TITLE: Nipah virus real-time RT-PCR (NiV-TM2018)
AUTHORS: Ian Mackay, Judy Northill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol aims to specifically amplify Nipah viruses (NiV) and not other viruses.</div><div class = "text-block">This is a modified version of a published assay. </div><div class = "text-block">Modifications were to account for mismatches underneath oligo target sites.</div><div class = "text-block">The assay targets the nucleoprotein (N) gene region and is designed as a qualitative test for investigating NiV infection of humans.</div></div>

[STEPS]
?. [Oligonucleotide sequences]
AB1NameSequence 5'-3'2NiV-N-TM2018_ForCTGGTCTCTGCAGTTATCACCATCGA3NiV-N-TM2018_RevACGTAYTTAGCCCATCTTCTAGTTTCA4NiV-N-TM2018_PrbFAM – CAGCTCCMGACACTGCCGAGGA– BHQ1MODIFICATIONS TO THE PUBLISHED ASSAY:Forward primer is unmodified from that described in the original paperReverse primer has a degeneracy added to the published versionProbe has a degenercay added and the 3' terminal T removed, compared to the published versionConditions, concentrations and reagents used all differ from those originally publishedAddition of in vitro transcribed synthetic template oligonucleotide controls (PRIMER and PROBE controls) THE DESIGN PHILOSOPHY:
?. [Reagents]
?. [Reaction set-up]
The assay has been used on a Rotor-Gene 6000 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 4.35 N/A   RRV E2 F 200pmol/µl 0.09 900nM   RRV E2 R3 200pmol/µl 0.09 900nM   BFV-FAM 100pmol/µl 0.03 150nM   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 water4.39N/A3NiV-N-TM2018_For 200pmol/µl0.05500nM4NiV-N-TM2018_Rev 200pmol/µl0.09900nM5NiV-N-TM2018_Prb 100pmol/µl0.03150nM62X 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
?. [Amplification]
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 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.