TITLE: Measles Vaccine Virus Taqman-MGB
AUTHORS: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This previously unpublished protocol aims to amplify genotype A measles virus (MeV) strains but not non-mealses viruses.</div><div class = "text-block">Mitchell Finger and Michael Lyon developed this in-house test in 2010.</div><div class = "text-block">The assay targets the intergenic region between the M (matrix) and F (fusion) genes, designed as a qualitative test for investigating measles vaccine virus (MVV) strains.</div><div class = "text-block">Numbering indicates the oligonucleotide location on the sequence with MeV strain Edmonston (Moraten vaccine), complete genome, GenBank accession number AF266287.</div></div>

[STEPS]
?. [Oligonucleotide sequences]
AB1NameSequence 5'-3'2Measles F 4729 VacAAACCCCCAGCAATTGGAA3Measles R 4795 VacGGTCACCTCGGTCGCTTGT
4Measles Probe 4757FAM - CCCTCTTCCTCAACACA - MGBNFQ
AB1NameSequence 5'-3'2Measles F 4729 VacAAACCCCCAGCAATTGGAA3Measles R 4795 VacGGTCACCTCGGTCGCTTGT
4Measles Probe 4757FAM - CCCTCTTCCTCAACACA - MGBNFQ
?. [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.MIX PREPARATION   Reagent Volume (µl) x1 Final reaction concentration   Nuclease-free water 4.42 N/A   Measles F 4729 Vac 150pmol/µl 0.04 300nM   Measles R 4795 Vac 150pmol/µl 0.04 300nM   Measles Probe FAM 100pmol/µl 0.06 300nM   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 well.  Add 5µL of template (extracted RNA, controls or NTC [nuclease-free water] ).  Total reaction volume is 20µL  ABC1ReagentVolume (µl) x1Final reaction concentration2Nuclease-free water4.42N/A3Measles F 4729 Vac 150pmol/µl0.04300nM4Measles R 4795 Vac 150pmol/µl0.04300nM5Measles Probe FAM 100pmol/µl0.06300nM62X 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 water4.42N/A3Measles F 4729 Vac 150pmol/µl0.04300nM4Measles R 4795 Vac 150pmol/µl0.04300nM5Measles Probe FAM 100pmol/µl0.06300nM62X 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.