Detection and genogrouping of noroviruses from children's stools by Taqman One-step RT-PCR
| Autor: | Robert H. Gilman, Sonia Apaza, Susana Pineda, Nancy M. Vu, Mayuko Saito, Fanny Herhold, Margaret Kosek, Susan Espetia, Sonia Montenegro, Romeo Pomari |
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| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Genotyping Techniques
General Chemical Engineering viruses Biology medicine.disease_cause Real-Time Polymerase Chain Reaction chemistry Genetic analysis General Biochemistry Genetics and Molecular Biology chemistry.chemical_compound Feces Open Reading Frames fluids and secretions virus RNA Rotavirus Virology Genotype medicine TaqMan Humans genetics Taq Polymerase Child calicivirus infection Caliciviridae Infections General Immunology and Microbiology isolation and purification General Neuroscience Norovirus virus diseases methodology virology Gastroenteritis Diarrhea classification real time polymerase chain reaction RNA Viral purl.org/pe-repo/ocde/ford#3.01.00 [https] RNA extraction medicine.symptom metabolism Taq polymerase |
| Zdroj: | Scopus-Elsevier |
| Popis: | 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. 1, 2, 3 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 1, 2, 3. 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. method4 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. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups. |
| Databáze: | OpenAIRE |
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