QC/QA Program: Pilot Study
June 1, 2018
Research and diagnostic laboratories across North America are running PCR tests looking for wild pathogens, including the chytrid fungi Batrachochytrium dendrobatidis and B. salamandrivorans. Although there are recommendations regarding protocols and the use of standards, methodology varies amongst laboratories.
The Canadian Wildlife Health Cooperative recently completed a pilot trial of validation for PCR tests for Bdand Bsal. Twelve randomized blind samples containing various concentrations of a DNA plasmid with sequences for Bd, Bdand Bsal, and blanks were sent to 19 participating laboratories from 4 countries: USA, Canada, Singapore and Australia. Tests were run as per each laboratory’s routine protocols and results reported as "positive" or "negative" and, if run by qPCR, included their respective Ct (Cq) values and estimated copy numbers. Results from each laboratory were compared against the known composition of the samples and against other laboratory's results; laboratories were informed of where their results stood in comparison to other laboratories, but the standings of individual laboratories remained confidential.
All laboratories detected medium and high concentrations, 7 laboratories failed to detect the smallest concentration (1.3 copies/μL). No laboratory reported false positive results. Great variability was found in the concentrations reported, with copies/μL sometimes as much as 4 orders of magnitude. These results reflect the great difference that exists in the calculation of pathogen load amongst laboratories and the need to explore a standardized methodology.
The trial also reflects the interest and willingness to collaborate amongst amphibian pathogen professionals: all past participants and several other laboratories have stated their commitment to participate in future trials.