RESUMO
Human noroviruses are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several norovirus food-related outbreaks. However, the extraction of norovirus RNA from frozen raspberries remains challenging. Recovery yields are low and PCR inhibitors limit the sensitivity of the detection methodologies. In 2017, 724 people from various regions of the Province of Quebec, Canada, were infected by noroviruses and the outbreak investigation pointed to frozen raspberries as a putative source. A new magnetic silica bead approach was used for the extraction of viruses from different outbreak samples. The RNA extracts were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and five samples were confirmed positive for norovirus by RT-qPCR amplicon sequencing. A multiplex long-range two-step RT-PCR approach was developed to amplify norovirus ORF2 and ORF3 capsid genes from the positive frozen raspberry RNA extracts and other sequencing strategies were also explored. These capsid genes were sequenced by Next-Generation Sequencing. Phylogenetic analyses confirmed the presence of multiple genotypes (GI.3, GI.6, and GII.17) and intra-genotype variants in some of the frozen raspberry samples. Variants of genotype GI.3 and GI.6 had 100% homology with sequences from patient samples. Similar strains were also reported in previous outbreaks. Confirmation approaches based on sequencing the norovirus capsid genes using Next-Generation Sequencing can be applied at trace level contaminations and could be useful to assess risk and assist in source tracking.
Assuntos
Infecções por Caliciviridae , Norovirus , Rubus , Infecções por Caliciviridae/epidemiologia , Surtos de Doenças , Genótipo , Humanos , Norovirus/genética , Filogenia , Quebeque/epidemiologia , RNA Viral/genéticaRESUMO
Consumption of leafy greens and to a lesser extent fresh herbs has been associated with several foodborne outbreaks including human norovirus (HuNoV). However, the extraction and detection of viruses from these matrices present multiple challenges such as low recovery yields and relatively high PCR inhibition. A new magnetic silica bead based (MSB) extraction protocol was developed and used to recover norovirus from leafy greens and fresh herbs. The performance results were compared to the ISO 15216-1:2017 standard. The HuNoV GII.4 and GI.5 recovery yields from spiked lettuce using the MSB extraction protocol range from 33 to 82%. There was a good correlation between murine norovirus (MNV) and HuNoV recovery yields from fresh herbs and leafy greens. No reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) inhibition was detected from leafy green extracts using the MSB methodology. The selected commercial RT-qPCR detection kit had a major impact on RT-qPCR inhibition levels detected in the ISO 15216-1:2017 RNA extracts. RNase treatment was used to estimate genome recovery from HuNoV with intact capsids. This treatment resulted in similar HuNoV and MNV recovery yields. Between 2019 and 2020, the MSB protocol was used to conduct a survey of HuNoV in domestic and imported leafy greens and fresh herbs sold at retail in Canada. All of the 280 samples tested were negative. Overall, the use of MSB was shown to be an efficient approach to recover HuNoV from leafy greens and certain types of fresh herbs and to conduct surveys.
Assuntos
Lactuca/virologia , Magnetismo/métodos , Norovirus/isolamento & purificação , Dióxido de Silício/química , Especiarias/virologia , Animais , Canadá , Contaminação de Alimentos/análise , Humanos , Fenômenos Magnéticos , Magnetismo/instrumentação , Norovirus/química , Norovirus/genética , Folhas de Planta/virologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Human noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.
Assuntos
Separação Imunomagnética/métodos , Norovirus/isolamento & purificação , Rubus/virologia , Dióxido de Silício/química , Frutas/virologia , Gastroenterite/virologia , Humanos , Separação Imunomagnética/instrumentação , Norovirus/química , Norovirus/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Different antibiotic resistance (AR) genes, such as Bla, Tet and NPTII, contaminate commercially available Taq polymerases. The specificity of the AR gene PCR can be increased when using a restriction enzyme-based decontamination of polymerase. The elimination of Taq polymerase contamination allows the use of PCR tests to screen seeds (corn) and processed food for the presence of genetically modified organisms (GMO) based on the detection of AR genes. Without a decontamination procedure for AR genes, PCR screening tests should be interpreted with caution.
