A comparison of two methods for detection of norovirus RNA in environmental swab samples

Norovirus is the principal cause of acute gastroenteritis globally, infecting and causing disease in people of all ages. Contaminated food and water are recognised as major routes of infection, often leading to outbreaks of gastroenteritis that can spread between countries and continents. Standardised molecular methods are available for the detection of norovirus from water and food items such as shellfish and fresh produce such as leafy greens and berry fruits, which are some of the most commonly identified food products as being contaminated at source. Detection of norovirus from stool samples also relies on similar molecular methods, but some differences exist between the nucleic acid extraction, reverse transcription, and amplification strategies recommended by the ISO 15216-1:2017 and related annexes, and those typically employed in clinical diagnostic or reference laboratories. Here we conduct a direct comparison of two methods for the detection and quantitation of norovirus directly from a stool sample and from swabs artificially contaminated with a dilution of the stool sample to simulate environmental sampling. We also compare the use of the linear dsDNA standard as recommended in the ISO 15216:2017 method against an in vitro transcribed single stranded RNA (ssRNA) for the estimation of the norovirus genome copy number present in a sample. Our results show that the two methods have comparable sensitivity for the detection of norovirus RNA from a clinical sample or from a contaminated 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 inefficiently 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.