TITLE: Measles virus TaqMan RT-PCR (F gene; no longer in regular use; see Guidelines)
AUTHORS: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">This real-time TaqMan-MGB RT-PCR protocol aimed to amplify measles virus (MeV) strains and not other viruses.</li><li style = "counter-reset:ol0;list-style-type:disc;">Michael Lyon and Mitchell Finger designed the assay in 2009 using Primer Express software.</li><li style = "counter-reset:ol0;list-style-type:disc;">The method was later published by Greg Smith in 2010 (see below).</li><li style = "counter-reset:ol0;list-style-type:disc;">The assay targets the fusion (F) gene region and is designed as a qualitative test for investigating MeV infection of humans.</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>This was a past assay that we no longer in use. For our favoured Measles virus TaqMan test, please refer to the </span><span style = "font-style:italic;">MeV N TaqMan</span><span> protocol.</span></li></ul></div></div>

[STEPS]
?. [Oligonucleotide sequences]
AB1NameSequence 5'-3'2Primer Measles MGB FPGCTCAAATTGCTCAGATACTATACAGAAA3Primer Measles MGB RPGCAGATATGGGGTCCCGTAA
4Probe Measles MGB ProbeFAM - CCTGTCATTATTTGGCC - MGBNFQ
AB1NameSequence 5'-3'2Primer Measles MGB FPGCTCAAATTGCTCAGATACTATACAGAAA3Primer Measles MGB RPGCAGATATGGGGTCCCGTAA
4Probe Measles MGB ProbeFAM - CCTGTCATTATTTGGCC - MGBNFQ
?. [Reagents]
?. [Reaction set-up]
The assay has been used on both a Rotor-Gene 6000 and a Rotor-Gene Q real-time thermocycler
  
 Prepare sufficient mix for the number of reactions.
 Include a suitable 'dead volume' as necessary if using a robotic dispenser.
  
   ABC1ReagentVolume (µl) x1Final reaction concentration2Nuclease-free water4.45N/A3Measles MGB FP 150pmol/µl0.04300nM4Measles MGB RP 150pmol/µl0.04300nM5Measles MGB Probe 100pmol/µl0.03155nM62X Reaction Mix1101X7SuperScript® III/Platinum® Taq Mix10.41X8ROX Reference Dye (25µM)0.040.05µM9Template5N/A10TOTAL20&nbsp;Dispense 15µL to each reaction well.Add 5µL of template (extracted RNA, controls or NTC [nuclease-free water] ).Total reaction volume is 20µL
ABC1ReagentVolume (µl) x1Final reaction concentration2Nuclease-free water4.45N/A3Measles MGB FP 150pmol/µl0.04300nM4Measles MGB RP 150pmol/µl0.04300nM5Measles MGB Probe 100pmol/µl0.03155nM62X Reaction Mix1101X7SuperScript® III/Platinum® Taq Mix10.41X8ROX Reference Dye (25µM)0.040.05µM9Template5N/A10TOTAL20&nbsp;
?. [Amplification]
ABC150°C5min1X295°C2min1X3&nbsp;&nbsp;&nbsp;495°C3sec|40X560°C30sec1|
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 curveFigure 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.