A Comparison of Two Methods for Detection of Norovirus RNA in Environmental Swab Samples

Abstract

Standardised molecular methods are available for the detection of norovirus from water and specific food items. Detection of norovirus from stool samples also relies on molecular methods, but differences exist between nucleic acid extraction, reverse transcription, and amplification strategies recommended by the ISO 15216-1:2017, and those employed in clinical laboratories. Here, we conduct a direct comparison of two methods for the detection and quantitation of norovirus from a stool sample and from artificially contaminated swabs. We also compare use of linear dsDNA standards as recommended in ISO 15216:2017 against an in vitro-transcribed single-stranded RNA (ssRNA) for estimation of norovirus genome copy number. Our results show that the two methods have comparable sensitivity for the detection of norovirus RNA from a clinical sample or swab. The use of a ssRNA standard revealed that quantitation performed against a linear dsDNA standard consistently underestimated the genome copy numbers by 1.5 to 2 log due to the relative inefficiency of the reverse transcription step. This has important implications for the estimation of the sensitivity of norovirus detection methods, comparability of results across sites, and assessment of viral loads that may be clinically significant or estimated to constitute infectious doses.