Amphibian Chytrid Fungi PCR Quality Control: Call for Participation

Eurycea bislineata

Several research and diagnostic laboratories across North America are running PCR tests looking for wild pathogens. Standardization of methodologies is difficult, and even more difficult is it for research laboratories to acquire a certification such as that granted by organizations like the American Association of Diagnostic Veterinary Laboratories or ISO committees. In an effort to provide a more accessible method for quality control/quality assurance (QC/QA) that will allow participating laboratories to confidentially evaluate the quality of their results, be it for diagnostic or research purposes, the Canadian Wildlife Health Cooperative is proposing a pilot ring test specific to selected amphibian pathogens. The methodology will follow the general guidelines of the Veterinary Laboratory Association (VLA) and, for this pilot run, will focus on the two amphibian chytrid fungi.

Briefly, a set of 12 blinded randomized samples will be shipped to each participating laboratory, at no cost, to undergo testing for B. dendrobatidis and B. salamandrivorans. The samples will be prepared by Pisces Molecular and will include plasmid DNA with the PCR/qPCR target sequences for one or both pathogens at varying concentrations, as well as blanks. Since Pisces will also be participating in the ring test, considerable thought and effort have been devoted to blinding the composition of the samples, so that Pisces is blinded as well: All test sample labeling as well as the exact number and type of positive and negative (blank) samples will be done at and by the CWHC, which is not directly involved in the PCR testing, and mailed to the participating laboratories. Each laboratory will agree to perform the tests (and absorb their reagent costs) on all samples and report back to CWHC within 3 weeks of receiving the material. Tests may be run singly or in triplicate (the latter being recommended), and results will be reported as “positive” or “negative” and, if run by qPCR, will include their respective Cq values and copy numbers. A formatted spread sheet will be circulated to all participating laboratories to facilitate reporting. Results will be compiled, tabulated and analyzed by CWHC in a report comparing each laboratory’s results against the known composition of the samples and against other laboratory’s results. Each laboratory will be informed of where its results stand in comparison to other laboratories, but the standings of individual laboratories will remain confidential.

The CWHC, working as an independent party, will coordinate the test distribution and cover the costs of the material and shipment to all interested laboratories. We expect this pilot ring test to serve as the first step towards establishing a peer-based validation system and to help the VLA in their efforts to set up a formal QC/QA program for PCR tests. Publication of the results (maintaining confidentiality regarding individual laboratories) as a diagnostic note will hopefully follow. Financially and logistically, the pilot run will require participation from 10-15 individual laboratories. We anticipate being able to send out the test samples by the end of January 2016.

Interested parties can contact María Forzán ( to sign up or to pose any questions or comments regarding the way in which the ring test is to be conducted. It is crucial that contact is made BEFORE JANUARY 9th, 2016, so the total number of participating laboratories can be determined and the appropriate number of testing tubes prepared.


Submitted by María Forzán, CWHC Atlantic

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