Development of a method for direct extraction of viral RNA from bivalve molluscs

H. Quang Le, E. Suffredini, D. Tien Pham, A. Kim To, D. De Medici

Research output: Contribution to journalArticle

Abstract

The detection of foodborne viruses in bivalve molluscs is a challenging procedure in relation to low virus concentration and to the presence of significant RT-PCR inhibitors. The aim of this study was the development of an efficient direct extraction method for foodborne viral RNA from bivalve molluscs. Using Mengovirus as a surrogate for foodborne viruses, five extraction methods based on RNA release by Trizol were compared on clams and oysters. A procedure consisting of Trizol, PureLink RNA Mini Kit, followed by Cetyltrimethylammonium bromide (CTAB) treatment and LiCl precipitation was found to provide RNA with the highest extraction efficiency and negligible inhibitory effect on real-time RT-PCR. This procedure was further compared to standard extraction method (ISO 15216) using clam, mussel and oyster samples spiked with Hepatitis A virus, Norovirus (NoV) GI and GII as well as bivalve samples naturally contaminated with NoV GI or GII. Results clearly demonstrated that the developed method provided, on average, a recovery 4·3 times higher than the standard reference protocol as well as good repeatability. Significance and Impact of the Study: A direct extraction procedure was developed to recover viral RNA from shellfish with improved efficiency in comparison to reference extraction method (ISO 15216). Without the need for specific equipment, this procedure offers an alternative for performing food safety controls and for risk assessment studies. Given the inclusion in this extraction method of several steps for the efficient removal of food components inhibiting PCR reaction, this approach could serve as a general scheme for the extraction of nucleic acids of other enteric viruses and/or from other food categories.

Original languageEnglish
Pages (from-to)426-434
Number of pages9
JournalLetters in Applied Microbiology
Volume67
Issue number5
DOIs
Publication statusPublished - Nov 1 2018

Keywords

  • food safety
  • PCR (polymerase chain reaction)
  • rapid methods
  • shellfish
  • viruses

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology

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