TITLE: Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay")
AUTHORS: Ian Mackay, Judy Northill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is my preferred, previously published [Ref 1], rhinovirus (RV) and enterovirus (EV) genotyping assay when seeking to identify the genotype of a respiratory picornavirus detected in a clinical sample extract. It is employed after use of a screening real-time RT-PCR has identified a respiratory picornavirus.</div><div class = "text-block"><span>I have not confirmed that it can detect every single RV genotype but I do know that it detects </span><span style = "font-style:italic;">many</span><span> from each of the three RV species (</span><span style = "font-style:italic;">Human rhinovirus A</span><span>, </span><span style = "font-style:italic;">Human rhinovirus B</span><span> and </span><span style = "font-style:italic;">Human rhinovirus C</span><span>) as well as at least some </span><span style = "font-style:italic;">Human enterovirus</span><span> (EV) genotypes.</span></div><div class = "text-block">The assay picks up EVs due to the shared genetic similarities in the 5'UTR target region. EVs can be discriminated using subgenomic sequencing (see  VP42 typing assay protocol), or simply described as 'respiratory EVs' since there is no specific-specific vaccine or treatment available anyway. </div><div class = "text-block">This is a robust primary subgenomic sequencing assay. It is more sensitive than any VP1 protocols because it targets more conserved primer target sites. It produces a more reliable typing result than does the 5'UTR region alone. </div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/u6kdyxe.png" /></div></div>

[STEPS]
?. [Oligonucleotide sequences...]
ABC1&nbsp;NameSequence (5'-3')2RT-PCR(Round 1)HRV_HEV VP42 OSCCGGCCCCTGAATGYGGCTAA3HRV_HEV VP42 OASACATRTTYTSNCCAAANAYDCCCAT4PCR(Round 1)HRV_HEV VP42 IS
ACCRACTACTTTGGGTGTCCGTG5HRV_HEV VP42 IAS
TCWGGHARYTTCCAMCACCANCC
ABC1&nbsp;NameSequence (5'-3')2RT-PCR(Round 1)HRV_HEV VP42 OSCCGGCCCCTGAATGYGGCTAA3HRV_HEV VP42 OASACATRTTYTSNCCAAANAYDCCCAT4PCR(Round 1)HRV_HEV VP42 IS
ACCRACTACTTTGGGTGTCCGTG5HRV_HEV VP42 IAS
TCWGGHARYTTCCAMCACCANCC
?. [Reagents]
?. [Reaction set-up]
ROUND 1 REACTION MIXIdeally, set up more reaction mixes than you'll need for both rounds at the same timeStrips of 8x 0.2ml tubes are good for this useFreeze in a frost-free freezer at -20°C until needed  ABC1ReagentVol (μl) 1xFinal reaction concentration2Nuclease-free water4.08N/A3Reaction Mix (2X)10.001X4HRV_HEV VP42 OS 200pmol/µl0.06600nM5HRV_HEV VP42 OAS 200pmol/µl0.06600nM6SuperScript® III RT/ Platinum® Taq Enzyme Mix0.20Unknown7Template5.00N/ADispense 15µL to each reaction tubeTotal reaction volume will be 20µlPCR (Round 2)   Reagent Vol (μl) 1x Final reaction concentration   Nuclease-free water 8.7 N/A   MyTaq Reaction Buffer (5X) 4 1X   Primers (μM)1 3.8 380nM   MgCl2 1.4 4.75mM   MyTaq HS DNA Polymerase (5U/uL) 0.2 Unknown   1st round amplicon 2 N/A   Dispense 18µL into each reaction tubeTotal reaction volume will be 20µl  ABC1ReagentVol (μl) 1xFinal reaction concentration2Nuclease-free water7.92N/A3MyTaq Reaction Buffer (2X)10.001X4HRV_HEV VP42 IS 200pmol/µl0.038380nM5HRV_HEV VP42 IAS 200pmol/µI0.0384.75mM61st round amplicon2.00N/ADispense 18µL into each reaction tubeTotal reaction volume will be 20µl
ABC1ReagentVol (μl) 1xFinal reaction concentration2Nuclease-free water4.08N/A3Reaction Mix (2X)10.001X4HRV_HEV VP42 OS 200pmol/µl0.06600nM5HRV_HEV VP42 OAS 200pmol/µl0.06600nM6SuperScript® III RT/ Platinum® Taq Enzyme Mix0.20Unknown7Template5.00N/A
ABC1ReagentVol (μl) 1xFinal reaction concentration2Nuclease-free water7.92N/A3MyTaq Reaction Buffer (2X)10.001X4HRV_HEV VP42 IS 200pmol/µl0.038380nM5HRV_HEV VP42 IAS 200pmol/µI0.0384.75mM61st round amplicon2.00N/A
?. [Amplification: ROUND 1, RT-PCR]
This reaction has been run using a SimpliAmp (Applied Biosystems) thermal cyclerTransfer 5µl of nucleic acid extract (extracted RNA, controls or NTC [nuclease-free water]) to defrosted reaction tubes and cycle  ABC155°C20 min1X294°C2 min1X3&nbsp;&nbsp;&nbsp;494°C30 sec|550°C45 sec| 4X668°C50 sec|7&nbsp;&nbsp;&nbsp;894°C30 sec|940°C45 sec| 25X1068°C50 sec|111268°C5 min1X1315°C∞&nbsp;
ABC155°C20 min1X294°C2 min1X3&nbsp;&nbsp;&nbsp;494°C30 sec|550°C45 sec| 4X668°C50 sec|7&nbsp;&nbsp;&nbsp;894°C30 sec|940°C45 sec| 25X1068°C50 sec|111268°C5 min1X1315°C∞&nbsp;
?. [Amplification: ROUND 2, PCR]
This reaction has been run using a SimpliAmp (Applied Biosystems) thermal cyclerTransfer 2µl of 1:100 pre-diluted Round 1 amplicon into defrosted 0.2ml reaction tubes and cycle  ABC195°C1 min1X2395°C30 sec|4 50°C45 sec|  4X5 72°C50 sec|6795°C30 sec|8 40°C45 sec|  25X972°C50 sec|101172°C5 min1X1215°C∞
ABC195°C1 min1X2395°C30 sec|4 50°C45 sec|  4X5 72°C50 sec|6795°C30 sec|8 40°C45 sec|  25X972°C50 sec|101172°C5 min1X1215°C∞
?. [Amplicon visualisation]