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

Summary

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children’s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

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. 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.

Protocol

1. Stool Samples

1. Stool samples should be stored frozen to preserve the RNA. To make a 10% fecal suspension, take approximately 0.1 g of thawed stool sample and complete to 1 ml with PBS.
2. Aliquot in 200 μl to avoid repeated freezing and thawing. Store the aliquots at -70 °C.
3. Thaw and centrifuge aliquots at 4,000 g for 10 min before use in extraction.

2. Preparation of Silica Particles for Extraction with Guanidine & Silica

1. At RT, suspend 30 g of silicon dioxide and complete with dH₂O up to 250 mL.
2. After 24 hrs of sedimentation, remove 215 mL of the superna....

Discussion

Using the economic in-house method for isolating nucleic acid from stool samples, we obtain equal results as with the commercial QIAmp Viral RNA kit from Qiagen, and together with the TaqMan RT-PCR developed in our laboratory we can detect a broad range of NoV genotypes belonging to the GI and GII genogroup. A recent publication of the region C protocol reported genotyping rates of 78%⁶. Since the diversity of the contemporary NoV strains has been increasing during previous years, the success rate will depend .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Guanidine isothiocyanate	Sigma-Adrich	G9277
Tris HCL	Sigma-Aldrich	T5941
EDTA	Sigma-Aldrich	E5134
Silica	Sigma-Aldrich	S5631
Triton X 100	VWR	14530
Diethylpyrocarbonate	Sigma-Aldrich	D-5758
QIAamp viral RNA Mini Kit (250)	Qiagen	52906
QuantiTec Probe RT-PCR kit (200)	Qiagen	204443
Qiagen One Step RT-PCR Kit (200)	Qiagen	210212
Rnase Inhibitor 2000 units	A. Biosystems	N808-0119	2000 unids/vial
Non-Stick Rnae-free Microfuge Tubes	Ambion	AM12450
UltraPure Agarose 1000	Invitrogen	16550-100