TITLE: Enterovirus (EV) D68 real-time RT-PCR (EV-D68-TM2018)
AUTHORS: Ian Mackay, Judy Northill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol aims to amplify enterovirus (EV) D68 viruses but not other viruses.</div><div class = "text-block">This protocol is modified from a previously published method cited below. Details of the modification are included in the method.</div><div class = "text-block">The oligonucleotides target the 5'UTR noncoding region. This is a qualitative test for investigating EV-D68 infection of humans.</div><div class = "text-block"><span>The test has identified both the historical EV-D68 strains and contemporary strains which has been confirmed by subgenomic sequencing of </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">partial 5'UTR-VP2</span></a><span> and partial VP1 regions. Culture of the virus is not required as this assay is capable of detecting EV-D68 direct from extracted clinical samples.</span></div></div>

[STEPS]
?. [Oligonucleotide sequences]
AB1NameSequence 5'-3'2EV-D68-For1TGTTYCCACGGTTGAAAAYAA3EV-D68-For2TTCCCACGGTTGAAARNYRAC4EV-D68-RevCAAGCTACACACGGGTTAGT5EV-D68-FAM-TM2018FAM - CCGTTAWCCGCTATAGTACTTCGAGAAACC - BHQ1MODIFICATIONS TO THE PUBLISHED ASSAY:Two modified forward primers, targeting the same region as the original assays single primer, included different configurations of degenerate basesAn entirely new reverse primer design and locationOne new FAM-labelled, degenerate, extended exonuclease or "TaqMan" oligoprobe replacing the 2x original assay's VIC-labelled TaqMan-MGB oligoprobesConditions, concentrations and reagents used all differ from those originally publishedAddition of in vitro transcribed synthetic template oligonucleotide controls (PRIMER and PROBE controls) DESIGN PHILOSOPHIES:Ensure that our assay could detect old and new variants of EV-D68Reduce the total number of degenerate positions in any single primer.
?. [Reagents]
?. [Reaction set-up]
The assay has been used on both a Rotor-Gene 6000 and a Rotor-Gene Q real-time thermocyclerPrepare sufficient mix for the number of reactions.Include a suitable 'dead volume' as necessary if using a robotic dispenser.
  
 MSuperscriptIX PREPARATION   Reagent Volume (µl) x1 Final reaction concentration   Nuclease-free water 4.35 N/A   RRV E2 F 200pmol/µl 0.09 900nM   RRV E2 R3 200pmol/µl 0.09 900nM   BFV-FAM 100pmol/µl 0.03 150nM   2X Reaction Mix1 10 1X   SuperScript® III/Platinum® Taq Mix1 0.4 1X   ROX Reference Dye (25µM) 0.04 0.05µM   Template 5 N/A   TOTAL 20       Dispense 15µL to each reaction vessel.Add 5µL of template (extracted RNA, controls or no-template control [NTC; nuclease-free water] ).Total reaction volume is 20µL
   ABC1ReagentVolume (µl) x1Final reaction concentration2Nuclease-free water4.28N/A3EV-D68-For1 200pmol/µl0.09900nM4EV-D68-For2 200pmol/µl0.09900nM5EV-D68-Rev 200pmol/µl0.09900nM6EV-D68-FAM-TM2018 100pmol/µl0.0150nM72X Reaction Mix1101X8SuperScript® III/Platinum® Taq Mix10.41X9ROX Reference Dye (25µM)0.040.05µM10Template5N/A11TOTAL20&nbsp;Dispense 15µL to each reaction vessel.Add 5µL of template (extracted RNA, controls or no-template control [NTC; nuclease-free water] ).Total reaction volume is 20µL
?. [Amplification]
ABC150°C5min1X295°C2min1X3&nbsp;&nbsp;&nbsp;495°C3sec|40X560°C30sec1|
?. [Result Analysis]
The definition used for a satisfactory positive result from a real-time fluorogenic PCR should include each of the following:A sigmoidal curve – the trace travels horizontally, curves upward, continues in an exponential rise and followed by a curve towards a horizontal plateau phaseA suitable level of fluorescence intensity as measured in comparison to a positive control (y-axis)A defined threshold (CT) value which the fluorescent curve has clearly exceeded (Fig.1 arrow), which sits early in the log-linear phase and is A flat or non-sigmoidal curve or a curve that crosses the threshold with a CT >40 cycles is considered a negative result.NTCs should not produce a curve
  
 
 Figure 1. Examples of satisfactory sigmoidal amplification curve shape when considering an assay’s fluorescent signal output. The crossing point or threshold cycle (CT) is indicated (yellow arrow); it is the value at which fluorescence levels surpass a predefined (usually set during validation, or arbitrary) threshold level as shown in this normalized linear scale depiction. LP-log-linear phase of signal generated during the exponential part of the PCR amplification; TP-a slowing of the amplification and accompanying fluorescence signal marks the transition phase; PP-the plateau phase is reached when there is little or no increase in fluorescent signal despite continued cycling.