Article

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

Laboratorio de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia.
Journal of Visualized Experiments 01/2012; DOI: 10.3791/3232
Source: PubMed

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.

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    ABSTRACT: Background. Human noroviruses are among the most common enteropathogens globally, and are a leading cause of infant diarrhea in developing countries. However, data measuring the impact of norovirus at the community level are sparse. Methods. We followed a birth cohort of children to estimate norovirus infection and diarrhea incidence in a Peruvian community. Stool samples from diarrheal episodes and randomly selected non-diarrheal samples were tested by polymerase chain reaction for norovirus genogroup and genotype. Excretion duration and rotavirus coinfection were evaluated in a subset of episodes. Results. Two hundred twenty and 189 children were followed to 1 and 2 years of age, respectively. By one year, 80% [95% confidence interval (CI):75-85] experienced at least one norovirus infection and by 2 years, 71% [CI:65-77] had at least one episode of norovirus-associated diarrhea. Genogroup (G)II infections were three time more frequent than GI. Eighteen genotypes were found; GII genotype 4 accounted for 41%. Median excretion duration was 34.5 days for GII versus 8.5 days for GI infection (p=0.0006). Repeat infections by the same genogroup were common but repeat infections by the same genotype were rare. Mean length-for-age Z-score at 12 months was lower among children with prior norovirus infection compared to uninfected children (coefficient: -0.33, 95%CI: -0.65 to -0.01, p=0.04); the effect persisted at 24 months. Conclusions. Norovirus infection occurs early in life and children experience serial infections with multiple genotypes, suggesting genotype-specific immunity. An effective vaccine would have a substantial impact on morbidity, but may need to target multiple genotypes.
    Clinical Infectious Diseases 01/2013; DOI:10.1093/cid/cit763 · 9.42 Impact Factor