How should the phylogenetic tree on a report be interpreted?
The phylogenetic tree is a way of visualizing the genetic distance between your isolate and its 10 closest matches. The distance measurement only tells you how your isolate compares to its closest matches, not how all of the matches are related to each other. In order to make an accurate identification, the distance measurement and the phylogenetic tree are taken into account.
How is the phylogenetic tree generated?
The phylogenetic tree generated for the purpose of determining the identity of an isolate is distance based. We use the Neighbor Joining (NJ) Tree on our identification reports. The first step in generating a tree is to pairwise align all of the sequences and calculate the genetic distance for each pair. The resulting data is stored in a distance matrix. Using the data from the distance matrix, the algorithm then determines the tree topology that best represents the pairwise distances between all combinations. The distance along the horizontal lines connecting two sequences is a close approximation of the sequence difference between all pairs.
What is a distance measurement and how is it calculated?
A distance measurement is a comparison of one sequence to another, and then a determination of the percentage of nucleotides that differ between the two sequences. First, the sequences are aligned to minimize the absolute number of differences between the two sequences. Gaps may be introduced into one or both of the sequences in order to achieve the optimal alignment. Next the sequences are compared at every nucleotide position (pair wise comparison) and the percentage difference is calculated.
Is there a single distance measurement cutoff that can be used to determine if an organism is a good species level match?
No. The species cutoff level varies from genus to genus. Sometimes the species that comprise a particular genus are very closely related (i.e., low genetic distance) as with the enterics, but in other genera the species are only distantly related (i.e., high genetic distance). This makes species interpretation using only one genetic distance cutoff impossible. One must first look at the genetic distance of the unknown to its closest match and then determine if that distance is on average equal to or less than the distance that separates the known species of the particular genus.
Can a confidence level for the ID be provided?
We provide a determination as to whether the confidence is a species match, a genus match, a family/order/class match, or a no match to the organisms in our database. There is no confidence interval associated with the reliability of the match. In our opinion, these numbers given by identification systems are rarely meaningful, and often cause a false sense of security of the results. Many systems will report a 99% confidence that the identification is correct - even when a gram-negative test card was used with a gram-positive organism. We feel that there is no substitute for critically examining the data.
A notification that no result was obtained was received. What would cause this? Can the test be repeated?
All samples that result in no sequence data are automatically processed a second time. If the second attempt results in no sequence data, we will send a notification report that we were unable to obtain a result. The most likely causes for failure are PCR inhibitors in the sample, failure to extract genomic DNA, or processing a yeast sample as a bacterial sample, or vice-versa.
What does a confidence level of "Genus", "Species", "Family", or "No match" mean on a report?
Identification reports receive an identification (confidence level) of species, genus, family/order/class (bacterial samples only), or no match. We will assign the highest level of taxonomic information possible for each sample.
A species level identification will be given a Genus and species name, as well as a confidence level of "Species" on the report.
A genus level identification will receive the Genus name of the organism - but a species designation could not be confidently assigned for this organism.
A family/order/class level identification indicates that although the sample could not be assigned to a particular genus, the sequence for the sample provided sufficient information to determine the family, order or class to which the organism belongs. The bacteria that are most closely related to your unknown determine the level of classification that we are able to assign.
A "no match" indicates that while we were able to sequence the organism successfully, we could not confidently assign a Genus Species or Genus name to the organism based on our current D2 Fungal library contents. This will only be assigned to fungal identifications.
If "no match" appears on a report, what else can be done to identify the organism?
A "no match" report may have resulted from a missing entry in our identification library or because the organism has never been described in the literature. If no match is obtained we will search GenBank upon request.
Can useful information be obtained from a "no match" report?
Absolutely. The DNA sequence of the organism can be used as an identifier for the isolate - especially in cases where it is important to recognize the isolate if it is seen again. In addition, if we report the isolate as a "no match", you can be confident that the isolate is not one of the organisms in our database, which is very useful for ruling out certain pathogens or undesirable organisms.
A gram-positive organism was sent, but the identification report received was for a gram-negative. How could this happen?
Accurate gram staining is often difficult and depends on many factors. In our studies we have seen that gram stains can be incorrect 10% of the time. If you have questions, please consider repeating the gram stain with a fresh culture. If the results are still discrepant please call customer support for further recommendations.
The GenBank database is so large - why isn't this used for identification?
The GenBank database does contain hundreds of thousands of 16S sequences
from microorganisms. However, there are several problems that must be
considered before using it. First of all, many of the sequences are from
new and unusual organisms isolated from extreme environments, or even
cloned 16S sequences of organisms that have never been cultured in the
laboratory. In those cases these sequences are not associated with a
particular genus and species name. There is little utility for the
purpose of identifying an organism, to show that the unknown is 96%
identical to a sequence cloned from the environment, that has never been
cultured or described. Also, GenBank tends to have sequences from many
unusual and extreme environments, but often has limited coverage for
routinely isolated organisms which can be misleading for the purpose of
identification.
One of the most important factors to consider is that
the GenBank database is not curated, or quality controlled, so any
individual can submit a 16S sequence to GenBank, with no assurance that
the data is correct. There is no process for verifying the accuracy of
the sequence, or that it was derived from the Genus and species name
associated with it. It is not GMP compliant. The size of the database
also limits its utility, because the algorithms that are used to search
large databases have been designed for rapid searching of large
databases, rather than accuracy.
On the ID Request Form, what do you mean by "Marketed" product and what samples should be indicated as being recovered from a marketed product?
If you have filed for licensure for a product, it should be considered
"Marketed." So "Marketed" product refers to any product that is either
already on the market or is post-filing (NDA, ANDA, etc.) with either
the FDA or any other regulatory agency.
An organism should be identified as originating from marketed product if
it is affiliated with a marketed product or is being used to make any
decisions about a marketed product. These are only guidelines for you
to use in deciding the status of your samples.
If the box is not checked, Accugenix assumes that it was not
isolated from marketed product.
How much cell material is needed to perform an identification?
A small colony that is visible to the naked eye contains more than enough DNA for our sequencing procedure.
Can PCR and comparative DNA sequencing be used to determine whether or not my sample is contaminated with bacteria or fungi?
PCR and sequencing based ID is a qualitative assay and should not be used to predict sterility of a product or sample.
Can a mixed culture be submitted for identification?
Our procedures require a pure culture, or a single colony well isolated from other organisms. If the plate contains multiple different colony morphologies, please be sure to indicate whether you would like only the dominant colony type identified, all distinct colony types identified, or only the circled colony identified. On occasion we receive plates where the colonies are not well separated, and must subculture to obtain pure isolates prior to processing. In this event, there will be a fee for subculturing, and the due date will be calculated from the day we are able to obtain a pure culture.
Blood, pigments, high DNA concentrations, phenolic compounds, humic acids.
Can the sequence of an isolate be compared to an organism previously submitted for identification?
Yes. Please call our customer support department and tell them which isolates you would like to compare.
Can comparative DNA sequencing determine if 2 isolates are the same strain or not?
In some instances the ultimate question is not what Genus and species the organism belongs to, but whether these isolates are the same or different. For example, is the S. aureus isolated from a media fill failure the same strain as what was found in some raw material, or is it the same as the strain found on the operator on the filling line? In these types of cases, we recommend submitting the isolates for testing on the RiboPrinter® system.
Can samples be sent for Saturday delivery?
We do not routinely accept samples for delivery on Saturdays. Should a Saturday delivery be required contact Customer Support. An additional processing fee may apply.
Is a permit from USDA or CDC needed to ship live bacterial or fungal cultures within the US?
No permits are required to ship unknown environmental samples within the United States; however, all live cultures should be packaged appropriately.
How should samples be packaged for shipment?
All cultures should be packaged with triple containment, whether or not you have reason to suspect they are pathogenic. Triple containment can be a plate or culture tube, within a sealed bag, placed within another bag containing absorbent material. This should then be placed within a sturdy box or container that will not be crushed during shipping. There are also many commercially available shipping packages available for live cultures. Please call customer support if you have any questions.
How does 16S rRNA sequence based identification compare to RiboPrinting?
For the purpose of bacterial and fungal identification, 16S rRNA sequencing is more accurate, reproducible, and cost effective. Sequencing is a PCR-based genetic test method, while the RiboPrinter® system uses a restriction fragment length polymorphism (RFLP) method. Questions regarding strain level differences, where the species assignment is known or unimportant, are best done by ribotyping on the RiboPrinter® system.
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