Ascochyta pisi (only identifiable
by genotypic methods) attacks
peas by causing lesions on leaves,
stems and pods. Initial symptoms
of ascochyta blight are small, dark,
irregular flecks on leaves and
tendrils. Over time, the lesions
expand and increase in number,
eventuallydestroying the plant.
FAQs
Determining Appropriate Test
DNA Sequencing
FunITS
Libraries
Results Analysis
Identification Request Form
Results Reporting
RiboPrinting
Sample Shipment
Determining Appropriate Test
Identification Request Form
Results Reporting
RiboPrinting
Sample Shipment
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 answered by ribotyping on the RiboPrinter® system.
Top
Which test is appropriate to 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 comparative 16S sequencing does not have the discriminatory power to differentiate bacterial strains. We recommend submitting the isolates for testing on the RiboPrinter® system.
Top
Why did Accugenix choose the Internal Transcribed Spacer (ITS) region as a target?
Accugenix is upgrading our fungal ID method to continue offering customers the latest in microbial ID technology. The ITS region is the most widely sequenced DNA region for fungi. The resulting sequences have a higher degree of variation between closely related species than the D2 region, enabling us to provide more accurate fungal identifications.
Top
What is the cost of FunITS?
The cost of FunITS is the same as FunSeq. Contact your Technical Support Specialist 800.886.9654 for pricing information.
Top
How will my company benefit from this new service?
Accugenix anticipates that you will receive more species level identifications. As with the FunSeq service, Accugenix will provide routine updates to reflect changes in taxonomy, as well as the addition of novel organisms.
Top
How will FunITS identifications compare to FunSeq (D2) identifications?
Accugenix is committed to providing you with the most accurate methods available. Your identifications should not change, but you may receive IDs that had a confidence level of 'Species*' change to 'Species.' Because FunITS provides an ID based on a target that has a higher degree of variation, you can expect more species level identifications. ITS does provide a higher degree of resolution than the D2 region; species tend to have a greater genetic distance between each other within the same genus. This may result in a genus level call rather than a previously named species.
Top
Should I anticipate discrepancies between D2 and ITS results? If so, could we request a D2 identification?
Effective January, 19, 2009, FunITS will replace FunSeq as the standard method for fungal sequence-based identifications. You may request FunSeq testing if required. Please contact your Technical Support Specialist at 800.886.9654.
Top
Is the FunITS process validated?
The FunITS service offering utilizes a cGMP compliant, validated method and library. Please contact our Quality Assurance department with any questions.
Top
Where is the target located?
The ITS target is located between the 5,8S (small subunit) and 28S (large subunit) of ribosomal DNA. Accugenix will amplify and sequence this target for identification.
Top
Do you have references supporting ITS as a preferred method?
Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A. Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Res. 2002 Dec;2(4):495-517. PMID: 12702266 [PubMed - indexed for MEDLINE]
Valente P, Ramos JP, Leoncini O. Sequencing as a tool in yeast molecular taxonomy. Can J Microbiol. 1999 Nov;45(11):949-58. Review. PMID: 10588043 [PubMed - indexed for MEDLINE]
Hinrikson HP, Hurst SF, De Aguirre L, Morrison CJ. Molecular methods for the identification of Aspergillus species. Med Mycol. 2005 May;43 Suppl 1:S129-37. PMID: 16110805 [PubMed - indexed for MEDLINE]
Li HC, Bouchara JP, Hsu MM, Barton R, Su S, Chang TC. Identification of dermatophytes by sequence analysis of the rRNA gene internal transcribed spacer regions. J Med Microbiol. 2008 May;57(Pt 5):592-600. PMID: 18436592 [PubMed - indexed for MEDLINE]
Top
DNA Sequencing
Libraries
How do your Bacterial and Fungal Libraries Updates benefit me?
We update our libraries annually in order to provide the most up-to-date information possible for our microbial identification services. With these updates, we routinely add many new species to our libraries in order to give more species level identifications for the organisms you send to us. We also routinely update the names of organisms in the database to reflect current taxonomy and review our library for any sequence or nomenclature errors.
Top
Will the test services you offer or the price for those services change with the release of the new libraries?
No. Neither the tests that we perform nor the prices for these tests will change as a result of library updates.
Top
When can I expect to see identification reports produced with the latest library updates?
Any identification reports produced on or after the library launch date will use the new library. The date and time (Eastern Time) of report generation is displayed beneath the sample name on each report.
Top
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 an identification result that was not to the "Species" level. With the addition of new organisms, there is a greater probability that your identification will be to the 'Species' level.
Top
How can I find out if an organism I need to have identified is included in your Bacterial or Fungal libraries?
Your Account Manager or Technical Support Specialist can provide that information for you.
Top
What do I do if an organism I need to identify is not represented in your libraries?
Please contact our Technical Support Department to explore other ways we can help you gain valuable information about your sample. The sequence data we generated for your organism can provide very useful information for you.
Top
I have often sent in a particular isolate that was identified to the Species Level, and now I have received a different Species level identification for the same isolate. Why did this happen?
There are a few reasons why this may happen. We have updated the names of many organisms in our libraries to reflect current taxonomy. Organism names can change when it is shown by phylogenetic analysis that an organism was 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 further questions, please contact our Technical Support Department.
Top
DNA Sequencing
Results Analysis
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 strain level differentiation. However, if two isolates have different 16S rRNA gene sequences, they can be considered to be different strains. This can be clarified by submitting the isolates for testing on the RiboPrinter® system.
Top
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 branching order and configuration of the phylogenetic tree are taken into account.
Top
How is the phylogenetic tree generated?
The phylogenetic tree, generated for the purpose of determining the identification of an isolate, is distance based. Accugenix uses the Neighbor Joining (NJ) Tree for data interpretation. The first step in generating a tree is a pairwise alignment of all of the sequences and calculation of 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 organisms is a close approximation of the sequence differences between all pairs.
Top
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 (pairwise comparison) and the percentage difference is calculated.
Top
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 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.
Top
Can a confidence level for the ID be provided?
Yes. We provide a confidence of species, genus, family, order, class or 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.
Top
I received a Customer Notification Form that indicated no result was obtained. What would cause this? Can the test be repeated?
A Customer Notification Form indicates that we were unable to obtain usable sequence data from your sample. All samples that do not produce sequence data are automatically processed a second time. If the second attempt results in no sequence data, we will send a Customer Notification Form. The most likely causes for unusable DNA sequence are PCR inhibitors in the sample, failure to extract genomic DNA, or processing a fungal sample as a bacterial sample or vice-versa. Customer Notification Forms are also generated for the following reasons: to indicate that the customer requested testing be stopped, when samples are received with no growth or no growth is obtained after subculture. A nominal processing fee is assessed to any sample that is processed but does not produce sequence data.
Once a Customer Notification Form is generated for a sample, processing has ended for that sample. A customer may re-submit an Identification Request Form for subsequent processing.
Top
What does a confidence level of 'Genus', 'Species', 'Family', or 'No match' mean on a report?
Identification reports contain an identification and 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 or class confidence level 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 the 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 or Species level name based on our current ITS Fungal library . This confidence level will only be assigned to fungal identifications.
Top
If 'no match' appears on a report, what else can be done to identify the organism?
A 'no match' report may result due to a missing library entry or because the organism has never been described in the literature. If "no match" is obtained, we will search a public database (GenBank) upon request.
Top
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. Additionally, 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.
Top
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 a high percentage 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 Technical Support for further recommendations.
Top
The GenBank database is so large. Why not use it 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 often has limited coverage for routinely isolated organisms, so it 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.
Top
How much cell material is needed to perform sequence-based identification?
A small colony that is visible to the naked eye contains more than enough DNA for our sequencing procedure. However, if possible, we encourage you to send a greater amount of cell material to allow for any further necessary testing.
Top
Can PCR and comparative DNA sequencing be used to determine whether or not my sample contains bacteria or fungi?
The sequence-based assay utilized by Accugenix is qualitative and should not be used to determine the presence or absence of organisms in or the sterility of a product or sample.
Top
Can a mixed culture be submitted for identification?
Yes. Mixed cultures can be submitted for identification. However, our procedures require a pure culture, or a single colony well isolated from other organisms.
If the plate contains multiple colony morphologies, please be sure to indicate on the ID Request Form 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 for isolation of the target colony prior to processing. Mixed cultures would produce multiple DNA sequences in one set of data which is not usable for our analysis. 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.
Top
What compounds inhibit PCR?
Some of the compounds that can inhibit PCR include blood, pigments, high DNA concentrations, phenolic compounds and humic acids.
Top
Can the sequence of an isolate be compared to an organism previously submitted for identification?
Yes. Please contact our Technical Support department and tell them which isolates you would like to compare. A sequence comparison report will be generated for your request.
Top
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 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.
Top
How is the due date for a sequencing ID report calculated?
Due dates are calculated from the date a pure culture (or culture with well isolated colonies) is received. The due date is determined in business days. Due dates must be extended if it is determined that the sample requires isolation of the target colony to proceed with the identification process. A Technical Support representative will communicate any change in report due date caused by the need to subculture the sample.
Top
How are reports provided?
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 following a Quality Assurance review of the entire submitted job. You may request overnight delivery of your hardcopy reports for an additional nominal fee. If you would prefer not to receive hard copies of results, please indicate this on the Identification Request Form.
Top
When should I use your Microbial Characterization service?
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 different. For example, if you are investigating the source of contamination found in a sterility failure, it may be necessary to determine the strain-relatedness of your isolates.
Top
How does the Microbial Characterization process at Accugenix work?
Accugenix uses the RiboPrinter® Microbial Characterization system. The RiboPrinter® system is an automated ribotyping system that generates a DNA fingerprint of the Ribosomal RNA genes, in a process that is similar to that of a southern blot.
First the bacterial genome is cut with a restriction enzyme and resulting DNA fragments are separated by gel electrophoresis. Then a DNA probe, chemiluminescent agent, and camera are used to visualize the DNA fragments of interest. A sample report is generated that lists your sample information and the sample fragment pattern. The resulting RiboPrint® pattern is then compared against the RiboPrint® patterns of other isolates in your unique customer pattern library to determine their similarity.
Top
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 Comparison Reports at no additional cost.
Top
What is included in a Bacterial Characterization Comparison Report?
The Comparison Report contains sample information and the Riboprint patterns for each sample requested. The first sample listed is the selected sample to which subsequent samples 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 provides a summary interpretation of the samples' similarity.
Top
How many samples can be compared in one Bacterial Characterization Comparison Report?
There is no limit to how many samples can be compared in one request, however, the report format is limited to 7 samples per page. The Comparison Report may be multiple pages as a result.
Due to the time required to generate and interpret the comparison, there is not a guaranteed turnaround time for comparison requests. Please discuss your needs with Technical Support when requesting the Comparison.
Top
Do you need a live organism to perform the Bacterial Characterization test?
Yes. A live organism is required for ribotyping. Accugenix prefers a culture less than 72 hours old. Turnaround time for your sample will begin on the day of receipt if the sample arrives at Accugenix less than 72 hours old. Any 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. A Technical Support representative will communicate any change in report due date caused by the need to subculture the sample.
Top
Can samples be sent for Saturday delivery?
We do not routinely accept samples for delivery on Saturdays. Should a Saturday delivery be required, contact Technical Support before Friday at 3 pm. An additional processing fee may apply.
Top
Is a permit from USDA or CDC required 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.
Top
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. Cushioning material should be used in the package to prevent sample damage in shipment. Using ice packs or blue ice should is not recommended, as the samples may freeze, disturbing the integrity of the organism to be identified. There are also many commercially available shipping packages available for live cultures. Please call Technical Support if you have any questions.
Top