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Detection and genogrouping of noroviruses from children's stools by Taqman One-step RT-PCR.

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dc.contributor.author Apaza, S.
dc.contributor.author Espetia, S.
dc.contributor.author Gilman, Robert Hugh
dc.contributor.author Montenegro, S.
dc.contributor.author Pineda, S.
dc.contributor.author Herhold, F.
dc.contributor.author Pomari, R.
dc.contributor.author Kosek, M.
dc.contributor.author Vu, N.
dc.contributor.author Saito, M.
dc.date.accessioned 2022-01-18T19:34:44Z
dc.date.available 2022-01-18T19:34:44Z
dc.date.issued 2012
dc.identifier.uri https://hdl.handle.net/20.500.12866/11202
dc.description.abstract Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate. For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups. en_US
dc.language.iso eng
dc.publisher MyJove Corporation
dc.relation.ispartofseries Journal of Visualized Experiments
dc.rights info:eu-repo/semantics/restrictedAccess
dc.rights.uri https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es
dc.subject Child en_US
dc.subject Humans en_US
dc.subject isolation and purification en_US
dc.subject genetics en_US
dc.subject metabolism en_US
dc.subject classification en_US
dc.subject methodology en_US
dc.subject Feces en_US
dc.subject real time polymerase chain reaction en_US
dc.subject Real-Time Polymerase Chain Reaction en_US
dc.subject virology en_US
dc.subject gastroenteritis en_US
dc.subject chemistry en_US
dc.subject virus RNA en_US
dc.subject Caliciviridae Infections en_US
dc.subject Genotyping Techniques en_US
dc.subject Norovirus en_US
dc.subject RNA, Viral en_US
dc.subject Gastroenteritis en_US
dc.subject Open Reading Frames en_US
dc.subject calicivirus infection en_US
dc.subject Taq Polymerase en_US
dc.title Detection and genogrouping of noroviruses from children's stools by Taqman One-step RT-PCR. en_US
dc.type info:eu-repo/semantics/article
dc.identifier.doi https://doi.org/10.3791/3232
dc.subject.ocde https://purl.org/pe-repo/ocde/ford#3.01.00
dc.relation.issn 1940-087X


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