Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 10/9/2003
Publication Date: 10/9/2003
Citation: Spackman, E., Suarez, D.L. 2003. An Assessment of Real-Time RT-PCR Robustness Using an Avian Influenza Virus Proficiency Panel. American Association of Veterinary Laboratory Diagnosticians.
Technical Abstract: The inter- and intra-laboratory reproducibility of a real-time RT-PCR (RRT-PCR) assay for avian influenza was assessed by the administration of a blind proficiency panel. The proficiency panel consisted of inactivated and stabilized avian influenza virus, and required bothl aspects of sample processing, including RNA isolation and RRT-PCR. Each sample on the panel corresponded to a specific virus titer. One test panel was administered to each individual technician in the participating laboratories who would routinely perform the assay with clinical samples. Eleven laboratories participated in the proficiency testing for a total of 20 test panels. Although a standardized procedure was recommended, due to differences in instrumentation, some laboratories used modified protocols. Results (cycle threshold values) of the tests were compared among all laboratories using the same procedure and with results from modified procedures to determine test variability and to determine the effects of instrumentation on assay sensitivity. Additionally, ten panels were run at our laboratory to evaluate sensitivity within a lab. This study helps to demonstrate the overall robustness of real-time PCR methods for diagnostics as well a method to assess user proficiency with all aspects of sample processing.