TITLE: Influenza A H3 virus TaqMan assay
AUTHORS: Ian Mackay, Judy Northill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This test is a modification to the World Health Organization's influenza A H3 TaqMan documented in 'WHO information for molecular diagnosis of influenza virus - update 1' (see file below). New primers were added and the WHO primers discarded.</div><div class = "text-block">The test targets the hemagglutinin (HA) gene.</div></div>

[STEPS]
?. 
?. [Oligonucleotides]
AB1Name5'-3' SEQUENCE2H3hFor1GGTACGGYTTCAGGCAT3H3hRev1TCAATCTGATGGAATTTCTCGTTG4H3h-1144dProbe6FAM-CTGCTGCTTGTCCTCTTCCCT-BHQ1New primers were designed to improve assay performance. http://www.who.int/entity/influenza/gisrs_laboratory/molecular_diagnosis_influenza_virus_humans_update_201403rev201505.pdf?ua=1
AB1Name5'-3' SEQUENCE2H3hFor1GGTACGGYTTCAGGCAT3H3hRev1TCAATCTGATGGAATTTCTCGTTG4H3h-1144dProbe6FAM-CTGCTGCTTGTCCTCTTCCCT-BHQ1
?. [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  Vol (µL) X1
  Final reaction concentration   Nuclease-free water  4.43
      H3hFor1 (200pmol/µL) 0.05 500nM    H3hRev1 (200pmol/µL)
  0.05 500nM    H3h-1144dProbe (100pmol/µL)
  0.03 150nM   2X Reaction Mix1 10.0 1X   ROX reference Dye (25µM)1,2 0.04 50nM   SuperScriptTM III/PlatinumTM Taq Mix1 0.4     TOTAL 15     Dispense 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 water4.43
&nbsp;3H3hFor1 (200pmol/µL)0.05500nM4H3hRev1 (200pmol/µL)
0.05500nM5H3h-1144dProbe (100pmol/µL)
0.03150nM62X Reaction Mix110.01X7ROX reference Dye (25µM)1,20.0450nM8SuperScriptTM III/PlatinumTM Taq Mix10.4&nbsp;9TOTAL15&nbsp;Dispense 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 water4.43
&nbsp;3H3hFor1 (200pmol/µL)0.05500nM4H3hRev1 (200pmol/µL)
0.05500nM5H3h-1144dProbe (100pmol/µL)
0.03150nM62X Reaction Mix110.01X7ROX reference Dye (25µM)1,20.0450nM8SuperScriptTM III/PlatinumTM Taq Mix10.4&nbsp;9TOTAL15&nbsp;
?. [Amplification]
RT-PCR  ABC150°C5min&nbsp;295°C2min&nbsp;3&nbsp;&nbsp;&nbsp;495°C3s40X560°C30s*&nbsp;
ABC150°C5min&nbsp;295°C2min&nbsp;3&nbsp;&nbsp;&nbsp;495°C3s40X560°C30s*&nbsp;
?. [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.