Abstract
Detection of norovirus (NoV) and hepatitis A virus (HAV) on fruits and vegetables using current standard methodologies can be inefficient. Method optimisation focussing on ease, rapidity and increased viral RNA recovery is needed for efficient reverse transcription (RT)-qPCR detection of viruses. A simple and quick direct lysis method for RNA extraction was optimised (method A) to achieve increased viral RNA recovery and minimised RT-qPCR inhibition by increasing the volume of lysis buffer and inclusion of pectinase, Plant RNA Isolation Aid and OneStep PCR Inhibitor Removal Kit. Method A and an internal method structurally comparable to the ISO 15216 standard (method B) were compared for their efficiencies to recover viral RNA from the process controls, mengovirus (MC0) and murine norovirus (MNV), spiked in 13 types of fruits, vegetables, compound foods or seeds/nuts. All extracts (> 61) were also analysed for RT-qPCR inhibition and for natural contamination of NoV and HAV. The overall mean extraction efficiencies of MC0 and MNV were 36 ± 31 and 44 ± 38%, respectively, for method A and 9 ± 16 and 5 ± 11%, respectively, for method B. Inhibition of RT-qPCR amplification of RNA from NoV genogroup (G)I, NoV GII, and HAV ranged from 5 ± 10 to 13 ± 14% for method A and 34 ± 36 to 48 ± 40% for method B. NoV GII was detected in samples of strawberries and seaweed processed by both methods. In conclusion, the new direct lysis method showed an overall better performance compared to the modified ISO 15216 standard and should be validated for implementation in analysis of viruses in foods of plant origin.
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Acknowledgements
We are grateful to Prof. A. Bosch, University of Barcelona, Spain for providing mengovirus, Prof. H. S. Virgin, Washington University, St. Louis, MO, USA for providing murine norovirus and Dr. Lowther, CEFAS, UK for providing dsDNA and RNA transcripts of NoV and HAV. We thank Resadije Idrizi and Professor Lisbeth Truelstrup Hansen at the Technical University of Denmark for excellent technical assistance and critical editing of the manuscript, respectively, as well as the Danish Veterinary and Food Administration and Technical University of Denmark for supporting this study.
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Conceptualization and methodology: SMR, ACS; Formal analysis and investigation: SMR, TJ, TBH and ACS; Writing—original draft preparation: SMR; Writing, reviewing and editing subsequent version: SMR, TJ, TBH and ACS; Funding acquisition: ACS; Resources: TJ and ACS; Supervision: ACS.
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Rajiuddin, S.M., Jensen, T., Hansen, T.B. et al. An Optimised Direct Lysis Method for Viral RNA Extraction and Detection of Foodborne Viruses on Fruits and Vegetables. Food Environ Virol 12, 226–239 (2020). https://doi.org/10.1007/s12560-020-09437-x
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DOI: https://doi.org/10.1007/s12560-020-09437-x