Abstract
This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3–57.2%) and 41.4% (95% CI 29.1–53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4–24.9%, and 12.3% GII, 95% CI 7.0–17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.
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Data will be made available on request.
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Acknowledgements
We thank Carlos Jose da Silva, Michel Vergne Felix Sucupira, and Marisa de Oliveira Ribeiro for laboratory assistance. We thank the cheese producers who participated in this study.
Funding
This study was funded by The Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ/SEI-260003/001753/2023), Inova Fiocruz/Oswaldo Cruz Foundation (VPPCB-008-FIO-18-2-67) and the Minas Gerais State Agency for Research and Development (FAPEMIG/2070.01.0002119/2020-24). This work was also supported by the Oswaldo Cruz Institute (PAEF-3/IOC/Fiocruz). T.M. Fumian, M.P. Miagostovich and J.P.G. Leite are FAPERJ and CNPq fellows.
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Conceptualization: Carina Pacheco Cantelli, Marcio Roberto Silva, and José Paulo Gagliardi Leite; sampling: André Almeida Santos Duch and Liliane Denize Miranda Menezes; methodology: Carina Pacheco Cantelli, Laís Marques Pimenta, Guilherme Caetano Lanzieri Tavares, Gabriel Assad Baduy, Alexandre Madi Fialho, and Adriana Gonçalves Maranhão; Funding acquisition: Carina Pacheco Cantelli, Marcio Roberto Silva, Marize Pereira Miagostovich, and José Paulo Gagliardi Leite; writing—original draft: Carina Pacheco Cantelli; writing—review and editing: Carina Pacheco Cantelli, Marcio Roberto Silva, André Almeida Santos Duch, Tulio Machado Fumian, Marize Pereira Miagostovich, and José Paulo Gagliardi Leite; supervision: José Paulo Gagliardi Leite. All authors have read and agreed to the published version of the manuscript.
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Preliminary results from this study were presented as a lecture at the 34º Congresso Brasileiro de Virologia and 8º Encontro do Mercosul de Virologia, 24 to 27 September, 2023, Ouro Preto, Minas Gerais, Brazil.
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Cantelli, C.P., Silva, M.R., Pimenta, L.M. et al. Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese. Food Environ Virol (2024). https://doi.org/10.1007/s12560-024-09588-1
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DOI: https://doi.org/10.1007/s12560-024-09588-1