Nov 28, 2017
Version 4
Human Parechovirus A real-time RT-PCR ["Nix assay"; 2017-] V.4
- 1The University of Queensland
Protocol Citation: Ian M Mackay 2017. Human Parechovirus A real-time RT-PCR ["Nix assay"; 2017-]. protocols.io https://dx.doi.org/10.17504/protocols.io.k2kcycw
Manuscript citation:
The oligonucleotides used in this assay have been previoulsy published.
Nix WA, Maher K, Johansson ES, Niklasson B, Lindberg AM, Pallansch MA, Oberste MS. Detection of all known parechoviruses by real-time PCR. J Clin Microbiol.46(8):2519-2524.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: November 28, 2017
Last Modified: March 27, 2018
Protocol Integer ID: 9004
Keywords: parechovirus, respiratory virus, RT-PCR, real-time PCR, rtPCR
Abstract
This version is based on a recipe (see forked version) my team and I used this assay between 2008-2015; we dubbed it the 'Nix assay'. It targets the 5'UTR and employs quite degenerate oligos.
In silico sequence alignments indicated the olignucleotides could theoretically detect aft least HPeV 1-7, 17 and 18. Further validation of this recipe is ongoing.
However during a period of assay comparison, another assay (see link below), the 'Benschop assay' (J.Clin.Virol. 2008. 41(2):69-74), was found to produce more sigmoidal and higher curves and 1-5 cycle improvements to CT values when compared among the sample sampe extract set.
Guidelines
- This protocol assumes the user is familiar with working in a laboratory, with PCR, the thermocycler and software used to run it
- This protocol should be re-evaluated if being used with different reagents, if the oligonucleotide sequences are changed or if the cycling conditions are altered
Materials
STEP MATERIALS
SuperScript™ III Platinum™ One-Step qRT-PCR KitLife TechnologiesCatalog #11732088
SuperScript™ III Platinum™ One-Step qRT-PCR KitLife TechnologiesCatalog #11732088
Protocol materials
SuperScript™ III Platinum™ One-Step qRT-PCR KitLife TechnologiesCatalog #11732088
SuperScript™ III Platinum™ One-Step qRT-PCR KitLife TechnologiesCatalog #11732088
SuperScript™ III Platinum™ One-Step qRT-PCR KitLife TechnologiesCatalog #11732088
Oligonucleotides...
Oligonucleotides...
| Name | 5'-3' oligonucleotide sequence | |
| AN345_panHPeV/LV (sense primer) | GTAACASWWGCCTCTGGGSCCAAAAG | |
| AN344_panHPeV/LV (antisense probe) | GGCCCCWGRTCAGATCCAYAGT | |
| AN257_HPeV/LV (probe) | FAM-CCTRYGGGTACCTYCWGGGCATCCTTC-BHQ1 |
Reagents
Reagents
SuperScript™ III Platinum™ One-Step qRT-PCR KitLife TechnologiesCatalog #11732088
Reaction setup...
Reaction setup...
Below is the reaction setup for a single RT-PCR reaction.
Ideally, this work is conduct in a laboratory separate to any space used to perform PCR, molecular cloning or the analysis or high concentration DNA.
This volume has been used in 0.1-0.2ml tubes or various other connected tube configurations such as 100-place rings.
Multiply this according to the number of reactions you will need, remembering to include a positive control and at least two non-template controls (NTCs)
You may also need to allow some extra volume, depending on the method used to pipette mix into tubes for the run. For example, some robot-loaded tubes can require two reaction 'dead volumes'.
| Reagent (stock concentration) | Vol (μL) / reaction | Final concentration | |
| Nuclease free water | 4.47 | N/A | |
| AN345_panHPeV/LV (200pmol/ul) | 0.03 | 300nM | |
| AN344_panHPeV/LV (200pmol/ul) | 0.03 | 300nM | |
| AN257_HPeV/LV FAM-BHQ1 (100pmol/ul) | 0.03 | 150nM | |
| 2X Reaction Mix1 | 10 | 1X | |
| Rox Reference Dye 25mM1 | 0.04 | 50nM | |
| SuperScript® III/Platinum® Taq Mix1 | 0.4 | 1X | |
| Template extract RNA | 5 | N/A | |
| Final volume | 20µl |
1SuperScript® III Platinum® One-Step qRT-PCR Kit, Cat No. 11732088
Amplification...
Amplification...
This assay has been optimized for use with a Rotor-Gene 6000 or Rotor-Gene Q thermal cycler.
The cycling conditions are as follows:
| RT-PCR | |
| 50°C 5min 95°C 2min 95°C 3s 40X 60°C 30s* |
*Florescence acquisition step
Result calling...
Result calling...
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, curves and reaches a horizontal plateau phase
- A 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
- A flat or non-sigmoidal curve or a curve that crosses the threshold with a CT value >40 cycles is considered a negative result
- No template contorls (NTCs; water instead of specimen extract) 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.