TITLE: Mayaro virus real-time RT-PCR - 2016 method
AUTHORS: Judy Northill, Ian Mackay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mayaro virus (MAYV) is an Alphavirus, transmitted by mosquitos in south and central America.</div><div class = "text-block">This assay is designed to detect a portion of the nsP1 region of MAYV .</div></div>

[STEPS]
?. [Oligonucleotide sequences]
AB1Name 
5'-3'
2MAYV2016TM-FGCATCAGGHGAAGTCGTTG3MAYV2016TM-RCTGATCTGTGTGAAGGCAAA4MAYV2016TM-P6FAM-AGACGACCTGCAGTCAGTGATGGC-BHQ1
AB1Name 
5'-3'
2MAYV2016TM-FGCATCAGGHGAAGTCGTTG3MAYV2016TM-RCTGATCTGTGTGAAGGCAAA4MAYV2016TM-P6FAM-AGACGACCTGCAGTCAGTGATGGC-BHQ1
?. [Reaction set-up]
Assay has been used on both a Rotor-Gene 6000 / Rotor-Gene Q 5-plex using 100-place rotor discs.Total reaction volume is 20µL.Prepare sufficient for number of reaction plus a 'dead volume' usually 2 extra. Adjust as necessary if using a robotic dispenser.  ABC1Reagent
Vol (µL) x1
Final reaction concentration
2Nuclease free water4.37&nbsp;
3MAYV2016TM-F (200pmol/ul)0.09900nM4MAYV2016TM-R (200pmol/ul)0.07700nM5MAYV2016TM-P (100pmol/ul)0.03150nM62 X Reaction mix*101X7Superscipt III/Platinum Taq enzyme mix*0.48ROX reference dye (25uM)*0.0450nM9TOTAL VOLUME
15
&nbsp;
Dispense 15µL to each reaction well. Add 5µL of template, extracted DNA, controls or NTC (nuclease-free water).Total reaction volume is 20µL
ABC1Reagent
Vol (µL) x1
Final reaction concentration
2Nuclease free water4.37&nbsp;
3MAYV2016TM-F (200pmol/ul)0.09900nM4MAYV2016TM-R (200pmol/ul)0.07700nM5MAYV2016TM-P (100pmol/ul)0.03150nM62 X Reaction mix*101X7Superscipt III/Platinum Taq enzyme mix*0.48ROX reference dye (25uM)*0.0450nM9TOTAL VOLUME
15
&nbsp;
?. [Amplification]
The assay has been optimised and validated for the Rotor-Gene 6000 and Rotor-Gene Q thermocyclers.PCR  AB11 cycle40 cycles250°C&nbsp;&nbsp;&nbsp;&nbsp; 5min95°C 3 seconds395°C&nbsp;&nbsp;&nbsp;&nbsp; 2min60°C 30 seconds*
AB11 cycle40 cycles250°C&nbsp;&nbsp;&nbsp;&nbsp; 5min95°C 3 seconds395°C&nbsp;&nbsp;&nbsp;&nbsp; 2min60°C 30 seconds*
?. [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) and 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 value >40 cycles is considered a negative resultNTCs 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.