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Norovirus Monitoring in Oysters Using Two Different Extraction Methods

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Abstract

Detection of noroviruses in bivalve shellfish is difficult because of the low concentration of norovirus and the presence of reverse transcription (RT)-PCR inhibitors. This study aimed to assess the presence of noroviruses in oysters extracted using a proteinase K extraction (ISO 15216 method) and an adsorption–elution method. Seventy oyster samples were extracted using the two extraction methods and evaluated using RT-nested PCR. The results showed norovirus detection rates at an equal frequency of 28.6%, of which a total of 48 (68.6%) samples had corresponding positive or negative results, while there were 22 (31.4%) samples with discrepant results. Norovirus genogroup (G)I, GII, and mixed GI and GII were detected in 20%, 4.3%, and 4.3% of samples, respectively, by the proteinase K extraction method, which comprised of GI.2, GI.5b, GI.6b, GII.4, and GII.17 genotypes. With the adsorption–elution method noroviruses were detected in 17.1%, 8.6%, and 2.9% of samples, respectively, which comprised of GI.2, GII.2, GII.4, and GII.17 genotypes. All norovirus-positive oyster samples were further estimated for genome copy number using RT-quantitative PCR. The oyster samples processed using the adsorption–elution method contained norovirus GI of 3.36 × 101–1.06 × 105 RNA copies/g of digestive tissues and GII of 1.29 × 103–1.62 × 104 RNA copies/g. Only GII (2.20 × 101 and 7.83 × 101 RNA copies/g) could be quantified in samples prepared using the proteinase K extraction method. The results demonstrate the different performance of the two sample-processing methods, and suggest the use of either extraction method in combination with RT-nested PCR for molecular surveillance of norovirus genotypes in oysters.

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Acknowledgements

This work was supported by a research grant from the Thai Government Budget through Mahidol University, Bangkok, Thailand, fiscal years 2015–2017. The proof reading of this manuscript was supported by the Editorial Office, Faculty of Graduate Studies, Mahidol University.

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Authors and Affiliations

  1. Department of Microbiology, Faculty of Public Health, Mahidol University, 420/1 Ratchawithi Road, Bangkok, 10400, Thailand

    Thamapan Tunyakittaveeward, Kitwadee Rupprom, Kannika Pombubpa & Leera Kittigul

  2. Department of Epidemiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand

    Nopporn Howteerakul

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  1. Thamapan Tunyakittaveeward
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  2. Kitwadee Rupprom
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Correspondence to Leera Kittigul.

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Tunyakittaveeward, T., Rupprom, K., Pombubpa, K. et al. Norovirus Monitoring in Oysters Using Two Different Extraction Methods. Food Environ Virol 11, 374–382 (2019). https://doi.org/10.1007/s12560-019-09396-y

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