Comparison and optimization of detection methods for noroviruses in frozen strawberries containing different amounts of RT-PCR inhibitors

• Published in: Food microbiology 2016-12, Vol.60, p.124-130
• Main Authors: Bartsch, Christina, Szabo, Kathrin, Dinh-Thanh, Mai, Schrader, Christina, Trojnar, Eva, Johne, Reimar
• Format: Article
• Language: English
• Subjects: Detection method; Food Microbiology - methods; Fragaria; Fragaria - chemistry; Fragaria - virology; Frozen Foods - virology; Limit of Detection; Norovirus; Norovirus - genetics; Norovirus - isolation & purification; Nucleic Acid Synthesis Inhibitors - analysis; PCR inhibitors; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral - genetics; RNA, Viral - isolation & purification; Strawberries
• ISSN: 0740-0020; 1095-9998
• DOI: 10.1016/j.fm.2016.07.005

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. •Frozen strawberries can contain different amounts of RT-PCR inhibitors.•Efficiency of norovirus extraction protocols from strawberries varies remarkably.•The ISO/TS15216 method shows good performance with frozen strawberries.•Additional RNA purification with Sephacryl®-based columns increases detection rate.•Use of these columns is suggested for norovirus detection in berry samples.