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.

1. Stool Samples
<ol>
 <li>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. </li>
 <li>Aliquot in 200 &mu;l to avoid repeated freezing and thawing. Store the aliquots at -70 &deg;C. </li>
 <li>Thaw and centrifuge aliquots at 4,000 g for 10 min before use in extraction. </li>
</ol>
2. Preparation of Silica Particles for Extraction with Guanidine &amp; Silica
<ol>
 <li>At RT, suspend 30 g of silicon dioxide and complete with dH2O up to 250 mL. </li>
 <li>After 24 hrs of sedimentation, remove 215 mL of the supernatant by suction. Add dH2O up to 250 mL, and suspend the silica pellet by shaking. </li>
 <li>After another 24 hrs, remove the supernatant by suction, and adjust the pH of the silica suspension to pH 2.0, using hydrochloric acid (HCl). Aliquot the silica suspension in glass bottles and autoclave. </li>
</ol>
3. Preparation of L6 Buffer for Extraction with Guanidine &amp; Silica
<ol>
 <li>At RT, prepare L6 buffer by dissolving 60 g of guanidinium thiocyanate (GuSCN) and 1.3 g of Triton X-100 in 50 mL of 0.1 M Tris hydrochloride, pH 6.4, and 11 mL of a 0.2 M EDTA, pH 8.0 solution. </li>
</ol>
4. Preparation of L2 Buffer for Extraction with Guanidine &amp; Silica
<ol>
 <li>At RT, prepare L2 buffer by dissolving 180 g of guanidinium thiocyanate (GuSCN) in 150 mL of 0.1 M Tris hydrochloride, pH 6.4. </li>
</ol>
5. Extraction Procedure 
<ol>
 <li>In each extraction a NoV positive stool sample, as positive control, and DEPC treated water, as negative control, should be included. </li>
 <li>In a micro-centrifuge tube mix the following; 1 mL of buffer L6, 20 &mu;L of silica particles, and add 200 &mu;L of the 10% fecal extract suspension. Vortex briefly and leave at RT for 15 min. </li>
 <li>Centrifuge the suspension at 6000 g for 10 s and wash the pellet with 1ml of buffer L2 twice, followed by two washes with 70% ethanol and one time with acetone. Dry the pellet in a dry heating block at 56 &deg;C for 5 min. </li>
 <li>Hydrate the RNA in the silica pellet by adding 50 &mu;L of RNase-free dH20. Add 1 &mu;L of RNasin, mix by inverting the tube and incubate at 56 &deg;C for 15 min. </li>
 <li>Centrifuge for 3 min at full speed, in a tabletop centrifuge, and collect 40 &mu;L of the supernatant containing the RNA. </li>
 <li>Quantify the extracted RNA samples using Nanodrop 2000 spectrophotometer (Thermo scientific). Then store at -70 &deg;C until use, up to 6 months. (Note: A better way to preserve total RNA intact is converting it into cDNA).</li>
</ol>
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:
<ol>
 <li>Pipet 560 &mu;L of Buffer AVL containing the carrier RNA into a 1.5 mL tube, and add 140 &mu;L of the 10% stool suspension. Mix by pulse-vortexing for 15 sec. Incubate at RT for 10 min. </li>
 <li>Add 560 &mu;L of ethanol (96-100%) to the sample and mix by pulse-vortexing for 15 sec, then briefly centrifuge. </li>
 <li>Apply 630 &mu;L of this solution to a spin column placed in a 2 mL collection tube. Centrifuge for 1 min at 6,000 g; then place the spin column into a clean 2 mL tube. </li>
 <li>Wash the column containing bound RNA with 500 &mu;L of Buffer AW1 and centrifuge at 6,000 g for 1 min. Place column in a clean 2 mL collection tube. </li>
 <li>Wash the column with 500 &mu;L of Buffer AW2 and centrifuge at full speed (20,000 g or 14,000 rpm) for 3 min. </li>
 <li>Place spin column in a clean 1.5 mL micro-centrifuge tube, add 40 &mu;L DEPC-treated water and elute the RNA at full speed. Repeat once for a final 80 &mu;L of eluted RNA. </li>
 <li>Quantify the extracted RNA samples using Nanodrop 2000 spectrophotometer (Thermo scientific). Then store at -70 &deg;C until use, up to 6 months. (Note: A better way to preserve total RNA intact is converting it into cDNA).</li>
</ol>
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
<ol>
 <li>Wipe and Clean all work surfaces, pipettes, and centrifuges with RNase AWAY to remove any potential RNase contamination. </li>
 <li>Pipet the NoV GI and GII screening master mixes according to Table 1. Primers and Taqman probes are listed in Table 2. </li>
 <li>Aliquot 10 &mu;L of appropriate master mix to each reaction tube. </li>
 <li>Add 5 &mu;L of undiluted unknown sample RNA, DEPC-treated water as negative control, or GI respectively GII positive control RNA, to the corresponding reaction wells. All samples should be run in duplicate. Positive controls and standards with concentration of 300 &mu;g/&mu;L and 500 &mu;g/&mu;L respectively were provided by National Calicivirus Laboratory Center for Disease Control and Prevention (CDC). </li>
 <li>Centrifuge the reaction plate at 6,000 g for 10 sec. </li>
 <li>In the experiment properties screen; define and select a type of experiment for the run. Make sure TaqMan reagents are displayed as the reagents type, and that Pre-PCR read and amplification are selected. </li>
 <li>Run 15 &mu;L reactions using the thermal profile in Table 3. </li>
 <li>Analyze the results. </li>
</ol>
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 
<ol>
 <li>Before genotyping, the genogroup (GI or GII) of the samples are determined by the screening RT-PCR described above. GI NoV RNA has to be amplified with the GI genotyping master mix and the GII NoV RNA with the GII genotyping master mix. </li>
 <li>Pipet the NoV GI and GII genotyping master mixes according to Table 4. Primers GI SKF/SKR and G2 SKF/SKR are listed in Table 5. </li>
 <li>Aliquot 45 &mu;L of the appropriate master mix to 0.2 mL reaction tubes. </li>
 <li>Add 5 &mu;L of DEPC-treated water to each negative control tube, and 5 &mu;L of pre-screened, NoV (GI and/or GII) positive RNA to the corresponding reaction tube. </li>
 <li>Run 50 &mu;L reactions using the thermal profile in Table 6. </li>
 <li>Send PCR products containing at least 100 ng/&mu;L of the amplified capsid region to Macrogen Company for purification and sequencing. (9700 Great Seneca Hwy. Rockville, MD 20850 and $10 per sample per sequencing). </li>
</ol>
9. Representative Results
<img src="/files/ftp_upload/3232/3232fig1.jpg" alt="Figure 1" /> 
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. <a href="https://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/3232/3232fig2.jpg" alt="Figure 2" /> 
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. 
 <table border="1">
 <tbody>
 <tr>
 <td>Master Mix </td>
 <td>Final Conc</td>
 <td>Volume (&mu;L)</td>
 </tr>
 <tr>
 <td>H20 PCR</td>
 <td>&nbsp;</td>
 <td>2.875</td>
 </tr>
 <tr>
 <td>Quantitect RT-PCR Master Mix</td>
 <td>1x</td>
 <td>6.250</td>
 </tr>
 <tr>
 <td>RT Mix</td>
 <td>1x</td>
 <td>0.125</td>
 </tr>
 <tr>
 <td>50 &mu;M Cog R primer</td>
 <td>1 &mu;M</td>
 <td>0.250</td>
 </tr>
 <tr>
 <td>50 &mu;M Cog F primer</td>
 <td>1 &mu;M</td>
 <td>0.250</td>
 </tr>
 <tr>
 <td>10 &mu;M Ring probe</td>
 <td>1 &mu;M</td>
 <td>0.125/0.250*</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>10.00</td>
 </tr>
 </tbody>
 </table>
 * 0.250 &mu;L of each probe was used for GI testing, while 0.125 &mu;L of each probe was used for GII testing.
Table 1. Qiagen Quantitect master mix for screening GI and GII (Trujillo et al., 2006).7
 <table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Geno- group</td>
 <td>Use</td>
 <td>Sequence (5' to 3')</td>
 </tr>
 <tr>
 <td>Cog 1R</td>
 <td>GI</td>
 <td>Primer</td>
 <td>CTT AGA CGC CAT CAT CAT TYA C</td>
 </tr>
 <tr>
 <td>Cog 1F</td>
 <td>GI</td>
 <td>Primer</td>
 <td>CGY TGG ATG CGN TTY CAT GA</td>
 </tr>
 <tr>
 <td>Cog 2R</td>
 <td>GII</td>
 <td>Primer</td>
 <td>TCG ACG CCA TCT TCA TTC ACA</td>
 </tr>
 <tr>
 <td>Cog 2F</td>
 <td>GII</td>
 <td>Primer</td>
 <td>CAR GAR BCN ATG TTY AGR TGG ATG AG</td>
 </tr>
 <tr>
 <td>Ring 1A</td>
 <td>GI</td>
 <td>Probe</td>
 <td>FAM-AGA TYG CGA TCY CCT GTC CA-BHQ-1</td>
 </tr>
 <tr>
 <td>Ring 1B</td>
 <td>GI</td>
 <td>Probe</td>
 <td>FAM-AGA TCG CGG TCT CCT GTC CA-BHQ-1</td>
 </tr>
 <tr>
 <td>Ring2-TP</td>
 <td>GII</td>
 <td>Probe</td>
 <td>FAM-TGG GAG GGC GAT CGC AAT CT-BHQ-1</td>
 </tr>
 </tbody>
</table>
Table 2. Primer and probe oligonucleotides used for real-time quantitative RT-PCR for genogroups I, II (Kageyama et al., 2003).8
 <table border="1">
 <tbody>
 <tr>
 <td>Step</td>
 <td>Temp (&deg;C)</td>
 <td>Time (min)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1</td>
 <td>50 </td>
 <td>30:00</td>
 <td>cDNA synthesis</td>
 </tr>
 <tr>
 <td>2</td>
 <td>95</td>
 <td>15:00</td>
 <td>HotStart Taq polymerase activation</td>
 </tr>
 <tr>
 <td>3</td>
 <td>95</td>
 <td>00:15</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>60</td>
 <td>01:00</td>
 <td>45X cycles</td>
 </tr>
 </tbody>
 </table>
Table 3. Thermal profile for One-step Taqman Real time RT-PCR (Trujillo et al., 2006).7
 <table border="1">
 <tbody>
 <tr>
 <td>Master Mix </td>
 <td>Final Conc</td>
 <td>Volume (&mu;L)</td>
 </tr>
 <tr>
 <td>H20 PCR</td>
 <td>&nbsp;</td>
 <td>29.50</td>
 </tr>
 <tr>
 <td>5X Qiagen RT-PCR Buffer</td>
 <td>1x</td>
 <td>10.00</td>
 </tr>
 <tr>
 <td>10 mM dNTP Mix</td>
 <td>0.4 mM</td>
 <td>2.00</td>
 </tr>
 <tr>
 <td>Enzyme Mix</td>
 <td>&nbsp;</td>
 <td>2.00</td>
 </tr>
 <tr>
 <td>40 U/&mu;L RNAsin</td>
 <td>20 U/&mu;L</td>
 <td>0.50</td>
 </tr>
 <tr>
 <td>10 &mu;M SKF</td>
 <td>0.1 &mu;M</td>
 <td>0.50</td>
 </tr>
 <tr>
 <td>10 &mu;M SKR</td>
 <td>0.1 &mu;M</td>
 <td>0.50</td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>45.00</td>
 </tr>
 </tbody>
 </table>
Table 4. Qiagen One-Step RT-PCR master mix for genotyping GI and GII.
 <table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Geno- group</td>
 <td>Use</td>
 <td>Sequence (5' to 3')</td>
 </tr>
 <tr>
 <td>G1SKF</td>
 <td>GI</td>
 <td>Primer</td>
 <td>5'- CTG CCC GAA TTY GTA AAT GA - 3</td>
 </tr>
 <tr>
 <td>G1SKR</td>
 <td>GI</td>
 <td>Primer</td>
 <td>5'- CCA ACC CAR CCA TTR TAC A -'3</td>
 </tr>
 <tr>
 <td>G2SKF</td>
 <td>GII</td>
 <td>Primer</td>
 <td>5'- CNT GGG AGG GCG ATC GCA A - 3</td>
 </tr>
 <tr>
 <td>G2SKR</td>
 <td>GII</td>
 <td>Primer</td>
 <td>5'- CCR CCN GCA TRH CCR TTR TA CAT- 3</td>
 </tr>
 </tbody>
 </table>
Table 5. Primers used for amplification of Region C of Capsid Region (Kojima et al., 2002).5
 <table border="1">
 <tbody>
 <tr>
 <td>Step</td>
 <td>Temp (&deg;C)</td>
 <td>Time (min)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1</td>
 <td>60</td>
 <td>30:00</td>
 <td>cDNA synthesis</td>
 </tr>
 <tr>
 <td>2</td>
 <td>96</td>
 <td>15:00</td>
 <td>HotStart Taq polymerase activation</td>
 </tr>
 <tr>
 <td>3</td>
 <td>94</td>
 <td>00:30</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>52</td>
 <td>01:00</td>
 <td>40X cycles</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>72</td>
 <td>00:30</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>4</td>
 <td>72</td>
 <td>10:00</td>
 <td>Annealing</td>
 </tr>
 </tbody>
 </table>
Table 6. Thermal profile for Taqman One-Step RT-PCR.

<ol>
	<li>Medici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+epidemiology+of+Norovirus+infections+in+sporadic+cases+of+viral+gastroenteritis+among+children+in+Northern+Italy.">Molecular epidemiology of Norovirus infections in sporadic cases of viral gastroenteritis among children in Northern Italy.</a> L. Medical Virology. 78, (2006).</li><li>Vidal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+recombinant+Norovirus+causing+outbreaks+of+gastroenteritis+in+Santiago,+Chile.">Novel recombinant Norovirus causing outbreaks of gastroenteritis in Santiago, Chile.</a> J. Clinica Microbiology. 4, (2006).</li><li>Xavier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+caliciviruses+associated+with+acute+infantile+gastroenteritis+in+Salvador,+an+urban+center+in+Northeast+Brazil.">Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil.</a> Braz. J. Med. Biol. Res. 42, (2009).</li><li>Boom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J. Clin. Microbiol. 28, 495-503 (1990).</li><li>Kojima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genogroup-specific+PCR+primers+for+detection+of+Norwalk-like+viruses.">Genogroup-specific PCR primers for detection of Norwalk-like viruses.</a> J. Virol. Methods. 100, 107-114 (2002).</li><li>Mattison, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+comparison+of+two+norovirus+ORF2-based+genotyping+protocols.">Multicenter comparison of two norovirus ORF2-based genotyping protocols.</a> J. Clin. Microbiol. 47, 3927-3932 (2009).</li><li>Trujillo, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+TaqMan+real-time+reverse+transcription-PCR+for+rapid+detection,+quantification,+and+typing+of+norovirus.">Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus.</a> J. Clin. Microbiol. 44, 1405-1412 (2006).</li><li>Kageyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broadly+reactive+and+highly+sensitive+assay+for+Norwalk-like+viruses+on+real-time+quantitative+RT-PCR.">A broadly reactive and highly sensitive assay for Norwalk-like viruses on real-time quantitative RT-PCR.</a> J. Clin. Microbiol. 41, 1548-1557 (2003).</li></ol>

No conflicts of interest declared.

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 ...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Guanidine isothiocyanate</td>
 <td>Sigma-Adrich</td>
 <td>G9277</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris HCL</td>
 <td>Sigma-Aldrich</td>
 <td>T5941</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E5134</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Silica</td>
 <td>Sigma-Aldrich</td>
 <td>S5631</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triton X 100</td>
 <td>BDH Chemicals</td>
 <td>14530</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Diethylpyrocarbonate</td>
 <td>Sigma-Aldrich</td>
 <td>D-5758</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>QIAamp viral RNA Mini Kit (250)</td>
 <td>QIAGEN</td>
 <td>52906</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>QuantiTec Probe RT-PCR kit (200)</td>
 <td>QIAGEN</td>
 <td>204443</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Qiagen One Step RT-PCR Kit (200)</td>
 <td>QIAGEN</td>
 <td>210212</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rnase Inhibitor 2000 units</td>
 <td>A.Biosystems</td>
 <td>N808-0119</td>
 <td>2000 unids/vial</td>
 </tr>
 <tr>
 <td>Non-Stick Rnae-free Microfuge Tubes</td>
 <td>Ambion</td>
 <td>AM12450</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>UltraPure Agarose 1000</td>
 <td>Invitrogen</td>
 <td>16550-100</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

detection and genogrouping of noroviruses from children's stools by taqman one-step rt-pcr

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.

Watch this Scientific Journal Video about Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR at JoVE.com

Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;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.

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

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

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17.1K Views.  Universidad Peruana Cayetano Heredia. 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.

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

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...