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.
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.
1. Stool Samples
2. Preparation of Silica Particles for Extraction with Guanidine & Silica
3. Preparation of L6 Buffer for Extraction with Guanidine & Silica
4. Preparation of L2 Buffer for Extraction with Guanidine & Silica
5. Extraction Procedure
6. Alternative Extraction Using the Commercial RNA QIAamp Viral RNA Kit
Full instructions are found in the booklet provided with the QIAGEN kit. Briefly the procedure is as follows:
7. Detection of Norovirus in Extracted RNA by One-step Real Time RT-PCR with Specific Taqman Probes
Instrument StepOnePlus Real Time PCR Systems (Applied Biosystems).
Methodology
8. Genotyping of NoV GI and GII Using Conventional RT-PCR and Sequencing
(It’s not mentioned in this video since it’s a common and widely used method.)
Instrument. Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems with the Quantitec template.
Methodology
9. Representative Results
Figure 1 shows representative results from the Taqman One-Step RT-PCR when used to assay RNA extracted from stool samples from diarrheal children. The threshold cycle (Ct) for a positive sample was set at less or equal to Ct 37 for GI and Ct 39 for GII. The Ct-values for the positive controls were found to be less than 27 and 18 of the ORF1-ORF2 junction for GI and GII, respectively. Click here to view larger figure.
Figure 2 shows a head-to-head comparison of Ct values of in silica method guanidinium and QiAmp Viral RNA kit. Data of both tests fall very close to the equality line (slope = 1 and intercept = 0). This is confirmed by the Regression Analysis and the Correlation Coefficient. All negative controls were evaluated and found to be 0 by both methods.
Master Mix | Final Conc | Volume (μL) |
H20 PCR | 2.875 | |
Quantitect RT-PCR Master Mix | 1x | 6.250 |
RT Mix | 1x | 0.125 |
50 μM Cog R primer | 1 μM | 0.250 |
50 μM Cog F primer | 1 μM | 0.250 |
10 μM Ring probe | 1 μM | 0.125/0.250* |
10.00 |
* 0.250 μL of each probe was used for GI testing, while 0.125 μL of each probe was used for GII testing.
Table 1. Qiagen Quantitect master mix for screening GI and GII (Trujillo et al., 2006).7
Name | Geno- group | Use | Sequence (5′ to 3′) |
Cog 1R | GI | Primer | CTT AGA CGC CAT CAT CAT TYA C |
Cog 1F | GI | Primer | CGY TGG ATG CGN TTY CAT GA |
Cog 2R | GII | Primer | TCG ACG CCA TCT TCA TTC ACA |
Cog 2F | GII | Primer | CAR GAR BCN ATG TTY AGR TGG ATG AG |
Ring 1A | GI | Probe | FAM-AGA TYG CGA TCY CCT GTC CA-BHQ-1 |
Ring 1B | GI | Probe | FAM-AGA TCG CGG TCT CCT GTC CA-BHQ-1 |
Ring2-TP | GII | Probe | FAM-TGG GAG GGC GAT CGC AAT CT-BHQ-1 |
Table 2. Primer and probe oligonucleotides used for real-time quantitative RT-PCR for genogroups I, II (Kageyama et al., 2003).8
Step | Temp (°C) | Time (min) | |
1 | 50 | 30:00 | cDNA synthesis |
2 | 95 | 15:00 | HotStart Taq polymerase activation |
3 | 95 | 00:15 | |
60 | 01:00 | 45X cycles |
Table 3. Thermal profile for One-step Taqman Real time RT-PCR (Trujillo et al., 2006).7
Master Mix | Final Conc | Volume (μL) |
H20 PCR | 29.50 | |
5X Qiagen RT-PCR Buffer | 1x | 10.00 |
10 mM dNTP Mix | 0.4 mM | 2.00 |
Enzyme Mix | 2.00 | |
40 U/μL RNAsin | 20 U/μL | 0.50 |
10 μM SKF | 0.1 μM | 0.50 |
10 μM SKR | 0.1 μM | 0.50 |
45.00 |
Table 4. Qiagen One-Step RT-PCR master mix for genotyping GI and GII.
Name | Geno- group | Use | Sequence (5′ to 3′) |
G1SKF | GI | Primer | 5′- CTG CCC GAA TTY GTA AAT GA – 3 |
G1SKR | GI | Primer | 5′- CCA ACC CAR CCA TTR TAC A -‘3 |
G2SKF | GII | Primer | 5′- CNT GGG AGG GCG ATC GCA A – 3 |
G2SKR | GII | Primer | 5′- CCR CCN GCA TRH CCR TTR TA CAT- 3 |
Table 5. Primers used for amplification of Region C of Capsid Region (Kojima et al., 2002).5
Step | Temp (°C) | Time (min) | |
1 | 60 | 30:00 | cDNA synthesis |
2 | 96 | 15:00 | HotStart Taq polymerase activation |
3 | 94 | 00:30 | |
52 | 01:00 | 40X cycles | |
72 | 00:30 | ||
4 | 72 | 10:00 | Annealing |
Table 6. Thermal profile for Taqman One-Step RT-PCR.
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 on the mismatches of primers in region C for the specimens being tested. The assay is useful for routine diagnostic as it eliminates post-amplification product processing, thus shortening the turn around time7. Apart from diagnosis, this protocol can also be used for clarifying the epidemiology of NoV infections, thus being useful for public health control of this disease.
When running the screening RT-PCR, negative control (NC) reactions for primer/probe sets should not exhibit fluorescence growth curves that cross the threshold line. If a false positive occurs with one or more of the primer/probe set’s NC reactions, sample contamination may have occurred, and in all such cases, the whole run was invalidated and repeated with stricter adherence to the guidelines.
Cross reaction for the screening RT-PCR was tested using the GI and GII positive standards provided by CDC. No cross reaction was observed between GI primers and probes with GII RNA and vice versa. The reproducibility of the screening RT-PCR was high as Ct-values where similar for repeated runs with RNA from the positive controls. RNA from children stool samples that had been found positive for NoV and had Ct-values between 20 and 33 where subsequently amplified by the conventional RT-PCR and sent for genotyping. Five positive samples with Ct-values between 35 and 38 were not sent for sequencing as the viral load where found to be too low for subsequent amplification using the conventional RT-PCR.
Currently, the availability of norovirus diagnosis is limited because the methods are not able to be implemented in local laboratories with only basic equipment. This delays diagnosis and treatment in individual diarrhea cases or in outbreaks. The kit method is reliable and fast yet requires a significant amount of time and resources to transport materials into countries. The cost is three times that of our method. We have shown an auxiliary method using readily-available in-country reagents for the detection of NoV that is equally simple, fast, and reliable. This cost-effective method bypasses reliance on materials purchased abroad, the problems of international and intra-national regulations on its transportation which ultimately will lead to an accessible diagnosis and rapid treatment of one the most common viral causes of gastroenteritis in children.
The authors have nothing to disclose.
The authors would like to thank the National Calicivirus Laboratory Center for Disease Control and Prevention (CDC) for the kind gift of a standard and control positives for NoV, and Laboratories of the School of Public Health at Johns Hopkins for providing the reagents.
Name of the reagent | Company | Catalogue number | Comments |
Guanidine isothiocyanate | Sigma-Adrich | G9277 | |
Tris HCL | Sigma-Aldrich | T5941 | |
EDTA | Sigma-Aldrich | E5134 | |
Silica | Sigma-Aldrich | S5631 | |
Triton X 100 | BDH Chemicals | 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 |