Interlaboratory Evaluation of a Method for Quantification of Norovirus RNA as an Alternative Use for ISO 15216-1:2017 to Conduct Japan Baseline Survey of Oysters

Very low likelihood that cultivated oysters are a vehicle for SARS-CoV-2: 2021–2022 seasonal survey at supermarkets in Kyoto, Japan

“…Previous studies on the detection of norovirus in the oyster midgut gland have, after PEG precipitation, further centrifuged the oyster sample to remove foreign substances derived from the midgut gland that can inhibit gene amplification, and removed fragmented RNAs in the midgut gland that can potentially lead to overestimation of the infection risk to humans, and used the resulting supernatant for RNA extraction ( Imamura et al, 2021 , MAFF 2018, 2019 ). However, some researchers have pointed out that the final centrifugation step causes SARS-CoV-2 and noroviruses to adsorb nonspecifically to the pellet containing foreign substances, reducing the virus extraction efficacy ( Brnić et al, 2022 ; Miura et al, 2018 ; Yamazaki, personal communication).…”

“…Given the variety of substances that inhibit PCR amplification, it can be difficult to identify the ones affecting an individual setup (Schrader et al, 2012; Wilson, 1997 ). A previous study using a common method adopted in Japan for detecting norovirus in oysters ( MAFF, 2019 ) reported that 188 (36.9%) of 510 fresh oyster samples purchased directly from producers had a recovery rate of less than 1% ( Imamura et al, 2021 ). The findings from this previous study infer that substances derived from the midgut gland can inhibit viral enrichment by PEG precipitation, real-time RT-PCR amplification efficiency, or both.…”

“…For samples packed in seawater, virus concentration and removal of midgut-gland contaminants were performed using a combination of existing authorized PEG precipitation methods reported by the Ministry of Agriculture, Forestry and Fisheries ( Imamura et al, 2021 ; MAFF, 2019 ), Ministry of Health, Labour and Welfare ( MHLW, 2003 ) and National Institute of Health Sciences ( NIHS, 2010 ) in Japan with minor modifications. Briefly, using sterile scissors and tweezers, the midgut gland was removed from 5 to 10 oysters in the same pack, with care taken to include as little of the surrounding white tissue as possible, and 3 g was transferred to a filtered stomacher bag.…”

“…While two countries in Europe have reported detecting SARS-CoV-2 in both natural and cultivated bivalves, there have been no such reports in Asia, including Japan ( Mancusi et al, 2022 ; Polo et al, 2021 ). Methods for detecting norovirus in oysters, other foods and fecal matter have been reported in Europe ( EFSA, 2019 ; ISO, 2017 ) and by government testing guidelines in Japan ( Imamura et al, 2021 ; MAFF, 2019 ; MHLW, 2003 ; NIHS, 2010 ). However, no method has been established for detecting SARS-CoV-2 in bivalves, with studies from European nations that reported detecting the virus having only modified the method used for norovirus ( Brnić et al, 2022 ; Desdouits et al, 2021 ; Le Guernic et al, 2022 ; Mancusi et al, 2022 ; Polo et al, 2021 ).…”

“…However, no method has been established for detecting SARS-CoV-2 in bivalves, with studies from European nations that reported detecting the virus having only modified the method used for norovirus ( Brnić et al, 2022 ; Desdouits et al, 2021 ; Le Guernic et al, 2022 ; Mancusi et al, 2022 ; Polo et al, 2021 ). Since both infectious viral particles and non-infectious fragmented RNAs accumulate in the midgut gland of oysters, methods used to detect RNA directly from oyster homogenates may overestimate the actual risk ( Imamura et al, 2021 , MAFF, 2019 , 2021 ; Polo et al, 2021 ).…”