title: Digital RT-PCR method for hepatitis A virus and norovirus quantification in soft berries.
abstract: Raw fruits may harbour many pathogens of public health concern including enteric viruses, which are the leading cause of foodborne outbreaks. Recently, consumption of soft berries has been associated with increasing reports of norovirus and hepatitis A virus outbreaks in Europe. Due to their low infectious doses and low concentrations in food samples, an efficient and sensitive analytical method is required for virus detection. In this study we explored two different ways to improve the reference method for the detection of enteric viruses in soft fruits (ISO/TS 15216-1; 15216-2): an additional purification step after RNA extraction; and the detection of enteric viral genome by an absolute quantification method (microfluidic digital RT-PCR). Both of these approaches led to an improvement of enteric virus detection in soft berries by greatly lowering PCR inhibition, raising viral extraction efficiencies and enabling validation of controls using pure RNA extracts. The PCR inhibitor removal step can be easily included in the routine method. Absolute quantification by digital RT-PCR may be a relevant alternative method to standardize quantification of enteric viruses in foodstuffs.
authors.affiliation: Université Paris Est, ANSES, Maisons-Alfort Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Fraisse
authors.affiliation: Université Paris Est, ANSES, Maisons-Alfort Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
authors.forename: Coralie
authors.initials: C
authors.lastname: Coudray-Meunier
authors.affiliation: Université Paris Est, ANSES, Maisons-Alfort Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Martin-Latil
authors.affiliation: Université Paris Est, ANSES, Maisons-Alfort Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Hennechart-Collette
authors.affiliation: Université Paris Est, ANSES, Maisons-Alfort Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
authors.forename: Sabine
authors.initials: S
authors.lastname: Delannoy
authors.affiliation: Université Paris Est, ANSES, Maisons-Alfort Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Fach
authors.affiliation: Université Paris Est, ANSES, Maisons-Alfort Laboratory for Food Safety, F-94701 Maisons-Alfort, France. Electronic address: sylvie.perelle@anses.fr.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Perelle
mesh_terms: D029798:Blueberry Plants / Q000821:virology; D005060:Europe; D005516:Food Microbiology / Q000379:methods*; D059022:Food Safety / Q000379:methods; D031985:Fragaria / Q000821:virology; D005638:Fruit / Q000821:virology*; D016679:Genome, Viral / Q000235:genetics; D030041:Hepatitis A virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D031965:Ribes / Q000821:virology; D065856:Rubus / Q000821:virology
chemical_list: D012367:RNA, Viral
keywords: Digital RT-PCR; Hepatitis A virus; Norovirus; PCR inhibition; RT-qPCR; Soft berries