Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

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. 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. method 4 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.

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

“…1. Extract nucleic acids from the biological sample according to a previously described protocol 15 and manufacturer's protocol.…”

Burden of Norovirus and Rotavirus in Children After Rotavirus Vaccine Introduction, Cochabamba, Bolivia

“…Norovirus genogroup was detected with an in-house protocol utilizing TaqMan RT-PCR targeting the highly conserved open reading frame (ORF) 1-ORF2 junction region. 23 A sample was considered norovirus positive when cycle threshold (Ct) values were < 39 cycles. For genotyping, we applied conventional RT-PCR using primers described by Kojima and others 24 that target the highly variable N-terminal shell of the capsid protein VP1 (Region C).…”

Minimally Invasive Saliva Testing to Monitor Norovirus Infection in Community Settings

“…RNA was extracted from a 10% wt/vol suspension of stool in phosphate-buffered saline using the Qiagen QIAmp viral RNA kit according to the manufacturer’s instructions and tested for norovirus GI and GII by RT-qPCR [24]. Samples were considered positive if the cycle threshold for the sample was ≤37 for norovirus GI and ≤39 for GII.…”