TITLE: Enterovirus (EV) D68 TaqMan 2018 (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 and 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 original 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 sequencing. 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-For2TTCCCACGGTTGAAARYRAC4EV-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) Some of the design philosophies behind these modifications included ensuring that our assay could detect old and new variants of EV-D68 and reducing 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-Rev0.09900nM6EV-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.