title: Comparison and optimization of detection methods for noroviruses in frozen strawberries containing different amounts of RT-PCR inhibitors.
abstract: Frozen berries have been repeatedly identified as vehicles for norovirus (NoV) transmission causing large gastroenteritis outbreaks. However, virus detection in berries is often hampered by the presence of RT-PCR-inhibiting substances. Here, several virus extraction methods for subsequent real-time RT-PCR-based NoV-RNA detection in strawberries were compared and optimized. NoV recovery rates (RRs) between 0.21 ± 0.13% and 10.29 ± 6.03% were found when five different artificially contaminated strawberry batches were analyzed by the ISO/TS15216-2 method indicating the presence of different amounts of RT-PCR inhibitors. A comparison of five different virus extraction methods using artificially contaminated strawberries containing high amounts of RT-PCR inhibitors revealed the best NoV RRs for the ISO/TS15216 method. Further improvement of NoV RRs from 2.83 ± 2.92% to 15.28 ± 9.73% was achieved by the additional use of Sephacryl(®)-based columns for RNA purification. Testing of 22 frozen strawberry samples from a batch involved in a gastroenteritis outbreak resulted in 5 vs. 13 NoV GI-positive and in 9 vs. 20 NoV GII-positive samples using the original ISO/TS15216 method vs. the extended protocol, respectively. It can be concluded that the inclusion of an additional RNA purification step can increase NoV detection by the ISO/TS15216-2 method in frozen berries containing high amounts of RT-PCR inhibitors.
authors.affiliation: Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Bartsch
authors.affiliation: Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Szabo
authors.affiliation: Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany.
authors.forename: Mai
authors.initials: M
authors.lastname: Dinh-Thanh
authors.affiliation: Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Schrader
authors.affiliation: Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Trojnar
authors.affiliation: Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany. Electronic address: reimar.johne@bfr.bund.de.
authors.forename: Reimar
authors.initials: R
authors.lastname: Johne
mesh_terms: D005516:Food Microbiology / Q000379:methods; D031985:Fragaria / Q000737:chemistry* / Q000821:virology*; D005628:Frozen Foods / Q000821:virology*; D057230:Limit of Detection; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D019384:Nucleic Acid Synthesis Inhibitors / Q000032:analysis; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D019384:Nucleic Acid Synthesis Inhibitors; D012367:RNA, Viral
keywords: Detection method; Norovirus; PCR inhibitors; Strawberries