Interlaboratory comparison of SARS-CoV2 molecular detection assays in use by U.S. veterinary diagnostic laboratories.

The continued search for intermediate hosts and potential reservoirs for SARS-CoV2 makes it clear that animal surveillance is critical in outbreak response and prevention. Real-time RT-PCR assays for SARS-CoV2 detection can easily be adapted to different host species. U.S. veterinary diagnostic laboratories have used the CDC assays or other national reference laboratory methods to test animal samples. However, these methods have only been evaluated using internal validation protocols. To help the laboratories evaluate their SARS-CoV2 test methods, an interlaboratory comparison (ILC) was performed in collaboration with multiple organizations. Forty-four sets of 19 blind-coded RNA samples in Tris-EDTA (TE) buffer or PrimeStore transport medium were shipped to 42 laboratories. Results were analyzed according to the principles of the International Organization for Standardization (ISO) 16140-2:2016 standard. Qualitative assessment of PrimeStore samples revealed that, in approximately two-thirds of the laboratories, the limit of detection with a probability of 0.95 (LOD95) for detecting the RNA was ≤20 copies per PCR reaction, close to the theoretical LOD of 3 copies per reaction. This level of sensitivity is not expected in clinical samples because of additional factors, such as sample collection, transport, and extraction of RNA from the clinical matrix. Quantitative assessment of Ct values indicated that reproducibility standard deviations for testing the RNA with assays reported as N1 were slightly lower than those for N2, and they were higher for the RNA in PrimeStore medium than those in TE buffer. Analyst experience and the use of either a singleplex or multiplex PCR also affected the quantitative ILC test results.

Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories.

The exquisite sensitivity of in vitro amplification assays such as real-time polymerase chain reaction (rtPCR) requires the establishment of thorough and robust laboratory practices. To this end, an American Association of Veterinary Laboratory Diagnosticians (AAVLD) committee of subject matter experts was convened to develop a set of best practices for performance of nucleic acid amplification assays. Consensus advice for the performance of preanalytical, analytical, and postanalytical steps is presented here, along with a review of supporting literature.