TITLE: Aichivirus real-time RT-PCR 2007 method
AUTHORS: Judy A. Northill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A real-time assay to detect </span><span style = "font-style:italic;">Aichivirus A</span><span> (AiV-A) in human samples.  It is based in the 3'UTR region and has been employed in our laboratory since 2007.</span></div></div>

[STEPS]
?. Oligonucleotide sequences  AB1Name5'-3'2AiV-F-8046TGCTTCGGCACGCTTAGTT3AiV-R-8151TGCARTACAACCAYGGCTTAGG4AiV-8082-FAM6FAM-CACTCCTCCATGGTGATATAAAGACCAC-TAMRA
AB1Name5'-3'2AiV-F-8046TGCTTCGGCACGCTTAGTT3AiV-R-8151TGCARTACAACCAYGGCTTAGG4AiV-8082-FAM6FAM-CACTCCTCCATGGTGATATAAAGACCAC-TAMRA
?. Reagents
?. Reaction Set-upAssay has been used on both a Rotor-Gene 6000 / Rotor-Gene Q 5-plex using 100-place rotor discs and a ABI 7500 Fast real-time machine.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.  ABC1ReagentVol μl (x1)Final Reaction concentration2Nuclease-free water4.413Primer AiV-F-8046 (100pmol/ul)0.06300nM4Primer AiV-R-8151 (100pmol/ul)0.06300nM5Probe AiV-8082-FAM (100pmol/ul)0.03150nM62 X Reaction mix*10.01X7ROX reference dye (25μM)*0.0450nM8Superscript III/Platinum Taq enzyme mix*0.49TOTAL15Dispense 15µL to each reaction well. Add 5µL of template, extracted RNA, controls or NTC (nuclease-free water).Total reaction volume is 20µL
ABC1ReagentVol μl (x1)Final Reaction concentration2Nuclease-free water4.413Primer AiV-F-8046 (100pmol/ul)0.06300nM4Primer AiV-R-8151 (100pmol/ul)0.06300nM5Probe AiV-8082-FAM (100pmol/ul)0.03150nM62 X Reaction mix*10.01X7ROX reference dye (25μM)*0.0450nM8Superscript III/Platinum Taq enzyme mix*0.49TOTAL15
?. Amplification  AB11 cycle40 cycles250°C 5 minutes95°C 3 seconds395°C 2 minutes60°C 30 seconds*
AB11 cycle40 cycles250°C 5 minutes95°C 3 seconds395°C 2 minutes60°C 30 seconds*
?. Result analysisThe 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.