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

Abstract

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.

Second round of the interlaboratory comparison (ILC) exercise of SARS-CoV-2 molecular detection assays being used by 45 veterinary diagnostic laboratories in the US

“…Research has reported one marker as more sensitive than the other or vice versa 29,30 . In the ILC1 20 , differences in ROD values between the two markers were very minor.…”

“…The results indicated that the ILC1 participants effectively detected SARS-CoV-2 RNA in MTM with their methods routinely used for testing clinical specimens. Two-thirds of the laboratories achieved nearly the theoretical optimum Level of Detection (LOD) of three copies 20 .…”

High Seroprevalence of SARS-CoV-2 in White-Tailed Deer (Odocoileus virginianus) at One of Three Captive Cervid Facilities in Texas

“…An aliquot of purified nucleic acid was tested for SARS-CoV-2 RNA by specific real time RT-qPCR to amplify the RdRp gene ( 25 ); a control plasmid containing a portion of the RdRp gene served as a positive control (Integrated DNA Technologies, Coralville, IA, USA). Using this same protocol, our lab has successfully completed the USDA COVID proficiency testing exercise (ICE-2) in the summer of 2021 ( 26 ).…”

RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step

“…This has resulted in the development of a range of different reverse transcriptase enzymes (RTases) with diverse performance characteristics [18] that enable each one to meet the needs of distinct applications [19]. For both research and diagnostic applications, RT reactions usually last between 15 and 30 min [10,11] using as high a temperature as possible [20]. This creates a substantial drawback for diagnostic tests aimed at rapid viral pathogen detection, especially when carried out in a point-of-care setting.…”

“…Combined with the subsequent discovery of Taq polymerase [3] and the theory [4] and practical realisation of the polymerase chain reaction (PCR) [5,6], reverse transcription (RT)-PCR was quickly adopted as a powerful method for the analysis of RNA [7,8]. Whilst early protocols included 60 min incubation times for the RT step [9], most contemporary protocols tend to be more rapid at between 15 [10] and 30 min [11], although markedly longer RT polymerisation times continue to be used [12]. This is generally not a problem for research applications but becomes an issue when RT-PCR is used in the context of rapid diagnostic testing.…”