Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories

Abstract Background Noroviruses (NoVs) are the leading cause of infectious gastroenteritis worldwide. Real-time reverse transcription PCR (real-time RT-PCR) is the preferred method of NoV detection for the majority of testing laboratories. Although the accepted target region for molecular detection assays is the conserved ORF1/ORF2 junction, multiple variations have been published with differences in primers, probes, reagents, multiplexing, etc. Objectives We assessed the detection limit for GII.4 NoV real-time RT-PCR assays as well as the ability to detect the non-GII.4 NoV genotypes in each participating laboratory. Study design A panel of 25 RNA samples was circulated to 18 testing laboratories for comparison of their real-time RT-PCR procedures for NoV detection. Results Multiple protocols with slight differences in reagents or conditions successfully detected 10 genome equivalents or fewer of NoV per reaction. Multiplex procedures were significantly associated ( p = 0.04) with false negative results, particularly for a GI.2 strain. Sensitive detection was associated with false positive results ( p = 0.03). Conclusions Overall, the data indicate that comparable results are produced under slightly different assay conditions.